CRISPR-based gene editing is presently being tried in many clinical trials

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Clinical Trials ◽  
Cell Function ◽  
Therapeutic Window ◽  
Development Environment ◽  
Drug Dosing ◽  
Protein Levels ◽  
Cell Tissue ◽  
Rnp Complex ◽  
Dosing Regimens

CRISPR is a bacterial host defense system that may work as "molecular scissors" in eukaryotic cells to permanently modify genetic coding. Some barriers to using CRISPR as a therapeutic include guaranteeing adequate delivery of the RNP complex to the proper cell/tissue and showing safe and effective editing. Off-target editing (i.e., unwanted modification in a non-target DNA location) may result in a range of safety problems impacting normal cell function. The degree of cell editing events, including off-target modifications, is known to be altered by in vitro dosage and time of exposure to active RNP complexes. The safety of these drugs relies heavily on preventing unwanted mutations, off-target mutations, and any genomic rearrangements, all of which may have harmful implications.In some illnesses, a slight general adjustment of positive and negative protein levels may be sufficient to have a therapeutic impact. Understanding this therapeutic window will enable researchers to modify drug dosing regimens, especially for in vitro use, to obtain optimum pharmacodynamics with the fewest potential adverse effects. Most of the bioanalytical endpoints outlined for CRISPR are simple methods performed in most labs. Development teams will need to manage resources by selecting key exposure endpoints that deliver the greatest value from pharmacokinetics/PD and safety evaluations. Two in vitro delivery strategies have entered clinical trials in immune-privileged locations. The drug development environment will have to be altered in close coordination with regulatory agencies to construct need-to-know endpoints and pivotal trials to successfully move medicines forward in a safe and controlled way.

scholarly journals Hydroxychloroquine in Patients with Rheumatic Disease Complicated by COVID-19: Clarifying Target Exposures and the Need for Clinical Trials

2020 ◽  
Vol 47 (9) ◽  
pp. 1424-1430 ◽  
Author(s):  
Stephen J. Balevic ◽  
Christoph P. Hornik ◽  
Thomas P. Green ◽  
Megan E.B. Clowse ◽  
Daniel Gonzalez ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Antiviral Activity ◽  
Rheumatic Disease ◽  
Rheumatic Diseases ◽  
Pharmacokinetic Model ◽  
Therapeutic Window ◽  
Total Serum ◽  
Viral Inhibition ◽  
Target Exposure

Objective.To characterize hydroxychloroquine (HCQ) exposure in patients with rheumatic disease receiving longterm HCQ compared to target concentrations with reported antiviral activity against the coronavirus disease 2019 caused by SARS-CoV-2 (COVID-19).Method.We evaluated total HCQ concentrations in serum and plasma from published literature values, frozen serum samples from a pediatric systemic lupus erythematosus trial, and simulated concentrations using a published pharmacokinetic model during pregnancy. For each source, we compared observed or predicted HCQ concentrations to target concentrations with reported antiviral activity against SARS-CoV-2.Results.The average total serum/plasma HCQ concentrations were below the lowest SARS-CoV-2 target of 0.48 mg/l in all studies. Assuming the highest antiviral target exposure (total plasma concentration of 4.1 mg/l), all studies had about one-tenth the necessary concentration for in vitro viral inhibition. Pharmacokinetic model simulations confirmed that pregnant adults receiving common dosing for rheumatic diseases did not achieve target exposures; however, the models predict that a dosage of 600 mg once a day during pregnancy would obtain the lowest median target exposure for most patients after the first dose.Conclusion.We found that the average patient receiving treatment with HCQ for rheumatic diseases, including children and non-pregnant/pregnant adults, are unlikely to achieve total serum or plasma concentrations shown to inhibit SARS-CoV-2 in vitro. Nevertheless, patients receiving HCQ long term may have tissue concentrations far exceeding that of serum/plasma. Because the therapeutic window for HCQ in the setting of SARS-CoV-2 is unknown, well-designed clinical trials that include patients with rheumatic disease are urgently needed to characterize the efficacy, safety, and target exposures for HCQ.

scholarly journals Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro†

2020 ◽  
Author(s):  
Aslihan Turhan ◽  
Miguel Tavares Pereira ◽  
Gerhard Schuler ◽  
Ulrich Bleul ◽  
Mariusz P Kowalewski
Keyword(s):  
Oocyte Maturation ◽  
Cell Function ◽  
Hypoxia Inducible Factor ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Negative Effects ◽  
Protein Levels ◽  
Maturation In Vitro

Abstract Various metabolic and hormonal factors expressed in cumulus cells are positively correlated with the in vitro maturation (IVM) of oocytes. However, the role of hypoxia sensing both during maturation of cumulus–oocyte complexes (COCs) as well as during the resumption of meiosis remains uncertain. HIF1alpha plays major roles in cellular responses to hypoxia, and here we investigated its role during bovine COC maturation by assessing the expression of related genes in cumulus cells. COCs were divided into the following groups: immature (control), in vitro matured (IVM/control), or matured in the presence of a blocker of HIF1alpha activity (echinomycin, IVM/E). We found an inhibition of cumulus cell expansion in IVM/E, compared with the IVM/control. Transcript levels of several factors (n = 13) were assessed in cumulus cells. Decreased expression of HAS2, TNFAIP6, TMSB4, TMSB10, GATM, GLUT1, CX43, COX2, PTGES, and STAR was found in IVM/E (P < 0.05). Additionally, decreased protein levels were detected for STAR, HAS2, and PCNA (P < 0.05), while activated-Caspase 3 remained unaffected in IVM/E. Progesterone output decreased in IVM/E. The application of PX-478, another blocker of HIF1alpha expression, yielded identical results. Negative effects of HIF1alpha suppression were further observed in the significantly decreased oocyte maturation and blastocyst rates from COCs matured with echinomycin (P < 0.05) or PX-478 (P < 0.05). These results support the importance of HIF1alpha for COC maturation and subsequent embryo development. HIF1alpha is a multidirectional factor controlling intercellular communication within COCs, steroidogenic activity, and oocyte development rates, and exerting effects on blastocyst rates.

scholarly journals Th17 Cell Enhances CD8 T-Cell Cytotoxicity via IL-21 Production in Emphysema Mice

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Min-Chao Duan ◽  
Ying Huang ◽  
Xiao-Ning Zhong ◽  
Hai-Juan Tang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Inflammatory Response ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
Cell Function ◽  
Cytotoxic T Cell ◽  
Protein Levels ◽  
The Impact

Emphysema is a T-cell mediated autoimmune disease caused predominantly by cigarette smoking. Th17 cells and related cytokines may contribute to this disorder. However, the possible implication of Th17 cells in regulating inflammatory response in emphysema remains to be elucidated. In the current study, we tested the protein levels of IL-17 and IL-21 in peripheral blood and lung tissues from cigarette-smoke- (CS-) exposed mice and air-exposed mice, analyzed the frequencies of CD4+IL-17+(Th17) cells, IL-21+Th17 cells, and CD8+IL-21R+T cells in peripheral blood and lung tissues of mice, and their relationship with emphysematous lesions, and explored the impact of IL-21 on cytotoxic CD8+T cells functionin vitro.It was found that the frequencies of Th17, IL-21+Th17, and CD8+IL-21R+T cells and the levels of IL-17 and IL-21 of CS-exposed mice were much higher than those of the air-exposed mice and correlated with emphysematous lesions. Additionally, the number of IL-21+Th17 cells positively correlated with the number of CD8+IL-21R+T cells. Thein vitroexperiments showed that IL-21 significantly augmented the secretion of perforin and granzyme B in CD8+T cells from CS-exposed mice. These data indirectly provide evidence that Th17 cells could be involved in the control of the local and system inflammatory response in emphysema by regulating CD8+cytotoxic T-cell function.

scholarly journals LSD1 as a Biomarker and the Outcome of Its Inhibitors in the Clinical Trial: The Therapy Opportunity in Tumor

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Clement Agboyibor ◽  
Jianshu Dong ◽  
Clement Yaw Effah ◽  
Emmanuel Kwateng Drokow ◽  
Waqar Pervaiz ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Clinical Trials ◽  
Clinical Results ◽  
Tumor Patients ◽  
Therapy Targets ◽  
Dosing Regimens ◽  
Novel Biomarkers ◽  
Selection Of

Tumors are the foremost cause of death worldwide. As a result of that, there has been a significant enhancement in the investigation, treatment methods, and good maintenance practices on cancer. However, the sensitivity and specificity of a lot of tumor biomarkers are not adequate. Hence, it is of inordinate significance to ascertain novel biomarkers to forecast the prognosis and therapy targets for tumors. This review characterized LSD1 as a biomarker in different tumors. LSD1 inhibitors in clinical trials were also discussed. The recent pattern advocates that LSD1 is engaged at sauce chromatin zones linking with complexes of multi-protein having an exact DNA-binding transcription factor, establishing LSD1 as a favorable epigenetic target, and also gives a large selection of therapeutic targets to treat different tumors. This review sturdily backing the oncogenic probable of LSD1 in different tumors indicated that LSD1 levels can be used to monitor and identify different tumors and can be a useful biomarker of progression and fair diagnosis in tumor patients. The clinical trials showed that inhibitors of LSD1 have growing evidence of clinical efficacy which is very encouraging and promising. However, for some of the inhibitors such as GSK2879552, though selective, potent, and effective, its disease control was poor as the rate of adverse events (AEs) was high in tumor patients causing clinical trial termination, and continuation could not be supported by the risk-benefit profile. Therefore, we propose that, to attain excellent clinical results of inhibitors of LSD1, much attention is required in designing appropriate dosing regimens, developing in-depth in vitro/in vivo mechanistic works of LSD1 inhibitors, and developing inhibitors of LSD1 that are reversible, safe, potent, and selective which may offer safer profiles.

Abstract 14736: Aquaporin 1 Modulates H 2 o 2 -mediated Endothelial Inflammaging

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Seyed Soheil Saeedi Saravi
Keyword(s):  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
Signaling Pathways ◽  
Cell Function ◽  
Immunofluorescence Assay ◽  
Endothelial Cell Function ◽  
Aquaporin 1 ◽  
Knock Down ◽  
Protein Levels

Background: Aquaporin 1 (AQP1), a key regulator of endothelial cell function, transports hydrogen peroxide (H 2 O 2 ) into the cells. H 2 O 2 -mediated inflammation has been implicated in endothelial dysfunction, yet specific roles of AQP1-mediated signaling pathways underlying age-dependent endothelial dysfunction remains incompletely understood. Methods: In these studies, we dissected AQP1-regulated inflammation modulation of eNOS signaling pathways in human aortic endothelial cells (HAEC) from passages 5 to 15 by exploiting siRNA approaches, live cell fluorescence imaging with genetically-encoded H 2 O 2 biosensor HyPer, biochemical and in vitro endothelial function assays. Results: We discovered that AQP1 expression remarkably increases in senescent HAEC (P.15) in association with significant increased SA-βgal activity compared to young cells (P.5). H 2 O 2 levels were increased in senescent cell cytosol revealed by fluorescence HyPer imaging. We found that senescence-associated increase in AQP1-mediated H 2 O 2 led to enhanced TNF-α, unlike HO-1, transcription (p<0.01). Moreover, immunofluorescence assay documented that AQP1 gene knock-down significantly ameliorated senescence-associated elevation of adhesion molecule ICAM-1. Immunoblot analyses demonstrated that increased AQP1 protein levels in senescent HAEC leads to significant increase in caveolin-1 phosphorylation (2-fold) and decreases in phosphorylation of AMPK (Thr172; 4-fold) and eNOS (Ser1177; 2-fold) compared to young ECs (for each, p<0.05, n>6). We discovered that AQP1 knock-down improved the reduced angiogenesis and wound healing capacity in association with eNOS down-regulation in senescent endothelial cells. Conclusion: These results establish that AQP1 plays a crucial role in the regulation of H 2 O 2 -mediated inflammation-associated endothelial senescence, and AQP1 deletion improves endothelial dysfunction by ameliorating ROS-modulated inflammaging.

scholarly journals Exploring Drug Dosing Regimens In Vitro Using Real-Time 3D Spheroid Tumor Growth Assays

2017 ◽  
Vol 22 (5) ◽  
pp. 537-546 ◽  
Author(s):  
Madhu Lal-Nag ◽  
Lauren McGee ◽  
Steven A. Titus ◽  
Kyle Brimacombe ◽  
Sam Michael ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Drug Discovery ◽  
Tumor Growth ◽  
Real Time ◽  
Chemotherapeutic Agents ◽  
Cancer Drug ◽  
Drug Dosing ◽  
Dosing Regimens

Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but only seem to reflect a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy, and resistance before preclinical and clinical studies.

scholarly journals Restoring Tumour Selectivity of the Bioreductive Prodrug PR-104 by Developing an Analogue Resistant to Aerobic Metabolism by Human Aldo-Keto Reductase 1C3

2021 ◽  
Vol 14 (12) ◽  
pp. 1231
Author(s):  
Maria R. Abbattista ◽  
Amir Ashoorzadeh ◽  
Christopher P. Guise ◽  
Alexandra M. Mowday ◽  
Rituparna Mittra ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Therapeutic Window ◽  
Phosphate Ester ◽  
Soluble Phosphate ◽  
Cycle Arrest ◽  
Myeloid Progenitor Cells ◽  
Clinical Models ◽  
Electron Reduction

PR-104 is a phosphate ester pre-prodrug that is converted in vivo to its cognate alcohol, PR-104A, a latent alkylator which forms potent cytotoxins upon bioreduction. Hypoxia selectivity results from one-electron nitro reduction of PR-104A, in which cytochrome P450 oxidoreductase (POR) plays an important role. However, PR-104A also undergoes ‘off-target’ two-electron reduction by human aldo-keto reductase 1C3 (AKR1C3), resulting in activation in oxygenated tissues. AKR1C3 expression in human myeloid progenitor cells probably accounts for the dose-limiting myelotoxicity of PR-104 documented in clinical trials, resulting in human PR-104A plasma exposure levels 3.4- to 9.6-fold lower than can be achieved in murine models. Structure-based design to eliminate AKR1C3 activation thus represents a strategy for restoring the therapeutic window of this class of agent in humans. Here, we identified SN29176, a PR-104A analogue resistant to human AKR1C3 activation. SN29176 retains hypoxia selectivity in vitro with aerobic/hypoxic IC50 ratios of 9 to 145, remains a substrate for POR and triggers γH2AX induction and cell cycle arrest in a comparable manner to PR-104A. SN35141, the soluble phosphate pre-prodrug of SN29176, exhibited superior hypoxic tumour log cell kill (>4.0) to PR-104 (2.5–3.7) in vivo at doses predicted to be achievable in humans. Orthologues of human AKR1C3 from mouse, rat and dog were incapable of reducing PR-104A, thus identifying an underlying cause for the discrepancy in PR-104 tolerance in pre-clinical models versus humans. In contrast, the macaque AKR1C3 gene orthologue was able to metabolise PR-104A, indicating that this species may be suitable for evaluating the toxicokinetics of PR-104 analogues for clinical development. We confirmed that SN29176 was not a substrate for AKR1C3 orthologues across all four pre-clinical species, demonstrating that this prodrug analogue class is suitable for further development. Based on these findings, a prodrug candidate was subsequently identified for clinical trials.

scholarly journals Effects of gypenosides on enteroendocrine L-cell function and GLP-1 secretion

2020 ◽  
Author(s):  
Chinmai Patibandla ◽  
Erin Campbell ◽  
Xinhua Shu ◽  
Angus M Shaw ◽  
Sharron Dolan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Function ◽  
Postprandial Glucose ◽  
Prohormone Convertase ◽  
Antioxidant Gene ◽  
Protein Levels ◽  
L Cells ◽  
Prohormone Convertase 2

AbstractGlucagon-like peptide 1 (GLP-1) is an incretin hormone produced in gut L-cells, which regulates postprandial glucose-dependent insulin secretion, also known as the incretin effect. GLP-1 secretion may be reduced in type 2 diabetes mellitus, impacting on glycaemic regulation. Thus, methods to enhance endogenous GLP-1 secretion by use of natural GLP-1 secretagogues may improve glucose control in diabetes. Gypenosides (GYP) extracted from the plant Gynostemma Pentaphyllum (Jiaogulan) are known for their glucose-lowering effects both in vitro and in vivo, although their effect on GLP-1 secretion is unknown. Our results showed that GYP enhanced cell viability and significantly upregulated antioxidant gene Nrf2, Cat and Ho-1 expression. GYP did not affect glucokinase expression but downregulated proglucagon gene expression over 24h, although, cellular GLP-1 content was unchanged. Prohormone convertase 1 (Pcsk1) gene expression was unchanged by GYP over 24h, although protein levels were significantly downregulated, while prohormone convertase 2 (Pcsk2) mRNA and protein levels were significantly upregulated. Acute exposure to gypenosides enhanced calcium uptake and GLP-1 release from GLUTag cells both at low and high glucose concentrations. These results suggest that anti-diabetic properties of gypenosides are partly linked to their ability to stimulate GLP-1 secretion. Gypenosides enhance antioxidant gene expression and may protect L-cells from excess oxidative stress.

scholarly journals P53 Contributes to Cisplatin Induced Renal Oxidative Damage via Regulating P66shc and MnSOD

2015 ◽  
Vol 37 (4) ◽  
pp. 1240-1256 ◽  
Author(s):  
Yanggang Yuan ◽  
Hui Wang ◽  
Yingyi Wu ◽  
Bo Zhang ◽  
Ningning Wang ◽  
...  
Keyword(s):  
Cell Injury ◽  
Kidney Injury ◽  
Therapeutic Window ◽  
Protein Levels ◽  
Cisplatin Nephrotoxicity ◽  
Mitochondrial Ros ◽  
Potential Strategy ◽  
Hk2 Cells

Background/Aims: Cisplatin is widely used to treat malignancies. However, its major limitation is the development of dose-dependent nephrotoxicity. The precise mechanisms of cisplatin-induced kidney damage remain unclear. Previous study demonstrated the central role of mitochondrial ROS (mtROS) in the pathogenesis of cisplatin nephrotoxicity. The purpose of this study was to explore the mechanism of mtROS regulation in cisplatin nephrotoxicity. Methods: p53, MnSOD and p66shc were detected at mRNA and protein levels by qPCR and western blot in HK2 cells. mtROS levels were determined by DCFDA and MitoSOX staining. Cell viability and cell apoptosis were accessed by CCK-8 assay, TUNEL assay and flow cytometry, respectivesly. siRNAs were used to knock down p53 and p66shc expression and subsequent changes were observed. In vivo assays using a mouse model of cisplatin-induced acute kidney injury were used to validate the in vitro results. Results: In HK2 cells, cisplatin exposure decreased the MnSOD and increased the expression of p53 and p66shc. MnTBAP, a MnSOD mimic, blocked cisplatin-induced the generation of mtROS and cell injury. P66shc and p53 siRNAs rendered renal cells resistant to cisplatin-induced mtROS production and cell death. Furthermore, knockdown of p53 restored MnSOD and inhibiting p66shc. Consistent with these results, we revealed that p53 inhibitor reduced cisplatin-induced oxidative stress and apoptosis by regulating MnSOD and p66shc in the kidney of cisplatin-treated mice. Conclusion: Our study identifies activation of p53 signalling as a potential strategy for reducing the nephrotoxicity associated with cisplatin treatments and, as a result, broadens the therapeutic window of this chemotherapeutic agent.

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