scholarly journals Development of an ObLiGaRe Doxycycline Inducible Cas9 system for pre-clinical cancer drug discovery

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Anders Lundin ◽  
Michelle J. Porritt ◽  
Himjyot Jaiswal ◽  
Frank Seeliger ◽  
Camilla Johansson ◽  
...  
Keyword(s):  
Gene Editing ◽  
Functional Integration ◽  
Cancer Drug ◽  
Cancer Drug Discovery ◽  
Mouse Cells ◽  
Genomic Editing ◽  
Human And Mouse ◽  
Mouse Genomic ◽  
In Cells

Abstract The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

scholarly journals ObLiGaRe doxycycline Inducible (ODIn) Cas9 system driving pre-clinical drug discovery, from design to cancer treatment

2020 ◽  
Author(s):  
Anders Lundin ◽  
Michelle J. Porritt ◽  
Himjyot Jaiswal ◽  
Frank Seeliger ◽  
Camilla Johansson ◽  
...  
Keyword(s):  
Gene Editing ◽  
Functional Integration ◽  
High Reproducibility ◽  
Mouse Cells ◽  
Genomic Editing ◽  
Human And Mouse ◽  
Mouse Genomic ◽  
Clinical Drug ◽  
In Cells

ABSTRACTThe CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. We have generated a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene. Targeted ObLiGaRe resulted in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse can be utilized for robust and tunable genome editing allowing for flexibility, speed and uniformity at reduced cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

scholarly journals Frontiers in Anti-cancer Drug Discovery: Challenges and Perspectives of Metformin as Anti-angiogenic Add-on Therapy in Glioblastoma

2021 ◽  
Author(s):  
Laura Guarnaccia ◽  
Stefania Elena Navone ◽  
Matteo Maria Masseroli ◽  
Melissa Balsamo ◽  
Manuela Caroli ◽  
...  
Keyword(s):  
Target Therapy ◽  
Cancer Drug ◽  
Cancer Drug Discovery ◽  
Average Survival ◽  
Anti Cancer ◽  
Tumor Neovascularization ◽  
Novel Therapeutic Strategies

Glioblastoma (GBM) is the most common primitive tumor in adult central nervous system (CNS), classified as grade IV according to WHO 2016 classification. GBM shows a poor prognosis with an average survival of approximately 15 months, representing an extreme therapeutic challenge. One of its distinctive and aggressive features is aberrant angiogenesis, which drives tumor neovascularization, representing a promising candidate for molecular target therapy. Although several pre-clinical studies and clinical trials have shown promising results, anti-angiogenic drugs have not led to a significant improvement in overall survival (OS), suggesting the necessity of identifying novel therapeutic strategies. Metformin, an anti-hyperglycemic drug of the Biguanides family, used as first line treatment in Type 2 Diabetes Mellitus (T2DM), demonstrated in vitro and in vivo antitumoral efficacy in many different tumors, including GBM. From this evidence, a process of repurposing of the drug has begun, leading to the demonstration of the inhibition of various oncopromoter mechanisms and, consequently, to the identification of the molecular pathways involved. Here, we review and discuss the potential metformin’s antitumoral effects on GBM, inspecting if it could properly act as an anti-angiogenic compound to be considered as a safely add-on therapy in the treatment and management of GBM patients.

scholarly journals An in vivo large-scale chemical screening platform using Drosophila for anti-cancer drug discovery

2012 ◽  
Vol 6 (2) ◽  
pp. 521-529 ◽  
Author(s):  
L. F. Willoughby ◽  
T. Schlosser ◽  
S. A. Manning ◽  
J. P. Parisot ◽  
I. P. Street ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Large Scale ◽  
Cancer Drug ◽  
Chemical Screening ◽  
Cancer Drug Discovery ◽  
Anti Cancer ◽  
Screening Platform

scholarly journals A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells.

1988 ◽  
Vol 106 (3) ◽  
pp. 545-556 ◽  
Author(s):  
J A Steitz ◽  
C Berg ◽  
J P Hendrick ◽  
H La Branche-Chabot ◽  
A Metspalu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
5S Rrna ◽  
5S Rna ◽  
Rna Molecules ◽  
La Protein ◽  
Rnp Complex ◽  
Mouse Cells ◽  
Protein Moiety ◽  
Human And Mouse

A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (approximately 7S) and contains most of the nonribosome-associated 5S RNA molecules in HeLa cells. The 5S RNA in the particle is matured at its 3' end, consistent with the results of in vivo pulse-chase experiments which indicate that this RNP represents a later step in 5S biogenesis than a previously described 5S*/La protein complex. The protein moiety of the 5S RNP has been identified as ribosomal protein L5, which is known to be released from ribosomes in a complex with 5S after various treatments of the 60S subunit. Indirect immunofluorescence indicates that the L5/5S complex is concentrated in the nucleolus. L5 may therefore play a role in delivering 5S rRNA to the nucleolus for assembly into ribosomes.

scholarly journals Frontiers in Anti-Cancer Drug Discovery: Challenges and Perspectives of Metformin as Anti-Angiogenic Add-On Therapy in Glioblastoma

Cancers ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 112
Author(s):  
Laura Guarnaccia ◽  
Giovanni Marfia ◽  
Matteo Maria Masseroli ◽  
Stefania Elena Navone ◽  
Melissa Balsamo ◽  
...  
Keyword(s):  
Target Therapy ◽  
Cancer Drug ◽  
Cancer Drug Discovery ◽  
Average Survival ◽  
Anti Cancer ◽  
Tumor Neovascularization ◽  
Novel Therapeutic Strategies

Glioblastoma is the most common primitive tumor in adult central nervous system (CNS), classified as grade IV according to WHO 2016 classification. Glioblastoma shows a poor prognosis with an average survival of approximately 15 months, representing an extreme therapeutic challenge. One of its distinctive and aggressive features is aberrant angiogenesis, which drives tumor neovascularization, representing a promising candidate for molecular target therapy. Although several pre-clinical studies and clinical trials have shown promising results, anti-angiogenic drugs have not led to a significant improvement in overall survival (OS), suggesting the necessity of identifying novel therapeutic strategies. Metformin, an anti-hyperglycemic drug of the Biguanides family, used as first line treatment in Type 2 Diabetes Mellitus (T2DM), has demonstrated in vitro and in vivo antitumoral efficacy in many different tumors, including glioblastoma. From this evidence, a process of repurposing of the drug has begun, leading to the demonstration of inhibition of various oncopromoter mechanisms and, consequently, to the identification of the molecular pathways involved. Here, we review and discuss metformin’s potential antitumoral effects on glioblastoma, inspecting if it could properly act as an anti-angiogenic compound to be considered as a safely add-on therapy in the treatment and management of glioblastoma patients.

scholarly journals Isolation and sequencing of AGO-bound RNAs reveals characteristics of mammalian stem-loop processing in vivo

2018 ◽  
Author(s):  
Ian M. Silverman ◽  
Sager J. Gosai ◽  
Nicholas Vrettos ◽  
Shawn W. Foley ◽  
Nathan D. Berkowitz ◽  
...  
Keyword(s):  
Mature Mirnas ◽  
Human Cells ◽  
Processing Efficiency ◽  
Stem Loop ◽  
Mature Micrornas ◽  
Mouse Cells ◽  
Low Levels ◽  
Short Hairpin Rnas ◽  
Human And Mouse

ABSTRACTMicroRNA precursors (pre-miRNAs) are short hairpin RNAs that are rapidly processed into mature microRNAs (miRNAs) in the cytoplasm. Due to their low abundance in cells, sequencing-based studies of pre-miRNAs have been limited. We successfully enriched for and deep sequenced pre-miRNAs in human cells by capturing these RNAs during their interaction with Argonaute (AGO) proteins. Using this approach, we detected > 350 pre-miRNAs in human cells and > 250 pre-miRNAs in a reanalysis of a similar study in mouse cells. We uncovered widespread trimming and non-templated additions to the 3’ ends of pre- and mature miRNAs. Additionally, we created an index for microRNA precursor processing efficiency. This analysis revealed a subset of pre-miRNAs that produce low levels of mature miRNAs despite abundant precursors, including an annotated miRNA in the 5’ UTR of the DiGeorge syndrome critical region 8 mRNA transcript. This led us to search for AGO-associated stem-loops originating from other mRNA species, which identified hundreds of putative pre-miRNAs derived from human and mouse mRNAs. In summary, we provide a wealth of information on mammalian pre-miRNAs, and identify novel microRNA and microRNA-like elements localized in mRNAs.

Replication of DNA containing apurinic sites in human and mouse cells probed with parvoviruses MVM and H-1

1987 ◽  
Vol 7 (7) ◽  
pp. 2620-2624
Author(s):  
J M Vos ◽  
J Rommelaere
Keyword(s):  
Mammalian Cells ◽  
Uv Light ◽  
Host Cells ◽  
Minute Virus Of Mice ◽  
Dna Viruses ◽  
Minute Virus ◽  
Mouse Cells ◽  
Viral Mutagenesis ◽  
Human And Mouse

We studied the effect of apurinic sites on DNA replication in mouse and human cells, using parvoviruses MVM (minute virus of mice) and H-1 as probes. Although apurinic sites are efficient blocks to the replication of these single-stranded DNA viruses in vivo, depurinated parvoviruses can be reactivated if host cells have been preexposed to a subtoxic dose of UV light. The target of this conditional reactivation process is the conversion of depurinated input DNA into double-stranded replicative forms; the concomitant increase in viral mutagenesis strongly suggests that apurinic sites can be bypassed in mammalian cells.

scholarly journals p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes

2013 ◽  
Vol 201 (4) ◽  
pp. 613-629 ◽  
Author(s):  
Albert R. Davalos ◽  
Misako Kawahara ◽  
Gautam K. Malhotra ◽  
Nicholas Schaum ◽  
Jiahao Huang ◽  
...  
Keyword(s):  
Growth Arrest ◽  
High Mobility ◽  
Cytokine Secretion ◽  
Blocking Antibody ◽  
Extracellular Milieu ◽  
Hmgb1 Expression ◽  
Mouse Cells ◽  
Human And Mouse ◽  
Oncogenic Stress

Cellular senescence irreversibly arrests proliferation in response to potentially oncogenic stress. Senescent cells also secrete inflammatory cytokines such as IL-6, which promote age-associated inflammation and pathology. HMGB1 (high mobility group box 1) modulates gene expression in the nucleus, but certain immune cells secrete HMGB1 as an extracellular Alarmin to signal tissue damage. We show that nuclear HMGB1 relocalized to the extracellular milieu in senescent human and mouse cells in culture and in vivo. In contrast to cytokine secretion, HMGB1 redistribution required the p53 tumor suppressor, but not its activator ATM. Moreover, altered HMGB1 expression induced a p53-dependent senescent growth arrest. Senescent fibroblasts secreted oxidized HMGB1, which stimulated cytokine secretion through TLR-4 signaling. HMGB1 depletion, HMGB1 blocking antibody, or TLR-4 inhibition attenuated senescence-associated IL-6 secretion, and exogenous HMGB1 stimulated NF-κB activity and restored IL-6 secretion to HMGB1-depleted cells. Our findings identify senescence as a novel biological setting in which HMGB1 functions and link HMGB1 redistribution to p53 activity and senescence-associated inflammation.

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