scholarly journals Expansion of Targetable Sites for the Ribonucleoprotein-Based CRISPR/Cas9 System in the Silkworm Bombyx Mori

2021 ◽  
Author(s):  
Yun-long Zou ◽  
Ai-jun Ye ◽  
Shuo Liu ◽  
Wen-tao Wu ◽  
Li-feng Xu ◽  
...  
Keyword(s):  
Gene Editing ◽  
Mutagenic Activity ◽  
Gene Mutations ◽  
Minor Effect ◽  
T7 Promoter ◽  
Multiple Gene ◽  
Silkworm Genome ◽  
Silkworm Bombyx Mori

Abstract Background:With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5’ GG motif for efficient initiation. The resulting transcripts bear a 5’ GG motif, which significantly constrains the number of targetable sites in the silkworm genome. Results:In this study, we used the T7 promoter to add two supernumerary G residues to the 5’ end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5’ GG mismatches depress the mutagenic activity of sgRNAs, and a single 5’ G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5’ GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5’ matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5’ GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation. Conclusions: In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

scholarly journals Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yun-long Zou ◽  
Ai-jun Ye ◽  
Shuo Liu ◽  
Wen-tao Wu ◽  
Li-feng Xu ◽  
...  
Keyword(s):  
Gene Editing ◽  
Mutagenic Activity ◽  
Gene Mutations ◽  
Minor Effect ◽  
T7 Promoter ◽  
Multiple Gene ◽  
Silkworm Genome ◽  
Silkworm Bombyx Mori

Abstract Background With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5′ GG motif for efficient initiation. The resulting transcripts bear a 5′ GG motif, which significantly constrains the number of targetable sites in the silkworm genome. Results In this study, we used the T7 promoter to add two supernumerary G residues to the 5′ end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5′ GG mismatches depress the mutagenic activity of sgRNAs, and a single 5′ G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5′ GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5′ matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5′ GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation. Conclusions In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

scholarly journals SCIDOT-40. THERAPEUTIC GENOME EDITING AS A NOVEL TREATMENT FOR GBM

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi280-vi280
Author(s):  
Daniel Rosenblum ◽  
Anna Gutkin ◽  
Dan Peer
Keyword(s):  
Gene Editing ◽  
Unmet Need ◽  
Gene Mutations ◽  
Infiltration Rate ◽  
Strand Break ◽  
Therapeutic Modality ◽  
Chromosomal Dna ◽  
Genomics And Proteomics

Abstract Glioblastoma multiforme (GBM), is the most aggressive form of glioma, a brain tumor that arises from glial cells. GBM is considered a complex malignancy with multiple gene mutations, aberrations, and overexpression together with high infiltration rate and resistance to apoptosis. The current treatment consists of surgical resection, aggressive radiation and chemotherapy regimen. This therapeutic strategy had not changed since 2005 when the chemotherapy Temozolomide was approved. This therapeutic approach extended GBM patients’ life expectancy to approximately 15 months since diagnosis. Although recent progress in genomics and proteomics has paved the way for identifying potential therapeutic targets for treating GBM, the majority of these leading drug candidates remain ineffective. Therefore, novel and effective treatment to GBM presents an unmet need. Lipid nanoparticles (LNPs) are the most clinically advanced delivery platform to date for systemic administration of RNA therapeutics, with the recent approval of Patisiran, siRNA encapsulating LNPs. In recent years, several gene editing technologies have been discovered in bacteria, enabling precise and permanent manipulations at the DNA level. The most advanced and versatile system is based on the CRISPR nuclease Cas9. The recognition of its target chromosomal DNA by Cas9 results in a site-specific double-strand break (DSB), that eventually results in gene disruption. This approach opens multiple venues for research and treatment of diseases including cancer. We have utilized CRISPR encapsulating LNPs to promote a therapeutic gene editing by disrupting key GBM survival genes in vitro in murine and human GBM cell lines as well as in vivo in an aggressive syngeneic GBM mouse model. Our results suggest that treatment with our LNPs based system can specifically and efficiently target GBM cells in vitro and in vivo. Our CRISPR-LNPs based platform could potentially mature to a clinical trial, and ultimately might become a new therapeutic modality in GBM.

Mutagenic Activity of p-Toluenesulfonhydrazide

1992 ◽  
Vol 8 (6) ◽  
pp. 369-376 ◽  
Author(s):  
David H. Blakey ◽  
Earle R. Nestmann ◽  
Janet M. Bayley ◽  
K. Laurie Maus ◽  
George R. Douglas
Keyword(s):  
Chinese Hamster Ovary ◽  
Mutagenic Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Gene Mutations ◽  
Metabolic Activation ◽  
High Volume ◽  
Ovary Cells

Toluenesulfonhydrazide (TSH) is a high volume production chemical for which there is relatively little toxicological data. In this study, the mutagenic activity of TSH was determined in the Salmonella/mammalian microsome assay and the in vitro chromosomal aberration assay using Chinese hamster ovary cells. TSH induced gene mutations both with and without metabolic activation in the Salmonella/mammalian microsome assay but that it did not induce chromosomal aberrations in Chinese hamster ovary cells. The results of this study indicate that TSH is an in vitro mutagen and should be assessed for in vivo mutagenicity.

scholarly journals Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

2021 ◽  
Author(s):  
Li-Nan Wang ◽  
Xiang-Lei Peng ◽  
Min Xu ◽  
Yuan-Bo Zheng ◽  
Yue-Ying Jiao ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Gene Deletion ◽  
Human Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
T7 Promoter ◽  
Vaccine Candidates ◽  
Rsv Infection ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

scholarly journals Diffuse intrinsic pontine glioma: current insights and future directions

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Dilakshan Srikanthan ◽  
Michael S. Taccone ◽  
Randy Van Ommeren ◽  
Joji Ishida ◽  
Stacey L. Krumholtz ◽  
...  
Keyword(s):  
Brain Tumor ◽  
Gene Mutations ◽  
Pediatric Brain Tumor ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Pontine Glioma ◽  
Current Standard ◽  
In Vivo Models ◽  
Landscape Models

AbstractDiffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor and the leading cause of brain tumor–related death in children. As several clinical trials over the past few decades have led to no significant improvements in outcome, the current standard of care remains fractionated focal radiation. Due to the recent increase in stereotactic biopsies, tumor tissue availabilities have enabled our advancement of the genomic and molecular characterization of this lethal cancer. Several groups have identified key histone gene mutations, genetic drivers, and methylation changes in DIPG, providing us with new insights into DIPG tumorigenesis. Subsequently, there has been increased development of in vitro and in vivo models of DIPG which have the capacity to unveil novel therapies and strategies for drug delivery. This review outlines the clinical characteristics, genetic landscape, models, and current treatments and hopes to shed light on novel therapeutic avenues and challenges that remain.

scholarly journals Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

1998 ◽  
Vol 42 (7) ◽  
pp. 1811-1814 ◽  
Author(s):  
Leonardo K. Basco ◽  
Rachida Tahar ◽  
Pascal Ringwald
Keyword(s):  
Dihydrofolate Reductase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Adult Patients ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Reductase Gene ◽  
Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

scholarly journals Mirtron-mediated RNA knockdown/replacement therapy for the treatment of dominant retinitis pigmentosa

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Harry O. Orlans ◽  
Michelle E. McClements ◽  
Alun R. Barnard ◽  
Cristina Martinez-Fernandez de la Camara ◽  
Robert E. MacLaren
Keyword(s):  
Retinitis Pigmentosa ◽  
Autosomal Dominant ◽  
Gene Mutations ◽  
Adeno Associated Virus ◽  
Treatment Development ◽  
Common Cause ◽  
Toxic Protein ◽  
Rna Knockdown

AbstractRhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a RhoP23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo.

Studies on the mutagenic activity of ascorbic acid in vitro and in vivo

1983 ◽  
Vol 117 (1-2) ◽  
pp. 183-191 ◽  
Author(s):  
E.P. Norkus ◽  
W. Kuenzig ◽  
A.H. Conney
Keyword(s):  
Ascorbic Acid ◽  
Mutagenic Activity

Overexpression of a plant cysteine synthase gene and biosynthesis of a plant specific metabolite, β-(pyrazol-1-yl)-L-alanine, in Escherichia coli

1994 ◽  
Vol 72 (1) ◽  
pp. 188-192 ◽  
Author(s):  
Kazuki Saito ◽  
Reiko Kanda ◽  
Makoto Kurosawa ◽  
Isamu Murakoshi
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Control ◽  
Spinacia Oleracea ◽  
Higher Plants ◽  
Total Soluble Protein ◽  
T7 Promoter ◽  
Cysteine Synthase ◽  
Mechanistic Investigation

Cysteine synthase (EC 4.2.99.8) in higher plants is responsible for biosynthesis of not only cysteine but also some nonprotein amino acids such as β-(pyrazol-1-yl)-L-alanine. The cDNA of a cysteine synthase from spinach (Spinacia oleracea) was inserted into pET8c (=pET3d) under the transcriptional control of strong T7 promoter to yield an overexpression vector pCEK1. The amount of the exogenous cysteine synthase was increased up to 40% of the total soluble protein of Escherichia coli transformed with pCEK1. β-(Pyrazol-1-yl)-L-alanine, a specific metabolite in plants of the Cucurbitaceae, was biosynthesized by overexpressed cysteine synthase from pyrazole in the presence of O-acetyl-L-serine and serine, in vitro and in vivo, respectively. The present study provides the system for mechanistic investigation of biosynthesis of cysteine and biogenetically related β-substituted alanines at molecular genetic level.

scholarly journals Promising potential of [177Lu]Lu-DOTA-folate to enhance tumor response to immunotherapy—a preclinical study using a syngeneic breast cancer model

2020 ◽  
Author(s):  
Patrycja Guzik ◽  
Klaudia Siwowska ◽  
Hsin-Yu Fang ◽  
Susan Cohrs ◽  
Peter Bernhardt ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Survival Time ◽  
Tumor Cells ◽  
Median Survival ◽  
Breast Tumor ◽  
Median Survival Time ◽  
Therapy Outcome ◽  
Minor Effect

Abstract Purpose It was previously demonstrated that radiation effects can enhance the therapy outcome of immune checkpoint inhibitors. In this study, a syngeneic breast tumor mouse model was used to investigate the effect of [177Lu]Lu-DOTA-folate as an immune stimulus to enhance anti-CTLA-4 immunotherapy. Methods In vitro and in vivo studies were performed to characterize NF9006 breast tumor cells with regard to folate receptor (FR) expression and the possibility of tumor targeting using [177Lu]Lu-DOTA-folate. A preclinical therapy study was performed over 70 days with NF9006 tumor-bearing mice that received vehicle only (group A); [177Lu]Lu-DOTA-folate (5 MBq; 3.5 Gy absorbed tumor dose; group B); anti-CTLA-4 antibody (3 × 200 μg; group C), or both agents (group D). The mice were monitored regarding tumor growth over time and signs indicating adverse events of the treatment. Results [177Lu]Lu-DOTA-folate bound specifically to NF9006 tumor cells and tissue in vitro and accumulated in NF9006 tumors in vivo. The treatment with [177Lu]Lu-DOTA-folate or an anti-CTLA-4 antibody had only a minor effect on NF9006 tumor growth and did not substantially increase the median survival time of mice (23 day and 19 days, respectively) as compared with untreated controls (12 days). [177Lu]Lu-DOTA-folate sensitized, however, the tumors to anti-CTLA-4 immunotherapy, which became obvious by reduced tumor growth and, hence, a significantly improved median survival time of mice (> 70 days). No obvious signs of adverse effects were observed in treated mice as compared with untreated controls. Conclusion Application of [177Lu]Lu-DOTA-folate had a positive effect on the therapy outcome of anti-CTLA-4 immunotherapy. The results of this study may open new perspectives for future clinical translation of folate radioconjugates.

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