scholarly journals CRISPR/Cas9-Mediated Hitchhike Expression of Functional shRNAs at the Porcine miR-17-92 Cluster

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 113 ◽  
Author(s):  
Chao Lu ◽  
Daxin Pang ◽  
Mengjing Li ◽  
Hongming Yuan ◽  
Tingting Yu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Rna Interference ◽  
Integration Site ◽  
Classical Swine Fever ◽  
Mirna Cluster ◽  
Transgenic Pigs ◽  
Blastocyst Development ◽  
Endogenous Gene ◽  
Fetal Fibroblasts

Successful RNAi applications depend on strategies allowing stable and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. In this study, we proposed an endogenous microRNA (miRNA) cluster as a novel integration site for small hairpin RNAs (shRNAs). We successfully integrated exogenous shRNAs at the porcine miRNA-17-92 (pmiR-17-92) cluster via a CRISPR/Cas9-mediated knock-in strategy. The anti-EGFP or anti-CSFV shRNAs could be stably and effectively expressed at the control of the endogenous promoter of the pmiR-17-92 cluster. Importantly, we confirmed that hitchhike expression of anti- classical swine fever (CSFV) shRNA had no effect on cell growth, blastocyst development and endogenous pmiR-17-92 expression in selected transgene (TG) porcine fetal fibroblasts (PFFs) clones. Moreover, these TG PFFs could inhibit the replication of CSFV by half and could be further used for generation of transgenic pigs. Taken together, these results show that our RNA interference (RNAi) expression strategy benefits numerous applications, from miRNA, genome and transgenic research, to gene therapy.

Using RNA interference to manipulate endogenous gene expression in Schistosoma mansoni sporocysts

2003 ◽  
Vol 128 (2) ◽  
pp. 205-215 ◽  
Author(s):  
Jon P Boyle ◽  
Xiao-Jun Wu ◽  
Chuck B Shoemaker ◽  
Timothy P Yoshino
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Schistosoma Mansoni ◽  
Endogenous Gene

ZIF-C for targeted RNA interference and CRISPR/Cas9 based gene editing in prostate cancer

2020 ◽  
Vol 56 (98) ◽  
pp. 15406-15409
Author(s):  
Arpita Poddar ◽  
Suneela Pyreddy ◽  
Francesco Carraro ◽  
Sudip Dhakal ◽  
Andrea Rassell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Gene Therapy ◽  
Rna Interference ◽  
Green Tea ◽  
Gene Editing ◽  
Metal Organic Frameworks ◽  
Host Gene ◽  
Host Gene Expression ◽  
Metal Organic

Metal–organic-frameworks for gene therapy in prostate cancer – ZIF-C based delivery of RNA interference and CRISPR/Cas9 causes host gene expression knockdown. Coating with a green tea phytochemical enhances uptake and increases cancer cytotoxicity.

The Clonal Inventory of Gene Corrected Hematopoiesis in Three Successful Clinical Gene Therapy Trials

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3733-3733
Author(s):  
Kerstin Schwarzwaelder ◽  
Manfred Schmidt ◽  
Annette Deichmann ◽  
Steven J. Howe ◽  
Marion G. Ott ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Sanger Sequencing ◽  
Integration Site ◽  
Cellular Gene ◽  
Post Transplantation ◽  
Cellular Gene Expression ◽  
Site Distribution ◽  
Clinical Gene Therapy

Abstract To enhance the safety and efficacy of future gene therapy trials using integrating vector systems it is necessary to analyse the clonality of the genetically modified cell pool. The comparative analyses of integration site distribution and cellular gene expression will further reveal causal mechanisms of in vivo clone selection. We followed the repopulation clonality of 21 patients which participated in 3 successful clinical gene therapy trials via linear amplification mediated PCR (LAM PCR). We identified the integration sites (IS) of pre and post transplantation samples by Sanger sequencing and accomplished RNA analyses. The comparative results from all trials showed that vector integration is favoured in gene coding regions, in particular transcriptional start sites. In both X-SCID trials significantly more post transplantation IS were located in or in the vicinity of genes encoding proteins with kinase or transferase activity. In pre transplantation samples no uniform gene class was overrepresented. In both trials we detected common insertion sites mainly post transplantation and the effect was more pronounced in the trial where 4 patients developed vector induced leukemia. Notably, we detected no significant differences regarding the IS distribution in leukemic versus non leukemic patients. The gene corrected repopulation of patient 1 and 2 of the X-CGD trial was polyclonal until 542 and 777 days after transplantation, respectively. 5 months after therapy dominant clones appeared. In patient 1, between 616 and 820 days post transplantation (post mortem time point) the number of participating clones and the contribution of a dominant clone decreased while the contribution of another dominant clone increased. In both patients the integrated vector induced the upregulation of the genes MDS1/EVI1, PRDM16 or SETBP1 and thus led to the in vivo expansion of affected cell clones. From these trials we sequenced >2000 unique IS by Sanger sequencing and several thousand via pyrosequencing (datamining is ongoing). Our data show that the integration site distribution was non random, that the integrated vector influenced the cellular gene expression which caused subtle to massive changes in the repopulation clonality and that it will soon be possible to define the clonal inventory of patients using next generation sequencing technologies.

scholarly journals 308 DEVELOPMENT OF TRANSGENIC LIVESTOCK WITH REDUCED MYOSTATIN EXPRESSION USING RNA INTERFERENCE

2009 ◽  
Vol 21 (1) ◽  
pp. 251
Author(s):  
K. Tessanne ◽  
T. Stroud ◽  
C. Long ◽  
G. Hannon ◽  
S. Sadeghieh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Lentiviral Vector ◽  
Muscle Growth ◽  
Negative Regulator ◽  
Large Animal ◽  
Large Animal Models ◽  
Transgenic Livestock ◽  
Fetal Fibroblasts ◽  
Wide Range

RNA interference (RNAi) is a means of regulating gene expression by targeting mRNA in a sequence-specific manner for degradation or translational inhibition. Short hairpin RNAs (shRNAs) and siRNAs have been extensively employed for manipulating gene expression in a wide range of species. However, the great majority of this work has involved in vitro studies with cells grown in culture. Our goal for this project is to produce transgenic livestock in which myostatin, a negative regulator of muscle growth, has been targeted for silencing by RNAi. In theory, livestock in which myostatin has been silenced should exhibit increased muscle growth and development. To that end, we designed shRNAs to target the bovine myostatin mRNA sequence. The shRNAs were cloned into a lentiviral vector that contains a cytomegalovirus promoter controlling green fluorescent protein and shRNA expression as well as neomycin resistance. Infective lentivirus was made in HEK293T cells through co-transfection of the lentiviral vector, a packaging plasmid, and a plasmid expressing the VSVG pseudotype. Bovine fetal fibroblasts were transduced, selected using Geneticin®, and nuclear transfer was utilized to produce cloned transgenic embryos. There were 186 fusion attempts resulting in 160 fused embryos (fusion rate = 86%). Of these, 54 reached the blastocyst stage (34%) and 10 embryos were transferred into 5 recipient females (2 embryos per recipient). At 40 days, ultrasound revealed 1 confirmed pregnancy. Current plans are to harvest this fetus at 90 days and analyze it for evidence of myostatin knockdown. The production of transgenic animals exhibiting myostatin knockdown through lentiviral-mediated RNAi will demonstrate the utility of RNAi in the study of gene function in large animal models without the need for homologous recombination techniques, which are currently inefficient in species other than mice.

scholarly journals Gene Therapy Progress and Prospects. Downregulating gene expression: the impact of RNA interference

Gene Therapy ◽  
2004 ◽  
Vol 11 (16) ◽  
pp. 1241-1248 ◽  
Author(s):  
N J Caplen
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Rna Interference ◽  
The Impact

Lentivirus-mediated RNA Interference of ppGalNAc-T2 Gene Expression Inhibit Proliferation and Migration of Jurkat Cell Line*

2011 ◽  
Vol 38 (8) ◽  
pp. 737-743
Author(s):  
Chun-Liang LIU ◽  
Dan-Dan LIN ◽  
Lan XU ◽  
Zhi JIANG ◽  
Shi-Liang WU
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Cell Line ◽  
Jurkat Cell ◽  
Jurkat Cell Line ◽  
And Migration ◽  
Proliferation And Migration

The Therapeutic Potential and Role of miRNA, lncRNA, and circRNA in Osteoarthritis

2019 ◽  
Vol 19 (4) ◽  
pp. 255-263 ◽  
Author(s):  
Yuangang Wu ◽  
Xiaoxi Lu ◽  
Bin Shen ◽  
Yi Zeng
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Extracellular Matrix ◽  
Inflammatory Reaction ◽  
Noncoding Rna ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Translation ◽  
Cochrane Library

Background: Osteoarthritis (OA) is a disease characterized by progressive degeneration, joint hyperplasia, narrowing of joint spaces, and extracellular matrix metabolism. Recent studies have shown that the pathogenesis of OA may be related to non-coding RNA, and its pathological mechanism may be an effective way to reduce OA. Objective: The purpose of this review was to investigate the recent progress of miRNA, long noncoding RNA (lncRNA) and circular RNA (circRNA) in gene therapy of OA, discussing the effects of this RNA on gene expression, inflammatory reaction, apoptosis and extracellular matrix in OA. Methods: The following electronic databases were searched, including PubMed, EMBASE, Web of Science, and the Cochrane Library, for published studies involving the miRNA, lncRNA, and circRNA in OA. The outcomes included the gene expression, inflammatory reaction, apoptosis, and extracellular matrix. Results and Discussion: With the development of technology, miRNA, lncRNA, and circRNA have been found in many diseases. More importantly, recent studies have found that RNA interacts with RNA-binding proteins to regulate gene transcription and protein translation, and is involved in various pathological processes of OA, thus becoming a potential therapy for OA. Conclusion: In this paper, we briefly introduced the role of miRNA, lncRNA, and circRNA in the occurrence and development of OA and as a new target for gene therapy.

scholarly journals Long-term lymphoid progenitors independently sustain naïve T and NK cell production in humans

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Natalia Izotova ◽  
Christine Rivat ◽  
Cristina Baricordi ◽  
Elena Blanco ◽  
Danilo Pellin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Nk Cell ◽  
Integration Site ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Lymphoid Progenitors ◽  
Clinical Gene Therapy ◽  
Cancer Immunotherapies

AbstractOur mathematical model of integration site data in clinical gene therapy supported the existence of long-term lymphoid progenitors capable of surviving independently from hematopoietic stem cells. To date, no experimental setting has been available to validate this prediction. We here report evidence of a population of lymphoid progenitors capable of independently maintaining T and NK cell production for 15 years in humans. The gene therapy patients of this study lack vector-positive myeloid/B cells indicating absence of engineered stem cells but retain gene marking in both T and NK. Decades after treatment, we can still detect and analyse transduced naïve T cells whose production is likely maintained by a population of long-term lymphoid progenitors. By tracking insertional clonal markers overtime, we suggest that these progenitors can support both T and NK cell production. Identification of these long-term lymphoid progenitors could be utilised for the development of next generation gene- and cancer-immunotherapies.

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