Novel non-integrating DNA Nano-S/MAR vectors restore gene function in patient-derived pancreatic tumour models I
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PROVIDER: PRJNA595930 | ENA |
REPOSITORIES: ENA
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Project description:Non-integrating minimally sized Nano-S/MAR DNA vectors can be used to genetically modify dividing cells in place of integrating vectors such as lentivirus and sleeping beauty. They represent a unique genetic tool, which avoids vector-mediated genetic damage cells and the activation of innate immune responses. Previous work has shown that DNA vectors comprising the mammalian scaffold/matrix attachment region (S/MAR) element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations which reduce their stability and can provoke cellular toxicity. Here, we present a new system, the Nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to their minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells by using Nano-S/MARs to stably restore the tumour suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that this class of DNA vector can be used to persistently modify a wide range of cells, providing sustained high levels of transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.
2021-12-12 | GSE142117 | GEO
Project description:Non-integrating minimally sized Nano-S/MAR DNA vectors can be used to genetically modify dividing cells in place of integrating vectors such as lentivirus and sleeping beauty. They represent a unique genetic tool, which avoids vector-mediated genetic damage cells and the activation of innate immune responses. Previous work has shown that DNA vectors comprising the mammalian scaffold/matrix attachment region (S/MAR) element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations which reduce their stability and can provoke cellular toxicity. Here, we present a new system, the Nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to their minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells by using Nano-S/MARs to stably restore the tumour suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that this class of DNA vector can be used to persistently modify a wide range of cells, providing sustained high levels of transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.
2021-12-12 | GSE142115 | GEO
Project description:Novel non-integrating DNA Nano-S/MAR vectors restore gene function in patient-derived pancreatic tumour models II
| PRJNA595934 | ENA
Project description:We describe herein non-integrating minimally sized nano-S/MAR DNA vectors, which can be used to genetically modify dividing cells in place of integrating vectors. They represent a unique genetic tool, which avoids vector-mediated damage. Previous work has shown that DNA vectors comprising a mammalian S/MAR element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations that can provoke cellular toxicity. Herein, we present a new system, the nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to the minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells to stably restore the tumor suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that these DNA vectors can be used to persistently modify a range of cells, providing sustained transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.
| S-EPMC7218229 | biostudies-literature
Project description:Analysis of the episomal backbone's influence on gene expression. The first hypothesis tested in the present study is that the episomal EBNA vectors, which rely on the EBNA-1 oncoprotein for episomal maintenance, have a greater influence on the cells' expression profiles than S/MAR vectors. The second hypothesis tested was that when bacterial sequences are removed from the episomal vector backbone, the gene disturbance is minimal.
2021-12-05 | GSE142299 | GEO
Project description:Methods: mRNA profiles of untransfected HeLa cells (wild-type; wt) were compared with mRNA profiles of HeLa cells stably maintaining an S/MAR-based episome. Results: We here report for the first time that episomally maintained S/MAR-based vectors do not alter gene expression profile of the host cell's genome. No global changes in gene expression in episome maintaining cells, compared to non-transfected cells could be observed. To identify differentially expressed genes, false discovery rate (FDR; q-value) cut off was set to 0.01. Significantly differentially expressed genes with q<0.01 and an absolute fold-change of 2 were not detected. For verification, we chose five genes with high fold-change and low q-values (q<0.05) and compared expression levels between untransfected HeLa and HeLa stably maintaining an S/MAR-based within three replicates in qPCR. Conclusions: S/MAR-based replicons used in this study do not code for viral proteins but tend to co-localise with promoter sequences and transcription start sites. Recent observations that cooperatively transcribed promoters can influence each other raise concerns that S/MAR-based replicons have the potential to alter endogenous gene expression. Therefore, we compared the transcriptome of untransfected HeLa cells with HeLa cells stably maintaining an S/MAR-based episome. Setting the FDR to <0.01, we found no significantly differentially expressed genes. This finding is of utmost importance for potential gene therapeutic application of S/MAR-based replicons.
2017-09-18 | GSE97725 | GEO
Project description:Human-induced pluripotent stem cells (iPSCs) are derived from differentiated somatic cells using defined factors and provide a renewable source of autologous cells for cell therapy. Many reprogramming methods have been employed to generate human iPSCs, including the use of integrating vectors and non-integrating vectors. Maintenance of the genomic integrity of iPSCs is highly desirable if the cells are to be used in clinical applications. Here, using the Affymetrix Cytoscan HD array, we investigated the genomic aberration profiles of 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors (“integrating iPSC lines”), 6 iPSC lines derived using non-integrating vectors (“non-integrating iPSC lines”), and the 2 parental cell lines from which the iPSCs were derived. The genome-wide copy number variation (CNV), loss of heterozygosity (LOH) and mosaicism patterns of integrating and non-integrating iPSC lines were investigated. The maximum sizes of CNVs in the genomes of the integrating iPSC lines were 20 times higher than those of the non-integrating iPSC lines. Moreover, the total number of CNVs was much higher in integrating iPSC lines than in other cell lines. The average numbers of novel CNVs with a low degree of overlap with the DGV and of likely pathogenic CNVs with a high degree of overlap with the ISCA database were highest in integrating iPSC lines. Different SNP calls revealed that, using the parental cell genotype as a reference, integrating iPSC lines displayed more single nucleotide variations and mosaicism than did non-integrating iPSC lines. This study describes the genome stability of human iPSCs generated using either a DNA-integrating or non-integrating reprogramming method, of the corresponding somatic cells, and of hESCs. Our results highlight the importance of using a high-resolution method to monitor genomic aberrations in iPSCs intended for clinical applications to avoid any negative effects of reprogramming or cell culture. 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors (“integrating iPSC lines”), 6 iPSC lines derived using non-integrating vectors (“non-integrating iPSC lines”), and the 2 parental cell lines from which the iPSCs were derived.
2015-06-08 | E-GEOD-69632 | biostudies-arrayexpress
Project description:Gene expression of murine hematopoietic stem and progenitor cells transduced with integrating vectors
| PRJNA430829 | ENA
Project description:Human-induced pluripotent stem cells (iPSCs) are derived from differentiated somatic cells using defined factors and provide a renewable source of autologous cells for cell therapy. Many reprogramming methods have been employed to generate human iPSCs, including the use of integrating vectors and non-integrating vectors. Maintenance of the genomic integrity of iPSCs is highly desirable if the cells are to be used in clinical applications. Here, using the Affymetrix Cytoscan HD array, we investigated the genomic aberration profiles of 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors (“integrating iPSC lines”), 6 iPSC lines derived using non-integrating vectors (“non-integrating iPSC lines”), and the 2 parental cell lines from which the iPSCs were derived. The genome-wide copy number variation (CNV), loss of heterozygosity (LOH) and mosaicism patterns of integrating and non-integrating iPSC lines were investigated. The maximum sizes of CNVs in the genomes of the integrating iPSC lines were 20 times higher than those of the non-integrating iPSC lines. Moreover, the total number of CNVs was much higher in integrating iPSC lines than in other cell lines. The average numbers of novel CNVs with a low degree of overlap with the DGV and of likely pathogenic CNVs with a high degree of overlap with the ISCA database were highest in integrating iPSC lines. Different SNP calls revealed that, using the parental cell genotype as a reference, integrating iPSC lines displayed more single nucleotide variations and mosaicism than did non-integrating iPSC lines. This study describes the genome stability of human iPSCs generated using either a DNA-integrating or non-integrating reprogramming method, of the corresponding somatic cells, and of hESCs. Our results highlight the importance of using a high-resolution method to monitor genomic aberrations in iPSCs intended for clinical applications to avoid any negative effects of reprogramming or cell culture.
2015-06-08 | GSE69632 | GEO
Project description:Analysis of gene expression of hESCs upon genetic modification with EBNA and S/MAR episomal vectors
| PRJNA596482 | ENA