Gene expression of murine hematopoietic stem and progenitor cells transduced with integrating vectors
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PROVIDER: PRJNA430829 | ENA |
REPOSITORIES: ENA
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Project description:Gene therapy has emerged as a promising therapeutic strategy, but efficient in vitro assessment of vector safety remains challenging. To create a predictor of vector induced genotoxicity, we developed the Surrogate Assay for Genotoxicity Assessment (SAGA), which uses genetic algorithm-enhanced machine learning to detect the deregulation of a specific gene expression signature in murine hematopoietic stem and progenitor cells (HSPC) transduced with potentially genotoxic vectors. For the SAGA assay, murine lineage negative (Lin-) HSPC are isolated and transduced with high multiplicities of infection (MOI) to reach at least 3 vector copies per cell. Post-transduction, bulk cultures are expanded for a total of 15 days in medium supplemented with cytokines promoting myeloid differentiation. Total RNA is prepared from the bulk cultures on day 15 and gene expression profiles are analyzed on microarrays.SAGA classifies samples based on their gene expression profile by using a support vector machine with a radial kernel on 9 features deduced by a genetic algorithm. SAGA is more robust, sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemia in clinical trials.
2019-01-01 | GSE109391 | 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: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:Overexpression of HOXB4 in hematopoietic stem cells (HSCs) leads to increased self-renewal without causing hematopoietic malignancies in transplanted mice. The molecular basis of HOXB4-mediated benign HSC expansion in vivo is not well understood. To gain further insight into the molecular events underlying HOXB4-mediated HSC expansion, we analyzed gene expression changes at multiple time points in Lin-Sca1+c-kit+ (LSK) cells from mice transplanted with bone marrow (BM) cells transduced with a MSCV-HOXB4-ires-YFP vector. A distinct HOXB4 transcriptional program was reproducibly induced and stabilized by 12 weeks after transplant. Dynamic expression changes were observed in genes critical for HSC self-renewal as well as genes involved in myeloid and B cell differentiation. Prdm16, a transcription factor associated with human acute myeloid leukemia (AML), was markedly repressed by HOXB4 but upregulated by HOXA9 and HOXA10, suggesting that Prdm16 downregulation was involved in preventing leukemia in HOXB4 transplanted mice. Functional evidence to support this mechanism was obtained by enforcing co-expression of sPrdm16 and HOXB4, which led to enhanced self-renewal, myeloid expansion, and leukemia. Altogether, these studies define the transcriptional pathways involved in HOXB4 HSC expansion in vivo and identify repression of Prdm16 transcription as a mechanism by which expanding HSCs avoid leukemic transformation. 5-FU treated bone marrow cells from female C57Bl/6L mice were transduced with concentrated supernatant from GPE+86-derived producer cells containing MSCV-HOXB4-ires-YFP or MSCV-ires-GFP retroviral vectors. Transduced cells were transplanted into lethally irradiated C57Bl/6L mice. HOXB4-YFP or GFP expressing LSK cells were sorted from transplanted mice at 6, 12 and 24 weeks post transplantation, of which RNA was extracted for microarray.
2014-10-01 | E-GEOD-59804 | biostudies-arrayexpress
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:In the context of lentiviral vector (LV)-mediated gene correction of hematopoietic stem and progenitor cells (HSPC), the viral origin of LV could trigger cellular responses with potential functional consequences that have not been investigated to date. Here, a time-course genome-wide transcriptional profiling of transduced human HSPC reveals a remarkably limited impact of LV on cellular gene expression, supporting some stealth features of the vector. In particular, LV efficiently escaped innate immune sensing that instead led to robust type I IFN signaling upon transduction with a gamma-retroviral vector. However, recognition of LV DNA prior to integration did trigger DNA damage responses that were activated also by non-integrating Adeno-associated vector DNA. As a consequence of this transient p53 activation, transduced HSPC showed increased apoptosis in vitro and reduced engraftment in vivo at limiting cell doses, but no long-term impact or competitive disadvantage were detected. Pharmacological inhibition of the LV signaling partially rescued both apoptosis and engraftment, potentially providing a novel strategy to further dampen the impact of ex vivo gene therapy on HSPC. Overall, our results shed light on the molecular mechanisms of viral vector sensing in HSPC and provide critical insight for the development of more stealth gene therapy strategies.
2017-06-28 | GSE92652 | GEO
Project description:Gene transfer vectors based on gamma-retroviruses target actively transcribing genes indicating that the cellular gene expression profile can be predictive of their integration pattern. Therefore, different culture conditions leading to different transcriptional activity may translate into differences in the profile of targeted genes in cells transduced with these vectors. Recent data from two gene therapy trials for SCID-X1 conducted in France and the UK suggested that small differences between in vitro stimulation conditions could explain the disparity in the frequency of common integrations sites observed in the two studies. We set out to compare the transcriptional activity of human CD34+ bone marrow-derived cells cultured under the French (S1) or British (S2) culture conditions.
2010-02-24 | GSE20505 | GEO
Project description: Not available
| S-EPMC2569169 | biostudies-literature
Project description:Expression data from cord blood CD34+ cells transduced with UBTF wildtype expression vectors, UBTF-TD expression vectors, or empty control vector.
| PRJNA784970 | ENA