Epigenetic Suppression of Transgenic T-cell Receptor Expression via Gamma-Retroviral Vector Methylation in Adoptive Cell Transfer Therapy
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PROVIDER: PRJNA644286 | ENA |
REPOSITORIES: ENA
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Project description:Transgenic T-cell receptor (TCR) adoptive cell therapies recognizing tumor antigens are associated with robust initial response rates, but frequent disease relapse. This usually occurs in the setting of poor long-term persistence of cells expressing the transgenic TCR, generated using murine stem cell virus (MSCV) -retroviral vectors. Analysis of clinical transgenic adoptive cell therapy products in vivo revealed that despite strong persistence of the transgenic TCR DNA sequence over time, its expression was profoundly decreased over time at the RNA and protein levels. Patients with the greatest degrees of expression suppression displayed significant increases in DNA methylation over time within the MSCV promoter region, as well as progressive increases in DNA methylation within the entire MSCV vector over time. These increases in vector methylation occurred independently of its integration site within the host genomes. These results have significant implications for the design of future viral-vector gene engineered adoptive cell transfer therapies.
2020-07-06 | GSE153820 | GEO
Project description:Transgenic T-cell receptor (TCR) adoptive cell therapies recognizing tumor antigens are associated with robust initial response rates, but frequent disease relapse. This usually occurs in the setting of poor long-term persistence of cells expressing the transgenic TCR, generated using murine stem cell virus (MSCV) γ-retroviral vectors. Analysis of clinical transgenic adoptive cell therapy products in vivo revealed that despite strong persistence of the transgenic TCR DNA sequence over time, its expression was profoundly decreased over time at the RNA and protein levels. Patients with the greatest degrees of expression suppression displayed significant increases in DNA methylation over time within the MSCV promoter region, as well as progressive increases in DNA methylation within the entire MSCV vector over time. These increases in vector methylation occurred independently of its integration site within the host genomes. These results have significant implications for the design of future viral vector gene-engineered adoptive cell transfer therapies. SIGNIFICANCE: Cellular immunotherapies' reliance on retroviral vectors encoding foreign genetic material can be vulnerable to progressive acquisition of DNA methylation and subsequent epigenetic suppression of the transgenic product in TCR adoptive cell therapy. This must be considered in the design of future generations of cellular immunotherapies for cancer.This article is highlighted in the In This Issue feature, p. 1611.
| S-EPMC7641915 | biostudies-literature
Project description:Adoptive T-Cell therapy is being considered as a promising method for cancer treatment. In this approach, patient's T cells are isolated, modified, expanded, and administered back to the patient. Modifications may include adding specific T cell receptors (TCR) or chimeric antigen receptors (CAR) to the isolated cells by using retroviral vectors. PG13 cells, derivatives of NIH3T3 mouse fibroblasts, are being used to stably produce retroviral vectors that transduce the T cells. PG13 cells are anchorage-dependent cells that grow in roller bottles or cell factories and lately also in fixed bed bioreactors to produce the needed viral vector. To scale up viral vector production, PG13 cells were propagated on microcarriers in a stirred tank bioreactor utilizing an alternating tangential flow perfusion system. Microcarriers are 10 µm - 0.5 mm beads that support the attachment of cells and are suspended in the bioreactor that provides controlled growth conditions. As a result, growth parameters, such as dissolved oxygen concentration, pH, and nutrients are monitored and continuously controlled. There were no detrimental effects on the specific viral vector titer or on the efficacy of the vector in transducing the T cells of several patients. Viral vector titer increased throughout the 11 days perfusion period, a total of 4.8 × 1011 transducing units (TU) were obtained with an average titer of 4.4 × 107 TU/mL and average specific productivity of 10.3 (TU) per cell, suggesting that this method can be an efficient way to produce large quantities of active vector suitable for clinical use.
| S-EPMC6028049 | biostudies-literature
Project description:Gene transfer of distinct cocktails of transcription factors enables the targeted modification of cell fate. Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) via ectopic expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows creation of pluripotent cells with the potential to differentiate into any cell type of the body. To achieve a high degree of biosafety for future application of converted cells, technologies are required to express the required transcription factors without affecting the target cell genome. Retroviral vectors are widely employed gene transfer vehicles and can be converted into transient gene delivery vehicles by mutation of the viral integrase, thus avoiding risks associated with retroviral integration. We improved the retroviral episome transfer system by identifying the most suitable vector architecture, by enhancing episomal expression and by tuning the activity of the transcription factor Oct4. Repeated episomal transductions allowed establishment of stable, fully reversible expression levels over time. Providing proof-of-principle in the reprogramming context, we were able to generate human iPSCs following retroviral episomal transfer of Oct4 in fibroblasts stably expressing Klf4, Sox2 and c-Myc, with a high percentage of clones not carrying residually integrated vector copies.
2015-05-06 | GSE57378 | GEO
Project description:Gene transfer of distinct cocktails of transcription factors enables the targeted modification of cell fate. Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) via ectopic expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows creation of pluripotent cells with the potential to differentiate into any cell type of the body. To achieve a high degree of biosafety for future application of converted cells, technologies are required to express the required transcription factors without affecting the target cell genome. Retroviral vectors are widely employed gene transfer vehicles and can be converted into transient gene delivery vehicles by mutation of the viral integrase, thus avoiding risks associated with retroviral integration. We improved the retroviral episome transfer system by identifying the most suitable vector architecture, by enhancing episomal expression and by tuning the activity of the transcription factor Oct4. Repeated episomal transductions allowed establishment of stable, fully reversible expression levels over time. Providing proof-of-principle in the reprogramming context, we were able to generate human iPSCs following retroviral episomal transfer of Oct4 in fibroblasts stably expressing Klf4, Sox2 and c-Myc, with a high percentage of clones not carrying residually integrated vector copies. RNA obtained from human embryonic stem cells, fibroblasts and induced pluripotent stem cell lines reprogrammed with the mentioned reprogramming protocol
2015-05-06 | E-GEOD-57378 | biostudies-arrayexpress
Project description:Endogenous retroviruses (ERVs) in the mammalian genome play diverse roles in embryonic development. These development-related ERVs are generally repressed in somatic cells and are therefore likely repressed in embryos derived from somatic cell nuclear transfer (SCNT). In this study, we sought to identify ERVs that are repressed in SCNT-derived morula embryos, which may consequently cause previously unexplained embryonic deaths shortly after implantation. Our transcriptome analysis revealed that, amongst ERV families, ERVK was specifically and strongly downregulated in SCNT embryos while other transposable elements including LINE and ERVL were unchanged. Among subfamilies of ERVK, RLTR45-int was most repressed in SCNT embryos despite its highest expression in control fertilized embryos. Interestingly, the nearby genes (within 5–50 kb, n = 19) of the repressed RLTR45-int loci were also repressed in SCNT embryos with a significant correlation between them. Furthermore, lysine H3K27 acetylation was enriched around the RLTR45-int loci. These findings indicate that RLTR45-int elements function as enhancers of nearby genes. Indeed, deletion of two sequential RLTR45-int loci on chromosome 4 or 18 resulted in downregulations of nearby genes at the morula stage. We also found that RLTR45-int loci, especially SCNT-low, enhancer-like loci, were strongly enriched with H3K9me3, a repressive histone mark. Importantly, these H3K9me3-enriched regions were resistant to histone demethylase Kdm4d in SCNT embryos. Thus, we identified ERVK subfamily RLTR45-int, putative enhancer elements, as a strong reprogramming barrier for SCNT. A large-scale downregulations of their regulating genes in SCNT morulae might cumulatively affect postimplantation development of SCNT embryos.
2022-10-08 | GSE214878 | GEO
Project description:Primary objectives: The main objective is to determine the feasibility and safety of adoptive cell therapy in combination with checkpoint inhibition across different metastatic solid cancers.
Primary endpoints: Toxicity: Registration of all AEs and unexpected events in relation to the treatment will be done in accordance with the common terminology criteria for adverse events (CTCAE) criteria as described in appendix 3.
| 2537065 | ecrin-mdr-crc
Project description:The γ-retroviral vector is a gene delivery vehicle that is commonly used in gene therapy. Despite its efficacy, its strong enhancers contributed to malignant transformations in some hematopoietic stem cell (HSC) gene therapy trials. A safer version without viral enhancers (SIN) is available, but its production is cumbersome, as high titers can only be obtained in transient transfection. Our aim was to develop a system that could easily generate high-titer SIN vectors from stable producer cells. The use of the cytomegalovirus enhancer-promoter sequence to generate the full-length genomic RNA combined to sequences that decrease transcriptional readthrough (WPRE and strong polyadenylation sequences) led to 6 × 106 infectious units (IU)/mL of a SIN GFP vector in transient transfection. The incorporation of a blasticidin selection cassette to the retroviral plasmid allowed the generation of stable clones in the 293Vec packaging cells that release 2 × 107 IU/mL and 1.4 × 107 IU/mL of a SIN GFP and a SIN PIGA vector, respectively. A titer of 1.8 × 106 IU/mL was obtained with a SIN vector containing the long 8.9-kb COL7A1 cDNA. Thus, an efficient process was established for the generation of stable 293Vec-derived retrovirus producer cells that release high-titer SIN vectors.
| S-EPMC6610700 | biostudies-literature
Project description:In vivo, the expansion of antigen experienced CD4+ T cells is limited by intrinsic factors. Using an in vivo genome-wide CRISPR-Cas9 screen, we identified SOCS1 as a major non-redundant checkpoint imposing a brake on antigen experienced CD4+ T-cell proliferation upon antigen challenge. Although SOCS1 has been previously described as an inhibitor of CD8+ T-cell effector function, we highlight here that it is abrogating the CD4+ T-cell response. SOCS1 integrates several cytokines receptor signals to actively silence TCR-induced polycytokine signaling pathways, restraining CD4+ T cell proliferation and functions. Targeting SOCS1 in mouse and human CD4+ and CD8+ T cells improved the therapeutic efficacy of antitumor adoptive T-cell therapies, enhancing intra-tumor T cell accumulation, persistence and effector functions. SOCS1 deletion in CAR-T cells enhances CD4+ CAR T-cell proliferation/survival and polyfunctionality in vivo, promoting long term rejection of established tumors. These findings identify SOCS1 as a major intracellular checkpoint inhibitor of re-stimulated CD4+ T cells, opening new possibilities to optimize adoptive T cell therapies.
2021-09-02 | GSE154791 | GEO