Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations.

ABSTRACT: Gamma-retroviral/lentiviral vectors (γRV/LV) with self-inactivating (SIN) long terminal repeats (LTRs) and internal moderate cellular promoters pose a reduced risk of insertional mutagenesis when compared with vectors with active LTRs. Yet, in a recent LV-based clinical trial for β-thalassemia, vector integration within the HMGA2 gene induced the formation of an aberrantly spliced mRNA form that appeared to cause clonal dominance. Using a method that we developed, cDNA linear amplification-mediated PCR, in combination with high-throughput sequencing, we conducted a whole transcriptome analysis of chimeric LV-cellular fusion transcripts in transduced human lymphoblastoid cells and primary hematopoietic stem/progenitor cells. We observed a surprising abundance of read-through transcription originating outside and inside the provirus and identified the vector sequences contributing to the aberrant splicing process. We found that SIN LV has a sharply reduced propensity to engage in aberrant splicing compared with that of vectors carrying active LTRs. Moreover, by recoding the identified vector splice sites, we reduced residual read-through transcription and demonstrated an effective strategy for improving vectors. Characterization of the mechanisms and genetic features underlying vector-induced aberrant splicing will enable the generation of safer vectors, with low impact on the cellular transcriptome.

SUBMITTER: Cesana D

PROVIDER: S-EPMC3336994 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Analysis of lentiviral vector splicing events with nanopore sequencing

Project description:All lentiviral vectors derived from HIV-1 have the major splice donor (SD1) in the 5’ leader region and splice acceptor 7 (SA7) within the env region. Splicing events decrease the amount of full-length RNA available for packaging into virions and could lead to packaging of genomes containing internal deletions. Because there are splice sites or splicing enhancer/silencer elements in both gag and env, we compared how deleting each region affected intracellular genomic RNA splicing in RNA isolated from transfected HEK293Tsa cells. Oxford Nanopore direct cDNA sequencing was used to characterize the splice variants because the long-read length allows full-length transcripts to be analyzed. We show that deleting 507 nt of env, including the SA7, decreased the number of splicing events per transcript and increased the proportion of unspliced genomic RNAs ~3-fold in the cell.

2021-01-30 | GSE165829 | GEO

Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts.

Project description:Retroviral vectors integrate in genes and regulatory elements and may cause transcriptional deregulation of gene expression in target cells. Integration into transcribed genes also has the potential to deregulate gene expression at the posttranscriptional level by interfering with splicing and polyadenylation of primary transcripts. To examine the impact of retroviral vector integration on transcript splicing, we transduced primary human cells or cultured cells with HIV-derived vectors carrying a reporter gene or a human ?-globin gene under the control of a reduced-size locus-control region (LCR). Cells were randomly cloned and integration sites were determined in individual clones. We identified aberrantly spliced, chimeric transcripts in more than half of the targeted genes in all cell types. Chimeric transcripts were generated through the use of constitutive and cryptic splice sites in the HIV 5? long terminal repeat and gag gene as well as in the ?-globin gene and LCR. Compared with constitutively spliced transcripts, most aberrant transcripts accumulated at a low level, at least in part as a consequence of nonsense-mediated mRNA degradation. A limited set of cryptic splice sites caused the majority of aberrant splicing events, providing a strategy for recoding lentiviral vector backbones and transgenes to reduce their potential posttranscriptional genotoxicity.

| S-EPMC3347495 | biostudies-literature

Analysis of lentiviral vector splicing events with nanopore sequencing

Project description:Analysis of lentiviral vector splicing events with nanopore sequencing

| PRJNA697985 | ENA

The Landscape of Aberrant Alternative Splicing Events in Steatotic Liver Graft Post Transplantation via Transcriptome-Wide Analysis.

Project description:The application of steatotic liver graft has been increased significantly due to the severe donor shortage and prevalence of non-alcoholic fatty liver disease. However, steatotic donor livers are vulnerable to acute phase inflammatory injury, which may result in cancer recurrence. Alternative splicing events (ASEs) are critical for diverse transcriptional variants in hepatocellular carcinoma (HCC). Here, we aimed to depict the landscape of ASEs, as well as to identify the differential ASEs in steatotic liver graft and their association with tumor recurrence after transplantation. The overall portrait of intragraft transcripts and ASEs were elucidated through RNA sequencing with the liver graft biopsies from patients and rat transplant models. Various differential ASEs were identified in steatotic liver grafts. CYP2E1, ADH1A, CYP2C8, ADH1C, and HGD, as corresponding genes to the common pathways involved differential ASEs in human and rats, were significantly associated with HCC patients' survival. The differential ASEs related RNA-binding proteins (RBPs) were enriched in metabolic pathways. The altered immune cell distribution, particularly macrophages and neutrophils, were perturbated by differential ASEs. The cancer hallmarks were enriched in steatotic liver grafts and closely associated with differential ASEs. Our work identified the differential ASE network with metabolic RBPs, immune cell distribution, and cancer hallmarks in steatotic liver grafts. We verified the link between steatotic liver graft injury and tumor recurrence at post-transcriptional level, offered new evidence to explore metabolism and immune responses, and provided the potential prognostic and therapeutic markers for tumor recurrence.

| S-EPMC10179559 | biostudies-literature

Aberrant Clonal Hematopoiesis following Lentiviral Vector Transduction of HSPCs in a Rhesus Macaque.

Project description:Lentiviral vectors (LVs) are used for delivery of genes into hematopoietic stem and progenitor cells (HSPCs) in clinical trials worldwide. LVs, in contrast to retroviral vectors, are not associated with insertion site-associated malignant clonal expansions and, thus, are considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis affecting the erythroid, myeloid, and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. Nine insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPCs, which is concerning, and suggests that strong constitutive promoters should not be included in LVs.

| S-EPMC6554657 | biostudies-literature

Characterization of viral RNA splicing using whole-transcriptome datasets from host species.

Project description:RNA alternative splicing (AS) is an important post-transcriptional mechanism enabling single genes to produce multiple proteins. It has been well demonstrated that viruses deploy host AS machinery for viral protein productions. However, knowledge on viral AS is limited to a few disease-causing viruses in model species. Here we report a novel approach to characterizing viral AS using whole transcriptome dataset from host species. Two insect transcriptomes (Acheta domesticus and Planococcus citri) generated in the 1,000 Insect Transcriptome Evolution (1KITE) project were used as a proof of concept using the new pipeline. Two closely related densoviruses (Acheta domesticus densovirus, AdDNV, and Planococcus citri densovirus, PcDNV, Ambidensovirus, Densovirinae, Parvoviridae) were detected and analyzed for AS patterns. The results suggested that although the two viruses shared major AS features, dramatic AS divergences were observed. Detailed analysis of the splicing junctions showed clusters of AS events occurred in two regions of the virus genome, demonstrating that transcriptome analysis could gain valuable insights into viral splicing. When applied to large-scale transcriptomics projects with diverse taxonomic sampling, our new method is expected to rapidly expand our knowledge on RNA splicing mechanisms for a wide range of viruses.

| S-EPMC5818608 | biostudies-literature

Transcriptome sequencing reveals aberrant alternative splicing in Huntington's disease.

Project description:Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG expansion in the gene-encoding Huntingtin (HTT). Transcriptome dysregulation is a major feature of HD pathogenesis, as revealed by a large body of work on gene expression profiling of tissues from human HD patients and mouse models. These studies were primarily focused on transcriptional changes affecting steady-state overall gene expression levels using microarray based approaches. A major missing component, however, has been the study of transcriptome changes at the post-transcriptional level, such as alternative splicing. Alternative splicing is a critical mechanism for expanding regulatory and functional diversity from a limited number of genes, and is particularly complex in the mammalian brain. Here we carried out a deep RNA-seq analysis of the BA4 (Brodmann area 4) motor cortex from seven human HD brains and seven controls to systematically discover aberrant alternative splicing events and characterize potential associated splicing factors in HD. We identified 593 differential alternative splicing events between HD and control brains. Using two expanded panels with a total of 108 BA4 tissues from patients and controls, we identified four splicing factors exhibiting significantly altered expression levels in HD patient brains. Moreover, follow-up molecular analyses of one splicing factor PTBP1 revealed its impact on disease-associated splicing patterns in HD. Collectively, our data provide genomic evidence for widespread splicing dysregulation in HD brains, and suggest the role of aberrant alternative splicing in the pathogenesis of HD.

| S-EPMC5179942 | biostudies-literature

Determining the impact of alternative splicing events on transcriptome dynamics.

Project description:BACKGROUND: The complete sequencing of the human genome and its subsequent analysis revealed a predominant role for alternative splicing in the generation of proteome diversity. Splice switching oligonucleotides (SSOs) are a powerful and specific tool to experimentally control alternative splicing of endogenous messenger RNAs in living cells. SSOs also have therapeutic potential to treat diseases that are caused by aberrant splicing. The assignment of biological roles to alternative splicing events of currently unknown function promises to provide a largely untapped source of potential new therapeutic targets. Here we have developed a protocol that combines high sensitivity microarrays with the transfection of SSOs to monitor global changes in gene expression downstream of alternate, endogenous splice events. RESULTS: When applied to a well-characterized splicing event in the Bcl-x gene, the application of high sensitivity microarrays revealed a link between the induction of the Bcl-xS isoform and the repression of genes involved in protein synthesis. CONCLUSION: The strategy introduced herein provides a useful approach to define the biological impact of any given alternative splicing event on global gene expression patterns. Furthermore, our data provide the first link between Bcl-xS expression and the repression of ribosomal protein gene expression.

| S-EPMC2584107 | biostudies-other

Signature of gene aberrant alternative splicing events in pancreatic adenocarcinoma prognosis.

Project description:Alternative splicing (AS), as an effective and universal mechanism of transcriptional regulation, is involved in the development and progression of cancer. Therefore, systematic analysis of alternative splicing in pancreatic adenocarcinoma (PAAD) is warranted. The corresponding clinical information of the RNA-Seq data and PAAD cohort was downloaded from the TCGA data portal. Then, a java application, SpliceSeq, was used to evaluate the RNA splicing pattern and calculate the splicing percentage index (PSI). Differentially expressed AS events (DEAS) were identified based on PSI values between PAAD cancer samples and normal samples of adjacent tissues. Kaplan-Meier and Cox regression analyses were used to assess the association between DEAS and patient clinical characteristics. Unsupervised cluster analysis used to reveal four clusters with different survival patterns. At the same time, GEO and TCGA combined with GTEx to verify the differential expression of AS gene and splicing factor. After rigorous filtering, a total of 45,313 AS events were identified, 1,546 of which were differentially expressed AS events. Nineteen DEAS were found to be associated with OS with a five-year overall survival rate of 0.946. And the subtype clusters results indicate that there are differences in the nature of individual AS that affect clinical outcomes. Results also identified 15 splicing factors associated with the prognosis of PAAD. And the splicing factors ESRP1 and RBM5 played an important role in the PAAD-associated AS events. The PAAD-associated AS events, splicing networks, and clusters identified in this study are valuable for deciphering the underlying mechanisms of AS in PAAD and may facilitate the establishment of therapeutic goals for further validation.

| S-EPMC8100795 | biostudies-literature

Detection of aberrant splicing events in RNA-seq data using FRASER.

Project description:Aberrant splicing is a major cause of rare diseases. However, its prediction from genome sequence alone remains in most cases inconclusive. Recently, RNA sequencing has proven to be an effective complementary avenue to detect aberrant splicing. Here, we develop FRASER, an algorithm to detect aberrant splicing from RNA sequencing data. Unlike existing methods, FRASER captures not only alternative splicing but also intron retention events. This typically doubles the number of detected aberrant events and identified a pathogenic intron retention in MCOLN1 causing mucolipidosis. FRASER automatically controls for latent confounders, which are widespread and affect sensitivity substantially. Moreover, FRASER is based on a count distribution and multiple testing correction, thus reducing the number of calls by two orders of magnitude over commonly applied z score cutoffs, with a minor loss of sensitivity. Applying FRASER to rare disease diagnostics is demonstrated by reprioritizing a pathogenic aberrant exon truncation in TAZ from a published dataset. FRASER is easy to use and freely available.

| S-EPMC7822922 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data