Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1076258 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27959700_1.fastq.gz | Fastqsanger.gz | |||
| SRR27959700_2.fastq.gz | Fastqsanger.gz | |||
| SRR27959701_1.fastq.gz | Fastqsanger.gz | |||
| SRR27959701_2.fastq.gz | Fastqsanger.gz | |||
| SRR27959702_1.fastq.gz | Fastqsanger.gz |
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Project description:The longitudinal kinetics of AAV5 vector integration profiles and evaluation of clonal expansion in mice
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Project description:Recombinant adeno-associated virus (AAV)-based vectors are used clinically for gene transfer and persist as extrachromosomal episomes. A small fraction of vector genomes can integrate into the host genome, but the theoretical risk of tumorigenesis may depend on vector regulatory features. A mouse model was used to investigate long-term kinetics and integration profiles of an AAV serotype 5 (AAV5) vector that mimics key features of valoctocogene roxaparvovec (AAV5-hFVIII-SQ), a gene therapy for severe hemophilia A. The majority (95%) of vector genome reads identified by target enrichment sequencing were derived from episomes, and mean integration frequency was 2.70 (standard deviation, 1.24) integrations per 1000 cells. Longitudinal integration analysis suggested AAV5 vector integrations occur primarily within 1 week, at low frequency, and their abundance was stable over time. Integration profiles were polyclonal, and only 5.46% of integrations had a common integration site order ≥5, suggesting random distributions when looking at a 50-kb genomic window. No integrations were associated with clonal expansion. Integrations were enriched near transcription start sites of genes highly expressed in the liver (P = 1x10−4) and less enriched for genes with low or no liver expression. We found no evidence of tumorigenesis or fibrosis caused by the vector integrations.
2024-02-01 | GSE254503 | GEO
Project description:We developed an in vivo hematopoietic stem and progenitor cell (HSPC) gene therapy approach that does not require HSC cell transplantation. To achieve therapeutically relevant numbers of corrected cells, we tested truncated HMGA2 instead of chemo-selection for enrichment of transduced HSPCs. We constructed HSC-tropic HDAd5/35++ vectors expressing a 3’UTR HMGA2 truncated gene and a GFP reporter gene. A SB100x transposase vector was used to mediate random integration of the tHMGA2/GFP transgene cassette. Mice were mobilized by subcutaneous injections of G-CSF and AMD3100/Plerixafor and intravenously injected with the integrating tHMGA2/GFP vector. This resulted in slow but progressive competitive expansion of GFP+ PBMCs, reaching about 50% by week 44 with further expansion in secondary recipients. Expansion occurred at the level of HSCs as well as at the level of myeloid, lymphoid, and erythroid progenitors within the bone marrow and spleen. Importantly, based on genome-wide integration site analyses, expansion was polyclonal, without any signs of clonal dominance. The results were validated in humanized mice. This is the first demonstration that simple subcutaneous and intravenous injections of mobilization drugs and HDAd vectors can trigger autocatalytic, self-limiting expansion of in vivo transduced HSPCs resulting in marking rates that, theoretically, are curative for hemoglobinopathies.
2024-10-08 | GSE268678 | GEO
Project description:<p>We analyzed clonal evolution in serial samples from five CLL patients who became resistant to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, using whole-exome and deep targeted sequencing. We observe a BTK-C481S mutation in one of five patients, and multiple PLCG2 mutations in a second patient. The other patients had an expansion of clones harboring del(8p) carrying additional driver mutations (EP300, MLL2, EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. We calculated the growth kinetics of ibrutinib-resistant subclones and estimated the size of the resistant clones at treatment initiation, which we validated by droplet-microfluidic technology. Haplo-insufficiency of TRAIL-R, a consequence of del(8p), led to TRAIL insensitivity which may contribute to development of ibrutinib resistance. These findings demonstrate that ibrutinib therapy has the potential to lead to clonal selection and expansion of rare cell populations already present at the time of treatment initiation. They also provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance, previously attributed solely to mutations in BTK and related pathway molecules.</p>
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Project description:Gene therapy has been thriving in recent years, as featured by general triumphs in several clinical trials to treat hemophilia. It highlights the significance of AAV mediated gene delivery to liver. AAV5 is a unique serotype with low neutralizing antibody prevalence. In this study, we generated an AAV5 mutant vector by inserting an oligopeptide into the variable region VIII of the AAV5, termed as AAVzk2, which displayed comparable yield with wild type serotypes. Mice intravenously injected with AAVzk2 demonstrated a stronger liver transduction than AAV5, which were grossly comparable with AAV8 and 9 treated mice. However, AAVzk2 negligibly transduced other tissues or organs such as heart, lung, spleen, kidney, brain and skeletal muscle, indicating a liver specific tropism. Further studies showed a superior human hepatocellular transduction of AAVzk2 to AAV5, 8 and 9, whereas the seroreactivity of AAVzk2 was as low as AAV5. Moreover, mass spectroscopy revealed great divergency in post-translational modification patterns between AAV5 and AAVzk2. Overall, we provide a novel AAV serotype that could not only facilitate a robust and specific liver gene delivery to a large population, but also offer valuable clues for future investigation of the nature of AAV5.
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Project description:<p>Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). In this study we sequenced the whole genome (~80X) and transcriptome of tumor and non-tumor samples from four HCC patients and identified over two hundred HBV integration sites. We found significant clonal expansion of HBV-integrated hepatocytes specifically in the tumor samples. We observed a diverse collection of genomic perturbations near viral integration sites, including gene disruption, viral promoter-driven human transcription, viral-human transcript fusion and DNA copy number alteration. We also sequenced one patient at ultra-high coverage (~240X) to build the most comprehensive HBV-integration landscape yet attempted. Our data suggest that the viral integration significantly expands carcinogenic opportunities in HBV-infected individuals.</p>
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Project description:Infiltrating T-lymphocytes from the peripheral blood into the central nervous system (CNS) play a dynamic role in the development of a neurological immune-mediated diseases. HAM/TSP is a chronic progressive inflammatory neurological disorder associated with human T-cell lymphotropic virus type I (HTLV-I) infection. In this chronic myelopathy, virus-infected circulating T-cells infiltrate the CNS and an immune response is initiated against the components of CNS. As the HTLV-I proviral load (PVL) has been used as the best clinical marker for patient diagnostic with HAM/TSP, we hypothesized there might be a signature on T-cell receptor (TCR) clonal repertoire in these patients, which could distinguish HAM/TSP patients from the healthy population, as well as from patients with a more heterogeneous CNS-reactive inflammatory disease as multiple sclerosis (MS). With this in mind, we applied an innovative unbiased molecular technique – unique molecular identifier (UMI) library-strategy to investigate with high accuracy the TCR clonal repertoire by high throughput sequencing (HTS) technology. cDNA-TCR β-chain libraries were sequenced from 2 million peripheral mononuclear cells (PBMCs) in 14 HAM/TSP patients, 34 MS patients and 20 healthy controls (HC). To address whether the clonal expansion correlates with the patient’s PVL level, analysis of longitudinal TCR repertoire was performed in 2 HAM/TSP patients. Over 5.6 million TCR sequences were generated per sample on HiSeq 2500 Illumina system and analyzed through the molecular identifier groups-based error correction pipeline (MiGEC). Bioinformatic analysis showed that clones with more than 8 reads had a lower coefficient of variation (CV) and then could be used with confidence to evaluate the TCR clonal expansion. While HAM/TSP patients showed the higher clonal T-cell expansion compared to MS and HC, increase of the TCR clonal expansion was inversely correlated with the diversity of TCR repertoire in all subject’s group. In addition, correlation of the PVL with TCR clonal expansion was observed in HAM/TSP patients at longitudinal time-points. Surprisingly, MS patients showed a higher diversity of TCR repertoire along with a very slight clonal T-cell expansion in comparison to either HAM/TSP patients or HC. Despite of the higher TCR clonal expansion in HAM/TSP patients, a non-shared or “private” TCR repertoire was observed in these patients. No clones that shared the same CDR3 amino acid sequences were seen in HC and MS patients. However, a cluster of related CDR3 amino acid sequences were observed for 18 out of 34 MS patients when evaluated by phylogenetic tree analysis. It suggestes that a TCR-repertoire signature might characterize patients with MS. Our findings suggest that even though a unique TCR-b repertoire shapes the immune response in patients with neurological immune-mediated disease, a relatedness on clonal T-cell repertoire exist in MS.
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