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Project description:Liver gene therapy with adeno-associated viral (AAV) vectors is under clinical investigation for hemophilia A (HemA), the most common inherited X-linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti-F8 antibodies which neutralize F8 activity. Taking advantage of split inteins-mediated protein trans-splicing, we divided the coding sequence of the large and highly secreted F8-N6 variant in two separate AAV split-inteins vectors whose co-administration to HemA mice results in F8 protein reconstitution and expression of therapeutic levels of F8 over time without anti-F8 antibodies development.
2022-03-31 | PXD031884 | Pride
Project description:Hemophilia B, or the 'Royal Disease', arises from mutations in the Coagulation Factor IX (F9) gene. Mutations within the F9 promoter are associated with a remarkable form of the disease, termed Hemophilia B Leyden, in which symptoms ameliorate after puberty. Mutations at the -5/-6 site account for the majority of Leyden cases, and have been postulated to disrupt the binding of a transcriptional activator, the identity of which has remained elusive for more than twenty years. Here we show that the ONECUT transcription factors (ONECUT1/HNF6 and ONECUT2/HNF6B) bind to the -5/-6 site. The various Hemophilia B Leyden mutations that have been reported in this site inhibit ONECUT binding to varying degrees that correlate well with their associated clinical severities. In addition, expression of F9 is crucially dependent on ONECUT factors in vivo and as such, mice that are deficient in either ONECUT1, ONECUT2 or both, exhibit depleted levels of F9. Taken together, our findings establish the ONECUT transcription factors as the missing regulators of Hemophilia B Leyden that operate through the -5/-6 site.
2013-03-06 | E-MTAB-890 | biostudies-arrayexpress
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:AAV gene therapy has recently been approved for clinical use and shown to be efficacious and safe in a growing number of clinical trials. However, the safety of AAV as a gene therapy has been challenged by a few studies that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. The association between AAV and HCC has been difficult to reconcile and is the subject of intense debate because numerous AAV studies have not reported toxicity. Here, we report a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the Rian locus and the over-expression of a proximal gene, Rtl1, were associated with HCC. In addition, we identify a number of genes with differential expression that maybe useful in the study, diagnosis and treatment of HCC. We demonstrate that AAV vector dose, enhancer-promoter selection, and the timing of gene delivery are the defining factors in AAV-mediated insertional mutagenesis. Our results help explain the AAV-mediated genotoxicity previously observed and have important implications for the design of both safer AAV vectors and gene therapy studies. To investigate the possibility that insertional mutagenesis by AAV contributed to the development of HCC, we collected normal and tumor tissues from adult mouse livers that received AAV injection at a neonatal stage.
2015-01-01 | E-GEOD-57597 | biostudies-arrayexpress
Project description:AAV gene therapy has recently been approved for clinical use and shown to be efficacious and safe in a growing number of clinical trials. However, the safety of AAV as a gene therapy has been challenged by a few studies that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. The association between AAV and HCC has been difficult to reconcile and is the subject of intense debate because numerous AAV studies have not reported toxicity. Here, we report a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the Rian locus and the over-expression of a proximal gene, Rtl1, were associated with HCC. In addition, we identify a number of genes with differential expression that maybe useful in the study, diagnosis and treatment of HCC. We demonstrate that AAV vector dose, enhancer-promoter selection, and the timing of gene delivery are the defining factors in AAV-mediated insertional mutagenesis. Our results help explain the AAV-mediated genotoxicity previously observed and have important implications for the design of both safer AAV vectors and gene therapy studies. To investigate the possibility that insertional mutagenesis by AAV contributed to the development of HCC, we collected normal and tumor tissues from adult mouse livers that received AAV injection at a neonatal stage.
2015-09-01 | E-GEOD-61632 | biostudies-arrayexpress
Project description:AAV gene therapy has recently been approved for clinical use and shown to be efficacious and safe in a growing number of clinical trials. However, the safety of AAV as a gene therapy has been challenged by a few studies that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. The association between AAV and HCC has been difficult to reconcile and is the subject of intense debate because numerous AAV studies have not reported toxicity. Here, we report a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the Rian locus and the over-expression of a proximal gene, Rtl1, were associated with HCC. In addition, we identify a number of genes with differential expression that maybe useful in the study, diagnosis and treatment of HCC. We demonstrate that AAV vector dose, enhancer-promoter selection, and the timing of gene delivery are the defining factors in AAV-mediated insertional mutagenesis. Our results help explain the AAV-mediated genotoxicity previously observed and have important implications for the design of both safer AAV vectors and gene therapy studies.
2015-09-01 | GSE61632 | GEO
Project description:Targeting PRMT5 in canine lymphoma: we characterized expression patterns of PRMT5 in canine lymphomas and correlated these with histological subtypes using tissue microarrays. We characterized expression of PRMT5 in three canine lymphoma-derived cell lines and primary lymph node biopsies. We have demonstrated that inhibition of PRMT5 leads to growth suppression and induction of apoptosis in canine lymphoma cell lines and primary canine lymphoma cells in a time and dose-dependent manner, while selectively decreasing global marks of symmetric dimethylarginine (SDMA) and histone H4 arginine 3 symmetric di-methylation. We performed ATAC-sequencing with pathway enrichment analysis to characterize genome-wide changes in chromatin accessibility after PRMT5 inhibition. We performed gene expression microarrays with pathway analysis to characterize whole transcriptome changes in canine lymphoma cell lines treated with PRMT5 inhibitors. This work validates PRMT5 as a promising therapeutic target for canine lymphoma and supports the continued used of the spontaneously occurring canine lymphoma model for the preclinical development of PRMT5 inhibitors for the treatment of human NHL.
2021-04-20 | GSE156084 | GEO
Project description:We have shown that intravenous injection of HDAC3 floxed mice with adeno-associated virus (AAV) expressing Cre depletes hepatic HDAC3, upregulates lipogenic gene expression, and causes fatty liver. When AAV-Flag-HDAC3 wild-type (WT) is co-injected along with AAV-Cre, the exogenous HDAC3 is expressed at endogenous levels and can completely rescue fatty liver phenotype. Here we profile transcriptome of the rescued WT livers in comparison with HDAC3-depleted (KO) livers. 4-months old C57BL/6 male mice were co-injected with AAV-Cre or AAV-Cre plus AAV-Flag-HDAC3. Mice were fed ad libitum and harvested at 5 pm (ZT10) at 2-weeks post-injection. Liver total RNA was extracted and hybridized to Affymetrix Mouse Gene 1.0ST array.
2013-11-25 | E-GEOD-49386 | biostudies-arrayexpress
Project description:Hemophilia mice were treated for one month with ASOs targeting either Upf1 or Upf3b. The liver RNAs were extracted and analyzed
2017-11-03 | GSE106436 | GEO
Project description:This is an ATAC sequencing experiment to explore chromatin accessibility change in mouse skeletal muscle treated with either AAV-GFP or AAV-CAAHR (a constitutively active mutant aryl hydrocarbon receptor). Mice received intramuscular injection of the AAV 5 months before the harvest of muscle.
2024-04-30 | GSE255812 | GEO