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.
Project description:Differentiation and maintenance of cardiac muscle is a complex biological process. MEF2D appears to play an important role in the regulation of cardiomyocyte homeostasis. We knocked down expression of MEF2D in NRVMs, and assessed global expression pattern changes in MEF2D knockdown against a negative control. NRVMs were extracted from 1 to 2 day old rat pups and were allowed to recover for 24 hours before being transduced with shRNA viral vectors to knockdown expression of MEF2D or LacZ (negative control). After 3 days, total RNA was harvested for Affymetrix global gene expression microarray analysis. Each array was pooled RNA from six biological replicates.
Project description:Maddalena et al. showed that the limited DNA transfer capacity (~4.7kb) of adeno associated viral (AAV) vectors can be expanded up to 14kb with triple AAV vectors for the efficient expression of the therapeutic CDH23 (10.1kb) and ALMS1 (12.5kb) genes.
Project description:This study identifies the age related changes in glycan receptors of heparan sulfate (HS) proteoglycan (receptor for rAAV2), and/or N-glycans with terminal galactose (receptor for rAAV9) and proteomics for receptors and co-receptors of adeno-associated virus in the nigro-striatal region of the brain. We analyzed the striatum and substantia nigra for changes in HS, N-glycans and proteomic signatures in young versus aged rat brain nigro-striatum from histological sections. These studies provide insight into age- and brain region-specific changes in glycan receptors and proteomics that will inform design of improved viral vectors for Parkinson’s disease gene therapy.
Project description:Adeno-associated virus (AAV) vectors generated with five different capsids were intravenously injected into human livers undergoing normothermic machine perfusion (NMP). To assess the tropism of the five AAV vectors in the human livers, single-cell suspensions of hepatocytes and non-parenchymal cells (NPCs) were analyzed by single-cell RNA sequencing (scRNAseq). Differentially expressed genes were identified in AAV vector-transduced hepatocytes and unique cell populations.
Project description:Numerous studies have suggested a link between fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) signaling pathways; however the nature of this link has not been established. To evaluate this relationship we investigated VEGF signaling in endothelial cells with disrupted FGF signaling in vitro and in vivo. We find that endothelial cells lacking FGF signaling become unresponsive to VEGF due to down regulation of VEGFR2 expression caused by reduced Vegfr2 enhancer activation, which is in turn caused by reduced activation of Ets family transcription factors. In vivo this manifests in the loss of vascular integrity and morphogenesis. Thus, basal FGF stimulation of the endothelium is required for maintenance of VEGFR2 expression and the ability to respond to VEGF stimulation and accounts for the hierarchic control of vascular formation by FGFs and VEGF. Primary mouse lung endothelial cells were transduced with either Adeno-Null (empty) or Adeno- dominant negative FGF receptor 1 and harvested 24 hours after transduction. Total RNA was extracted and subjected to the analysis using SuperArray GEArray Q Series Mouse Angiogenesis Gene Array. Comparisons were made between treatments.
Project description:Purpose: Assess whether knocking out the UMLILO lncRNA altered the expression of genes transcribed within the CXCL chemokine TAD Outcome: To confirm whether the effect of UMLILO was limited to the CXCL TAD. Adeno-associated viral vectors (AAVs) were constructed that contain CRISPR/Cas9 and guides targeting UMLILO to delete the full length UMLILO transcript. RNAseq was performed on a transduced THP-1 population to verify genome-wide effects of UMLILO depletion. This revealed that IL8, CXCL1, 2, 3 transcription was abrogated, but a similar effect was not seen for genes located outside of the CXCL TAD boundary
Project description:We show that an excess of VEGF-B protects the heart via adaptive cardiac hypertrophy and increased coronary arterial reserve, and by inducing a shift from lipid to glucose metabolism. Six hearts transduced with AAV-VEGF-B were compared to six AAV-HSA (human serum albumin) controls