Efficient Transduction of Vascular Endothelial Cells with Recombinant Adeno-Associated Virus Serotype 1 and 5 Vectors

Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human α1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding β-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies

Adeno-associated virus vector-mediated gene delivery to the vasculature and kidney.

Relatively successful elsewhere, gene delivery aimed at the vasculature and kidney has made very little progress. In the kidney, the hurdles are related to the unique structure–function relationships of this organ and in the blood vessels to a variety of, mostly endothelial, factors making the delivery of transgenes very difficult. Among gene-therapeutic approaches, most viral gene delivery systems utilized to date have shown significant practical and safety-related limitations due to the level and duration of recombinant transgene expression as well as their induction of a significant host immune response to vector proteins. Recombinant adeno-associated virus (rAAV) vectors appear to offer a vehicle for safe, long-term transgene expression. rAAV-based vectors are characterized by a relative non-immunogenicity and the absence of viral coding sequences. Furthermore, they allow for establishment of long-term latency without deleterious effects on the host cell. This brief review addresses problems related to transgene-delivery to kidney and vasculature with particular attention given to rAAV vectors. The potential for gene therapy as a strategy for selected renal and vascular diseases is also discussed

Heme Oxygenase-1 Deficiency Promotes Epithelial-Mesenchymal Transition and Renal Fibrosis

Induction of heme oxygenase-1 (HO-1) is associated with potential antifibrogenic effects. The effects of HO-1 expression on epithelial-mesenchymal transition (EMT), which plays a critical role in the development of renal fibrosis, are unknown. In this study, HO-1−/− mice demonstrated significantly more fibrosis after 7 d of unilateral ureteral obstruction compared with wild-type mice, despite similar degrees of hydronephrosis. The obstructed kidneys of HO-1−/− mice also had greater macrophage infiltration and renal tubular TGF-β1 expression than wild-type mice. In addition, the degree of EMT was more extensive in obstructed HO-1−/− kidneys, as assessed by α-smooth muscle actin and expression of S100A4 in proximal tubular epithelial cells. In vitro studies using proximal tubular cells isolated from HO-1−/− and wild-type kidneys confirmed these observations. In conclusion, HO-1 deficiency is associated with increased fibrosis, tubular TGF-β1 expression, inflammation, and enhanced EMT in obstructive kidney disease. Modulation of the HO-1 pathway may provide a new therapeutic approach to progressive renal diseases

Transduction of human and mouse pancreatic islet cells using a bicistronic recombinant adeno-associated viral vector

Recent reports indicate successful transduction of pancreatic islets using recombinant adeno-associated viral (rAAV) vectors. This advance offers new possibilities in rendering islets resistant to rejection and recurrence of autoimmune destruction in the setting of islet transplantation as treatment of type 1 diabetes. Most gene delivery approaches using islets have thus far involved transduction with a single gene. However, the concomitant delivery of more than one gene encoding cytoprotective and/or immunoregulatory molecules may offer superior clinical utility. Here, we have generated a bicistronic rAAV (serotype 2) vector incorporating a viral internal ribosome entry site (IRES), derived from polio virus type 1, to allow for translation of two coupled cDNAs from a single mRNA transcript. Our study demonstrates the ability of this vector to produce significant expression of two reporter proteins in human and mouse islets in vitro. This expression did not interfere with beta-cell function. Transduction was maintained in vivo following transplantation of mouse islets. These data are the first report of efficient islet cell transduction with two genes using a single bicistronic rAAV vector and have direct implications for strategies aimed at enhancing islet transplant survival

Suppression by CD4+CD25+ Regulatory T Cells Is Dependent on Expression of Heme Oxygenase-1 in Antigen-Presenting Cells

Heme oxygenase-1 (HO-1) has been viewed as a cytoprotective protein, ameliorating the effects of inflammatory cellular damage, and as beneficial in allograft protection from acute and chronic rejection, suggesting important functions in both innate and adaptive immune responses. Mice deficient in HO-1 exhibit defective immune regulation characterized by a proinflammatory phenotype. We examined if impaired regulatory T cell (Treg) function contributes to the immunoregulatory defects observed in HO-1−/− mice. HO-1−/− mice exhibited a significantly higher proportion of Foxp3-expressing cells among total CD4+ and CD4+CD25+ cells in comparison to HO-1+/+ mice, and HO-1−/− Treg cells were at least as effective as HO-1+/+ Treg cells in suppressing proliferation of effector T cells in vitro from either HO-1+/+ or HO-1−/− mice. However, the absence of HO-1 in antigen-presenting cells abolished the suppressive activity of Treg cells on effector T cells. These findings demonstrate that HO-1 activity in antigen-presenting cells is important for Treg-mediated suppression, providing an explanation for the apparent defect in immune regulation in HO-1−/− mice