IFN-α gene therapy for woodchuck hepatitis with adeno-associated virus: differences in duration of gene expression and antiviral activity using intraportal or intramuscular routes

Abstract

Gene delivery of IFN-alpha to the liver may represent an interesting strategy to maximize its antiviral efficacy and reduce side effects. We used a recombinant adeno-associated virus (AAV) encoding woodchuck IFN-alpha (AAV-IFN) to treat animals with chronic woodchuck hepatitis virus infection. The vector was given by intraportal or intramuscular route. Long-term transgene expression was detected after intraportal administration of an AAV encoding luciferase. In contrast, in the majority of the animals that received AAV-IFN through the portal vein, the expression of IFN-alpha was transient (30-40 days) and was associated with a significant but transient decrease in viral load. One animal, in which hepatic production of IFN-alpha persisted at high levels, died because of bone marrow toxicity. The disappearance of IFN-alpha expression correlated with the disappearance of AAV genomes from the liver. Intramuscular administration of AAV-IFN resulted in prolonged but fluctuating expression of the cytokine with no significant antiviral effect. In summary, this report shows that long-term expression of IFN-alpha in muscle is feasible but higher interferon levels might be needed to control viral replication. On the other hand, IFN-alpha gene delivery to the liver using an AAV vector induces a significant but transient antiviral effect in the woodchuck model.

Treatment of Chronic Viral Hepatitis in Woodchucks by Prolonged Intrahepatic Expression of Interleukin-12

“…The PBMCs were cultured in a 96-well plate at a density of 2 ϫ 10 5 cells/well, and rWHcAg was added in triplicate at a concentration of 1 g/ml. Forty-eight hours later, the supernatants were harvested and the IL-2 concentration was determined using a CTL-L2 assay (2). SI values of Ն3.1 were considered positive for a specific PBMC response.…”

“…Serum WHV DNA was isolated using a High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Lewes, United Kingdom) and was quantified by real-time quantitative PCR (qPCR). The detection limit of the assay was 10 vg per l of serum (2). t-DNA and the cccDNA-enriched fraction were extracted from the liver as previously described (33).…”

“…The presence of serum WHV surface antigen (WHsAg) levels was determined by enzyme-linked immunosorbent assay (ELISA) (2,19). The WHV e antigen (WHeAg) concentration was determined using a commercial ELISA for HBVe antigen that recognizes woodchuck antigen (DiaSorin S.p.A., Saluggia, Italy).…”

“…The T-cell proliferative response to WHcAg stimulation was analyzed using freshly isolated peripheral blood mononuclear cells (PBMCs) as previously described (2). Briefly, freshly isolated PBMCs were cultured in 96-well U-bottom plates at a density of 2 ϫ 10 5 cells/well in complete RPMI medium.…”

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system

“…The EF1α promoter (EF1Pr) was amplified by PCR using primers 22 and 23 from AAV-Luc, kindly provided by Dr. Gloria Gonzalez-Aseguinolaza [26], obtaining a fragment of 1,182 bp that was subcloned into pBS-pac plasmid [27] with EcoRV and NotI restriction sites. In the newly generated plasmid (pBS-EF1-pac), pac expression is driven from EF1Pr.…”