Efficient Inhibition of Hepatitis B Virus Replication in Vivo Using Peg-Modified Adenovirus Vectors

Abstract

Achieving safe delivery of anti-hepatitis B virus (HBV) RNA interference (RNAi) effectors is an important objective of this gene-silencing technology. Adenoviruses (Ads) have a natural tropism for the liver after systemic administration, and are useful for delivery of expressed anti-HBV RNAi sequences. However, a drawback of Ad vectors is diminished efficacy and toxicity that results from stimulation of innate and adaptive immunity. To attenuate these effects we used monomethoxy polyethylene glycol-succinimidyl propionate (mPEG-SPA) to modify first-generation vectors that express an anti-HBV RNAi effector. Efficient hepatocyte transduction and knockdown of HBV replication were achieved after intravenous administration of 5 x 10(9) PEGylated or native recombinant Ads to HBV transgenic mice. After the first injection, circulating HBV viral particle equivalents (VPEs) remained low for 3 weeks and began to increase after 5 weeks. A second dose of PEGylated anti-HBV Ad caused a less sustained decrease in circulating VPEs, but no silencing after a second dose was observed in animals treated with unmodified vector. Release of inflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), interferon-gamma, interleukin-6, and tumor necrosis factor-alpha, was elevated in animals receiving unmodified vectors. However, only a modest increase in MCP-1 was observed in mice that received a second dose of PEG Ads. Also, polymer-conjugated vectors induced a weaker adaptive immune response and were less hepatotoxic than their unmodified counterparts. Collectively, these observations show that PEG modification of Ads expressing RNAi effectors improves their potential for therapeutic application against HBV infection.

Inhibition of Hepatitis B Virus Replication by Helper Dependent Adenoviral Vectors Expressing Artificial Anti-HBV Pri-miRs from a Liver-Specific Promoter

“…Pol III promoters have been commonly used to express anti-HBV RNAi activator sequences [30, 37]. However, powerful constitutive activity of the U6 Pol III promoter may result in saturation of the RNAi pathway with resultant hepatotoxicity [9].…”

Efficient silencing of gene expression with modular trimeric Pol II expression cassettes comprising microRNA shuttles

“…Propagation of pTZ pri-miR-122/5 has been described previously (3). To produce U6-driven pri-miR expression plasmids (pTZ U6-pri-miR-31/5, pTZ U6-pri-miR-31/8 and pTZ U6-pri-miR-31/9), the pri-miR-31 shuttle sequences were excised with NheI and ScaI then inserted into equivalent sites downstream of the U6 promoter in the pTZ-U6 vector (12). Pol II-driven miR expression plasmids (pCI-pri-miR-31/5, 31/8 and 31/9) were constructed by excising the pri-miR-31 shuttle sequences with SalI and XbaI and ligating these fragments to XhoI and XbaI sites of pCI-neo (Promega, WI, USA).…”

“…Mice were injected using the hydrodynamic injection procedure with a combination of 5 µg pCH-9-3091 (14), 5 µg of RNAi expression vector, 5 μg of control U6 (pTZ-U6 vector (12)) or CMV (pCI-neo, Promega, WI, USA) promoter-containing backbone plasmid and 5 µg of psiCHECK2.2. Blood was collected 3 and 5 days post-injection.…”

Gene and cell therapy in South Africa: Current status and future prospects

“…While this approach does not achieve a sterilising cure, it is able to produce a functional cure for hepatitis B, a previously undreamt of goal in anti-HBV therapeutics. The unit has also explored the use of recombinant adenoviruses, [24] lentiviruses [25] and AAVs, [9,10] given its core competency in viral vectorology. Considerable progress has been made and a number of candidates have been identified for further development.…”