Benitec using RNAi to focus on hep C

A senior scientist from Brisbane-based biotech company Benitec (ASX:BLT) has told a conference that there is "incredible excitement" in the biomedical research community about the human therapeutic potential of RNA interference (RNAi) gene-silencing technology.

Speaking at CSIRO's Horizons in Livestock Science conference, Dr Mick Graham said that, since acquiring Californian RNAi therapeutics company Avocel last May, Benitec had rapidly transformed itself into a gene-therapy company.

When Benitec acquired Avocel, the US company was already testing multi-targeted RNAi gene constructs as a potential therapy for hepatitis C, and a delivery system based on an adeno-associated virus.

Graham said hepatitis C is one of the world's most serious health problems, with 170 million people currentl infected. Cirrhosis of the liver, resulting from chronic, hepatitis C virus (HCV) infections, are the major cause of liver-transplant operations in the US, and a major risk factor for lethal hepatocelular carcnoma..

Current therapies employing interferon are effective in clearing HCV only 30 to 40 per cent of cases, and have severe side-effects.

Benitec researchers in Australa and the US are testing RNAi constructs that multiple genes in the hepatitis C virus (HCV), and Graham said the constructs were already showing exciting potential to prevent HCV replication in vitro.

"You need to knock out multiple gene products in the pathogen to counter its very high mutation rates, and the high level of natural strain variation in the virus, otherwise it's not going to be an effective therapy," he said.

Graham said there was high hope that RNAi might even clear an HCV infection completely because, although the virus chronically in infects liver tissue, it does not integrate its genome into liver cells like retroviruses.

Graham said Benitec's Australian researchers and their Californian colleagues were experimenting with synthetic genes comprising a single transcriptional unit with up to five hairpin RNAs embedded in it, targeting up to five different HCV genes.

The RNA-induced silencing complex (RISC), the molecular 'machine' that identifies and cleaves messenger RNAs from target genes, does not have an unlimited capacity to process mRNAs, and begins to saturate if more than three genes are targeted simultaneously. Two- or three-hairpin constructs may be optimal.

Graham said Benitec had been experimenting with multi-hairpin constructs targeting transgenes linked togreen fluorescent protein (GFP) or firefly luciferase sequences.

Reduced fluorescence or visible light emissions from cells treated with these hairpin constructs confirms that the target gene(s) have been successfully knocked down -- 100 per cent silencing is rarely achieved with the current state of the technology.

Researchers are also experimenting to determine the minimal length of nucleotide sequence in the constructs that will achieve gene-silencing, without invoking the interferon response, which is keyed to double-stranded RNA molecules above a particular size.

Benitec researchers have been aligning DNA sequences from the virus' multiple serotypes to identify highly conserved regions that could be effectively targeted with a multi-hairpin construct.

Graham said Benitec was experimenting with a "seriously interesting vector" -- adeno-associated virus strain 8 (AAV8) which he said had given gene therapy a new lease on life this year. AAV8 persists episomally, and infects up to 98 per cent of hepatocytes in a mouse model. "We're getting high expression in liver tissue," he said. "The question is whether we can do the same in humans."

He said Benitec was as advanced as any company in the world working on HCV, and was excited by the progress it was making.

But Graham said the enormous variability between strains of the virus remaned a probem: even if hairpin RNAi constructs targeted multiple, conserved sequences shared by multiple strains of HCV, no single construct was likely to be effective against more than about 70 per cent of virus isolates.

It was likely that patients would be treated with RNAi constructs desgined specifically for their particular strain of the virus.