Benitec develops new knockout method for animal genes

By Melissa Trudinger
Monday, 16 September, 2002

Queensland company Benitec has developed a new method of knocking out genes in animals, combining its proprietary RNA interference (RNAi) technology with standard transgenic techniques.

The company has used the technique to shut down two endogenous genes in mice, demonstrating that RNA interference techniques can be used to permanently silence normal genes in live animals.

Benitec managing director James Anderson said that until now, nearly all gene silencing had been demonstrated in cell culture.

"This is a pretty big step given where the technology is going to lead," he said. "Benitec was the first to show that RNA interference worked in mammalian cells, and now it is the first to show it silences endogenous genes of transgenic animals."

Anderson explained that the silencing was accomplished by injecting DNA encoding the RNAi construct into the nucleus of a fertilised mouse egg, essentially the same method used to make a transgenic mouse.

The RNAi construct consists of a DNA sequence followed by its antisense sequence as an inverted repeat, with the appropriate flanking control sequences. When the cell transcribes the RNAi construct, the resulting RNA molecule folds back on itself to form a double-stranded RNA that interferes with the expression of the target gene.

Benitec used the method to knock down expression of two genes in mice, the galT (alpha-1,3-galactosyltransferase) gene, which is a gene of interest in xenotransplantation studies, and the tyrosinase gene, which is associated with black coat colour.

"It's a very clean and efficient way of knocking out genes," said Anderson.

Benitec has filed patent applications for the new method and sees significant commercial potential in the technology.

"This result reinforces Benitec's position as the world leader in gene silencing and RNA interference as it applies to humans and animals and also reinforces our dominant intellectual property position in gene silencing using DNA technology," said CEO John McKinley.

Anderson said the immediate application of the new method would be to efficiently create animal models of disease by knocking out relevant genes, in a way that was less laborious than the current methods used.

He noted that a number of companies in Australia and overseas were using animal models to identify gene function, and said that this would provide them with a very powerful tool to use to identify causative genes and new drug targets.

In addition, Anderson said, the technique could lead to new therapeutic approaches being developed for the treatment of human diseases, including cancers, autoimmune diseases and viral diseases. RNAi has already been demonstrated to inhibit viral infections in cultured cells.

A recent result from a research team led by Professor Jo Milner and Dr Ming Jiang at the University of York in the UK has shown that RNAi can be used to restore p53 function in human papillomavirus (HPV)-infected cervical cancer cells by silencing the HPV E7 gene. The restored gene then triggers apoptosis in the cancer cells.

In another study by Mark Kay at Stanford University, RNAi was used to interfere with the expression of human hepatitis C virus in mice transgenic for the virus.

Among non-human applications, Benitec believes that the technology may be useful for developing disease resistant livestock, by knocking down the expression of certain genes.

According to Anderson, the company is now focusing on developing methods to control expression of the RNAi construct, to enable conditional silencing. Among other uses, this would allow researchers to identify genes involved in development which are lethal when knocked out.

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