ddRNAi: expressed RNA interference technology

Benitec Biopharma Ltd, 1-15 Barr St Balmain (Sydney) NSW Australia             P.+ 61 (0) 2 9555 6986           E. info@benitec.com

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The dream of gene regulation to control or cure disease was given a boost in 1998, with the discovery of RNA interference (RNAi ) as a way to turn off or silence ‘unwanted’ genes. As a result, RNAi soon became a major focus for medical researchers worldwide.

Gene silencing by RNAi
The process of RNAi occurs naturally in cells and can be triggered either by synthetic small interfering RNA (siRNA) or DNA-directed RNAi, most commonly in the form of shRNA.  The siRNA approach has a number of disadvantages for treatment of chronic disease when compared to ddRNAi.                                                                               
The gene silencing process using RNAi is this:

1. Identify the target disease or condition
2. Identify the target protein that is strongly associated with the condition
3. Identify the gene sequence that codes for that protein
4. Make a dsRNA molecule to target a small section of the gene sequence
5. Deliver the dsRNA molecule to the target cell in the target organ.

If the level of mRNA and the protein both decrease by at least 70%, it is likely that a successful gene-silencing molecule has been developed. Then the challenge is to deliver it to the target organ, where the disease or condition is focused and the target gene expressed.

Gene silencing using siRNA
siRNA utilises synthetic dsRNA molecules, the sequence of which are designed to specifically cleave the target gene mRNA and so prevent production of the specific target protein. Delivering the siRNA molecule to the target organ has proven to the most difficult aspect of siRNA-based therapeutics.
According to a review of drug delivery by Rao et al (Advanced Drug Delivery Reviews 2009), siRNA has a number of limitations:

1. it is rapidly degraded in body fluids, so keeping it stable during in vivo delivery is problematic without modification of its structure;   
2. modifications of siRNA can alter its key performance characteristics;
3. siRNA needs to be delivered to specific disease-involved cells or tissues or its effective concentration will be significantly diluted;
4. its loading efficiency into cells is generally low, so higher doses of the siRNA molecule are needed;
5. the need for high doses can lead to potentially higher toxicity and ‘off target effects’. 

Gene silencing by ddRNAi
Expressed RNAi overcomes most of the challenges of siRNA-based methods because it is DNA-directed RNAi (ddRNAi).  Benitec Biopharma holds a dominant patent position globally for expressed RNAi approaches, including shRNA. The difference lies in the process by which the double stranded silencing RNA molecules are produced. The ddRNAi approach results in the cell producing dsRNA itself, rather than introducing synthetic RNA. The specific dsRNA (usually in the form of shRNA) is coded for in the nucleus as a result of transfecting the cell with a DNA-based construct. Thus in ddRNAi the silencing is literally directed by DNA.

ddRNAi  techniques rely on introduction of a DNA sequence directly into the cell’s nucleus via well-characterized gene therapy vectors, including viral and non-viral vectors such as lentivirus, adenovirus, AAV and modified polyethylenimine. These proven delivery mechanisms for DNA overcome many of the difficulties faced by siRNA.  The DNA sequence codes for specific shRNAs which are processed to siRNAs and complete the usual RNAi cycle in the cytoplasm. This highlights another major difference:  when the DNA constructs enter cells they can persist either as integrated DNA or extrachromosomal episomes. The dose of DNA required is therefore small and possibly a single molecule is sufficient to silence a gene,, yet the genetic change is long-lasting because the shRNA continues to be expressed for long periods, up to years (Sci Trans Med 2010).

In summary, Benitec Biopharma’s approach to gene silencing is DNA-directed (ddRNAi), which is significantly longer-lasting, requires significantly ower doses and has low toxicity compared to synthetic siRNA approaches. (See siRNA vs shRNA)

Importantly, as it is a proprietary technology platform rather than a single therapeutic molecule, Benitec Biopharma’s ddRNAi can be licensed broadly worldwide to groups or organisations involved in developing shRNA-based therapeutics for a multitude of human diseases.