Technology

"For decades, RNA molecules were dismissed as little more than drones, taking orders from DNA and converting genetic information into proteins. But a string of recent discoveries indicates that a class of RNA molecules called small RNAs operates many of the cell's controls. They can turn the tables on DNA, shutting down genes or altering their levels of expression. Science hails these electrifying discoveries, which are prompting biologists to overhaul their vision of the cell and its evolution, as 2002's Breakthrough of the Year."

J. Couzin, "Small RNAs Make Big Splash", Science 298:2296-7, 20 December2002.

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Technology overview

RNA Interference

Hailed the 'Breakthrough of the Year" in 2002 by Science magazine and representing a staple research tool in industry and academia, RNAi technology has gone from proof of principle in animal models to human clinical trials in less than 3 years. As an acknowledgement of the importance of this breakthrough the Nobel Prize was awarded to Fire and Mello in 2006, a mere 8 years after the publication of their seminal paper.

As a technology, several key events shaped the field of RNA interference as we know it today. The phenomenon of RNA interference was first reported in plants1 by Richard Jorgensen in 1990, although the exact mechanism was not understood until a few years later. Jorgensen's group reported the curious phenomenon that petunia pigment genes were shut down when they inserted extra copies of the genes in an attempt to deepen the purple color. In 1998, Andrew Fire and Craig Mello2 reported that dsRNAs (double stranded RNA molecules) injected into the nematode Caenorhabditis elegans silenced the corresponding genes containing complementary sequences. Benitec's Graham et al. demonstrated the universality of RNAi and invented DNA constructs to trigger the RNAi process in human and mammalian cells3 . Tuschl and colleagues4 provided a biochemical understanding of the RNAi pathway and showed that the functional units of RNAi are likely represented by dsRNAs shorter than 30 base pairs. McCaffrey et al. published the first in vivo evidence of RNA interference in adult mice5 , verifying the work of Graham et al., allowing therapeutic RNAi companies to capitalize on insights derived from the past decade of gene therapy research.

The RNAi pathway is present in every cell of virtually every multi-cellular organism and possibly evolved as an innate mechanism for cellular defense against double-stranded RNAviruses, in addition to interferon-regulated antiviral pathways. The dsRNA molecules are processed into small interfering RNA (siRNA) by a cytoplasmic enzyme called Dicer, which helps guide the siRNA to the RNA-induced silencing complex (RISC). RISC mediates sequence-specific binding to its corresponding messenger RNA (mRNA) and catalyzes the cleavage and destruction of the mRNA, leading to gene silencing. RNAi drugs are designed to exploit this natural mechanism and destroy rogue genes or viral genomes.

Benitec's ddRNAi Technology

RNAi can be introduced into cells by either the "expressed" or the "delivered" route, depending on whether the RNAi molecules are expressed in cells or whether they are delivered exogenously. Benitec's patented DNA-directed RNA interference (ddRNAi) technology uses the "expressed" pathway. It involves inserting a DNA construct into a cell, triggering the production of double stranded RNA that is immediately cleaved into small interfering RNA (siRNA), which then enters the cellular RNAi pathway resulting in the destruction of the rogue gene or viral genomes. Using this approach Benitec researchers were the first to demonstrate RNAi in mammals.

Benitec's siRNA Technology

Benitec has a cross-licensing agreement with Alnylam Pharmaceuticals Inc. which gives it rights to five siRNA targets.

Separate therapeutic applications for ddRNAi and siRNA

Technically the RNAi field has two major hurdles: i) validation of RNAi as a therapeutic, suitable for human use; ii) delivery of the RNAi-based therapeutic effectively to a target organ. To date, no company has successfully brought an RNAi-based technology through the clinical trial process although Sirna (Merck) has a therapeutic molecule for age-related macular degeneration about to enter phase II trials. The commercial decisions must ultimately reflect a balance of opportunity and risk which this document will attempt to define as well as recommending a way forward.

DNA-directed RNAi or ddRNAi is used to produce a dsRNA inside the cell. By introducing a DNA construct into a cell, Benitec's ddRNAi technology triggers the production of double stranded RNA (dsRNA), which is then cleaved into small interfering RNA (siRNA) by Dicer, a specific type of RNAse III, as part of the RNAi process. Consequently both strategies operate using a similar mechanism however both applications have strengths and weaknesses and consequently different therapeutic applications.

ddRNAi

ddRNAi produces effective gene silencing with a lower dose than siRNA because dsRNA production is effectively catalytic. This attribute may prove critical when targeting infectious agents such as viruses because the virus could potentially replicate so quickly that concentration of a siRNA molecule could become rate-limiting.
ddRNAi has the ability to control the degree of silencing within a cell; it can also be potentially regulated for inducible (transient or cyclical) applications although these applications should be seen as 2nd and 3rd generation products.
ddRNAi has the capability for simultaneous delivery of multiple therapeutic agents into target cells, an essential requirement in treating highly-mutable disease statescaused by viruses and cancers.
ddRNAi can deliver the construct as i) a plasmid in liposome, ii) using viral vectors, iii) using stem cells. However the size of ddRNAi vectors will result in less efficient delivery by liposomes when compared with siRNA.
As a therapeutic model, ddRNAi is analogous to gene therapy with many of the same delivery and regulatory issues.

siRNA

siRNA-based therapies are expected to result in a higher cost of goods because of modifications required to produce more stable RNA, while minimizing undesirable events. Further new modifications have recently been defined that helps to minimize off-target events - this is further expected to increase the cost of the siRNA molecules (as well impacting negatively on the royalty stack).
Liposome delivery is more efficient with siRNA - purely due to mass-action effects - however it is unlikely, in the short-term, that the delivery of siRNA will approach the efficiency of small membrane permeable molecules (drugs). Some compartments within the body, such as the brain, are likely to remain difficult to effectively deliver siRNA-based therapeutics.
As a therapeutic model, siRNA is very similar to biologics - with a relatively long half-life (when compared with traditional drugs) which must be delivered either systemically or topically.

References:

Hence while siRNA and ddRNAi have similar mechanisms of action, the application of these therapies are quite distinct and should be treated a two different therapeutic applications (with some areas of overlap). Benitec's ddRNAi technology has considerable advantages in for a number of key therapeutic applications: infectious disease, neurological disease, some cancers etc.

1 Napoli C, Lemiex C and Jorgenson RA. (1990). Introduction of a chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279-289.
2 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811.
3 Graham MW. Genetic constructs for delaying or repressing the expression of a target gene, US patent 6,573,099.
4 Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498.
5 McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ and MA Kay (2002). RNA interference in adult mice. Nature 418:38-39.