ARBUTUS - EXHIBIT 2016
`Moderna Therapeutics, Inc. v. Arbutus Biopharma Corporation
`IPR2019-00554
`
`

`

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`
`“RNAI will be used to
`
`
`
`Woonmen
`Z
`\
`
`ment,” explains Christian Petersen, a researcher
`new drugsto the market. According to Luc Van
`Parijs, an immunologist at MIT, the cost of pro-
`in Sharp's laboratory. “It’s a natural way to
`ducing RNAi-based therapeutics is high, side
`silence genes.”
`effects are potentially severe, and physicaldeliv-
`In RNAi experiments, specific siRNAs are
`designed to bind with their mRNA counterpart.
`ery of the drugs to diseased cells is extremely
`challenging. Nevertheless, RNAi protagonists are
`Then, once introduced to cells, the synthetic
`bullish about the technology’s future. “The more
`siRNAs are recognized by the intracellular
`machinery and guided toward specific proteins
`we learn about RNAi, the more useful it be-
`comes,” says Alnylam co-founder Phillip Zamore,
`(Figure 1). The siRNAs bind with these proteins,
`dissect signaling
`who is also an associate professor at the
`and the resultant complexes migrate through-
`pathways, fo discover
`out the cell, disabling one mRNAafter another
`University of Massachusetts Medical School
`(www.umassmed.edu). “We've only scratched
`genes important to
`in ways that are poorly understood. Exactly
`the surface of its potential.”
`how many mRNAsasingle siRNA-protein com-
`embryonic
`plex can disable remains unclear. Extrapolating
`development, and to
`from in vitro experiments, Zamore suggests
`elucidate the function
`that thousands might be disabled. Clearly, there-
`fore, RNAi may have a major advantage overanti-
`of novel genesin
`sense in terms of drug potency.
`other fundamental
`Apart
`from drug development, Ruth
`biological processes,”
`Ruprecht, an immunologist at the Dana Farber
`CancerInstitutes and professor at the Harvard
`Ruprecht says.
`Medical School (www.med.harvard.edu), sug-
`gests that RNAi will also have an immediate impact on functional
`genomics. Silencing genes quickly and easily could give scien-
`tists new windowsinto how genesparticipate in cellular processes.
`Scientists could simply turn the genes off and then see what hap-
`pens. This is an important advance for biology because even
`
`A NEW TRICK
`FOR AN OLD IDEA
`RNAj’s therapeutic premise—that RNAs involved
`in disease can be selectively blocked with
`drugs—isn’t new. For more than 10 years, sci-
`entists have tried to use “antisense” techniques
`to silence genes that contribute to disease. Anti-
`sense drugs are single-stranded chemically mod-
`ified DNA molecules that are designed to bind directly to
`disease-related mRNA molecules and disable them (Figure 1).
`However, antisense has long been a problematic technology, and
`only one antisense drug—Vitravene, produced by Isis
`Pharmaceuticals (www.isip.com) to treat eye infections in AIDS
`patients—has won U.S. FDA
`approval. In most cases, anti-
`sense compounds degrade
`rapidly, so their effects are
`transitory and their potency is
`low. Furthermore, no one
`really knows how the process
`sora dshNA
`works. Antisense has perpet-
`teeteretrerept
`Dicer
`ually frustrated researchers:
`Antisense RNA
`enzyme
`The efficacy of new com-
`eT
`pounds can seem arbitrary,
`and gene-silencing effects are
`typically obtained only with
`considerabletrial and error.
`
`Ter Tor Te Tr siRNAs
`
`RISC
`complex
`
`ly >
`
`sotatr
`
`|
`
`Like antisense, RNAi
`works by interfering with
`mRNA,but there are impor-
`tant differences. To begin
`with, whereas antisense com-
`pounds are wholly synthetic,
`relies on double-
`stranded short interference
`RNA molecules (siRNAs) that
`exist naturally in the cells of
`many species. “Therefore,
`their functioning is somehow
`connected to normal endoge-
`
`No translation
`
`mRNA
`digestion
`
`ee
`
`Meet Ope
`
`wher
`
`
`Figure 1. Silence and antisense. Gene expression can be silenced in many ways. In antisanse technology(left), an
`oligoribonucleotide binds with the mRNA molecule to preventprotein translation. With RNAi technology, however, the
`Dicer enzyme cleaves double-stranded (ds) RNA molecules into short interference (si) RNA molecules, which are shut-
`tled by the RISC protein complex to the mRNA molecule. Once bound, the complex digests the mRNA molecule, pre-
`
`

`

`though most of the human genome has been decoded,little is
`known about whatvast regions of the genomeactually do. “RNAi
`will be used to dissect signaling pathways,to dis-
`cover genes important to embryonic development,
`and to elucidate the function of novel genes in
`other fundamental biological processes,” Ruprecht
`
`several genes involved in the ceramide pathway, which iscriti-
`cal to programmedcell death. The researchers then inhibited the
`functions of human versions of these genes in tumorcells and
`found that they could disruptcell proliferationin at
`least two cell lines.
`
`
`
`
`
`THE ROAD To RNAi DISCOVERY
`The term RNAI was coined in 1998 by researchers
`Andrew Fire, then at the Carnegie Institution of
`Washington (www.ciw.ed«), and Craig Mello from the
`University of Massachusetts Medical School upontheir
`discovery that double-stranded RNA molecules were
`remarkably potent inhibitors of a targeted gene in C,ele-
`gans (2). The technique was later shownto workinflies
`and plants but not in mammalian cells. This is mainly because
`the RNAs were destroyed by mammalian immune systems.
`Later, Thomas Tuschl, a biochemist then at the Max Planck
`Institute for Biophysical Chemistry (www.mpibpe.gwdg.de) and
`now at Rockefeller University (www.rockefeller. com), overcame
`this problem by trimming the
`RNA molecules to 21-23
`
`Table 1
`
`Exploiting RNAi
`
`Therapeutics
`
`Company
`
`Website
`
`AGYTherapeutics, Inc.
`
`www.agyinc.com
`
`Alnylam
`Pharmaceuticals
`
`Cenix BioScience
`
`Devgen
`OS! Pharmaceuticals
`
`Nucleonics Inc.
`
`www.alnylam.com
`
`Www.cenix-
`bioscience.com
`
`www.devgen.com
`
`www.osip.com
`www.nucleonics
`inc.com
`
`Ribopharma AG
`
`www.ribopharma.de
`
`Ribozyme
`Pharmaceuticals
`
`www.rpi.com
`
`Target
`CNS disorders
`
`Diabetes,
`cancer, arthritis
`
`Cancer
`
`CNS disorders
`
`Cancer
`
`HIV, hepatitis
`
`Cancer
`
`Hepatitis
`
`nucleotides. Tuschl's tiny
`siRNAs evaded the immune
`
`system to disable mRNAsin
`mammalian cells. Tuschl’s dis-
`
`covery, announced at the RNA
`Society’s annual meeting in
`Banff, Alberta, Canada, in May
`2001, stunned researchers, who
`had essentially given up on RNA
`targets (3). Thus enabled, RNAi
`quickly leapt to the forefront of
`biomedical research.
`The next key event took
`place in Sharp’s laboratory at
`MIT. Collaborating with Judy
`Lieberman and Premlata Shankar
`
`of the Harvard University Center
`for Blood Research, Sharp
`demonstrated that siRNAs could
`
`stop HIV infections in cell cul-
`tures, thereby demonstrating
`the technology’s clinical poten-
`tial (4). Independently, Mario
`Stevenson at the University of
`Massachusetts Medical School
`
`confirmed these findings (5).
`Lieberman then showed that
`
`massive doses of siRNAinjected
`directly into the bloodstreams
`of mice protected liver cells
`from the effects of chemically
`induced fulminating hepatitis
`(6). In this case, the siRNAs tar:
`
`RNAi techniques in these more functional stud-
`ies are analogous to the gene knockout methods
`used to identify and validate drug targets (see “A
`knockouts tale”, Modern Drug Discovery, June 2003,
`p 26). If, for example, silencing a particular gene can
`cure a diseased animal, then the protein encoded by that
`geneis an attractive target for drug development. The
`standard technique in these experimentsinvolves breed-
`ing transgenic animal species suchas the p53 knockout mouse,
`a mutatedstrain in which this tumor-suppressor gene has been
`inactivated. But the process of breeding transgenics is expensive
`and time-consuming. RNAi could dramatically accelerate this
`process, researchers say.
`“Say you have 100 kinases
`and you want to know which
`one in that pathway could be a
`druggable molecule,” Zamore
`explains. “Using RNAi, a mid-
`size academiclaboratory could
`knock those 100 kinases down
`in a month, That's a whole lot
`faster than breeding 100 knock-
`outs.” RNAi might also allow
`researchers to “interrogate”
`several genes simultaneously,
`Zamore adds. This is crucial
`because disease typically
`involves complex networks of
`genes working in tandem
`through a range of intercon-
`nected pathways.
`At the recent Applications of
`RNA Interference conference
`held in San Diego, researcher
`Thomas Kidd described some
`of the work using RNAi tech-
`nology that was being done at
`Exelixis, Inc. (www. exelixis.com)
`(J). Using RNAi to knock down
`expression of genes
`throughout the genomes of
`Drosophila, Caenorhabditis ele-
`gans, and zebrafish—standard
`model organisms—an Exelixis
`team disrupted the function of
`more than 2700 druggable gene
`targets in just 16 months,
`
`Regeneron Pharmaceuticals Website
`
`
`
`Screening
`
`Company
`
`Amgen Inc.
`Benitec
`
`Deltagen
`
`Eos Biotechnology
`Exelixis Pharmaceuticals
`
`Immusol Inc.
`
`Millennium Pharmaceuticals
`
`Novartis Research Foundation
`
`Www.amgen.com
`
`www.benitec.com.au
`
`www.deltagen.com
`www.eosbiotech.com
`
`www.exelixis.com
`
`www.immusol.com
`
`www.minm.com
`
`http://web.gnf.org
`www.regeneron.com
`
`

`

` between the two approaches), antisense
`
`the apoptosis of liver cells when over-
`expressed. HumanFas is also involved
`in liver diseases.
`
`Recently, Francis Chisari and col-
`leagues at The Scripps Research
`Institute (wwwscripps.edu) tested the
`capability of RNAi to inhibit hepatitis
`C virus (HCV) RNA replication (7).
`The researchers developed a series of
`siRNAsagainst various host and viral
`genes and transfected the RNA mole-
`cules into cultured HCV-infected cells,
`looking for signs of viral RNA repli-
`cation using reverse transcriptase real-
`time PCR and Northern blot analysis.
`The researchers found that several of
`the siRNAs inhibited viral replication
`within two days of transfection, sug-
`gesting that RNAi might provide a
`valuable therapeutic tool. Further-
`more, they speculated that RNAi might
`play a role in viral clearance during nat-
`ural HCV infections and that “thera-
`
`peutic induction of RNAi, either alone
`or in combination with [interferon]
`treatment, might represent an alter-
`native approach for the treatment of
`chronic HCVinfection.”
`
`Table 2
`
`Some suppliers of RNAi tools and reagents
`Website
`
`Company
`
`Amaxa Biosystems
`Ambion
`
`Compugen
`Dharmacon
`
`Eurogentec
`
`Galapagos
`Genomics
`
`Gene Therapy
`Systems
`
`GenScript Corp.
`
`Imgenex Corp.
`
`Intradigm Corp.
`InvivoGen
`
`WWW.amaxa.com
`
`www.ambion.com
`
`www.cgen.com
`
`www.dharmacon.com
`
`WwWw.eurogentec.com
`
`www.galapagos
`genomics.com
`
`www.genetherapy
`systems.com
`
`www.genscript.com
`
`www.imgenex.com
`
`www.intradigm.com
`
`www.invivogen.com
`
`Invitrogen Corp.
`
`www.invitrogen.com
`
`Qiagen
`
`www.giagen.com/siRNA
`
`Mirus Corp.
`Molecula Research Labs
`
`http://genetransfer.com
`www.molecula.com
`
`MWGBiotechInc.
`
`Spring Bioscience
`
`Stratagene
`
`New England
`Biolabs,Inc.
`
`Novagen
`
`Oligoengine
`
`Proliga LLC
`
`Promega Corp.
`
`Sequitur, Inc.
`
`THE ROAD AHEAD
`Although promising,these findings also
`illustrate how far RNAi hasto go before
`it yields human drugs.Theinjectionsin
`this case, given in three bolus doses,
`were equivalent to nearly half the ani-
`mal’s total blood volume. When asked
`what major challenges lie ahead, Zamore
`said, “Delivery, delivery, delivery.”
`Presently, researchers are embed-
`ding siRNAs in cationic lipids that
`pass through cell membranes and
`deliver their products into the interior of the cell. These meth-
`ods are fine for culturedcells, says Zamore, but he adds, “I would-
`n’t want anyoneinjecting cationic lipids into my bloodstream.”
`An alternative method currently being developed by Van Parijs
`involves attenuated lentiviruses that deliver genes encoding for
`siRNAs. Van Parijs says that this approach could be especially
`useful for designing gene-knockoutspecies for functional stud-
`ies or drug target validation. But he’s skeptical that the technique,
`which is analogous to gene therapy, could be applied to making
`RNAi-based drugs. “Whenever you talk about introducing a
`genetic element using a retrovirus, you have to be concerned about
`screwing up the genome and producing cancer,” he explains.
`“That's a valid concern and one that has to be addressed.”
`
`thesizing the required RNA molecules.
`It costs thousandsof dollars to produce
`enough RNAfor experimentsin tissue
`cultures, he says. “In humans, every
`injection might run you $1000.” He
`adds that the costs could conceivably be
`reduced by economiesof scale: Should
`the technology prove therapeutically
`successful, pharmaceutical plants could
`make RNAin sufficient volumeto bring
`down costs.
`As its protagonists point out (in
`what seems to be a growingturfbattle
`
`is farther along in terms of drug devel-
`opment than is RNAi. Frank Bennett,
`vice president for antisense researchat
`Isis, says the company now has a group
`of 12 new antisense drugs in various
`stages of clinical trials. “It’s not relevant
`to say one approachis better than the
`other,” Bennett argues. “The reality is
`that RNAi is a new and exciting area in
`biology, and it has demonstrated value
`in target validation as have otheranti-
`sense mechanisms. Our conclusion
`
`from investigating these mechanismsis
`that there is no single mechanism that
`is vastly superiorto the others. Theyall
`have potential utility.”
`On this point, however, Alnylam’s
`Maraganore begsto disagree. “RNAi
`is acatalytic process that usesthe cell's
`own machinery to disable mRNA,”
`he says. “So the process is very dif-
`ferent, and so is the potency, which is
`up to a thousandfold greater. This is
`an important aspect of how wethink
`RNAi therapeutics are going to be
`developed and made successful.”
`
`www.mwgbiotech.com
`www.neb.com
`
`WWw.novagen.com
`
`www.oligoengine.com
`
`www.proligo.com
`
`WWw.promega.com
`
`www.sequiturinc.com
`
`www.springbio.com
`
`www.stratagene.com
`
`REFERENCES
`(1) Winkler, H.; Powell, 5S. Targets 2003, 2, 42-44.
`(2) Fire, A; et al. Nature 1998, 391, 806-810.
`(3) Garber, K. Technology Rev., December 2002, pp 42=48,
`(4) Novina, C. D.; et al. Nat. Med. 2002, 8, 681-686.
`(5) Jacque, J«M.; et al. Nature 2002, 418, 435-438.
`(6) Fong, E.; et al. Nat. Med. 2003, 9, 347-351.
`(7) Kapadia, 5. B.; et al. Proc. Natl. Acad. Set. U.S.A, 2003, 100, 2014-
`2018.
`
`Charles W. Schmidt is a freelance science writer from Portland, ME.
`Send your comments or questions aboutthis article to mdd@acs.org
`or to the Editorial Office address on page 3. @
`
`high throughput, screening
`
`KEY TERMS:cell biology, clinical, drug delivery, genomics,
`
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