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

`

`new drugs to the market. According to Luc Van
`Parlis, an immunologist at MIT, the cost of pro-
`ducing RNAi—based therapeutics is high. side
`effects are potentially severe, and physical deliv-
`ery of the drugs to diseased cells is extremely
`challenging. Nevertheless, RNAi protagonists are
`bullish about the technology's future. "The more
`we learn about RNAi, the more useful it be-
`comes," says Alnyiam co-founder Phillip Zamore,
`who is also an associate professor at the
`University of Massachusetts Medical School
`(WWW-amassmed.edu). “We‘ve only scratched
`the surface of its potential.”
`
`A new TRICK
`FOR AN OLD IDEA
`
`
`
`"RNAI will be used to
`
`dissect signaling
`
`pathway-5. to discover
`
`genes Important to
`
`embryonic
`
`development. and to
`
`olucldate the function
`
`of novel genes in
`
`other fundamental
`
`biological processes,"
`
`Ruprecht sags.
`
`ment," explains Christian Petersen, a researcher
`in Sharp‘s laboratory. “It’s a natural way to
`silence genes."
`In RNAi experiments. specific siRNAs are
`designed to bind with their mRNA counterpart.
`Then, once introduced to cells, the synthetic
`siRNAs are recognized by the intracellular
`machinery and guided toward specific proteins
`(Figure 1). The siRNAs bind with these proteins,
`and the resultant complexes migrate through-
`out the cell, disabling one mRNA after another
`in ways that are poorly understood. Exactly
`how many mRNAs a single siRNA—protein com-
`plex can disable remains unclear. Extrapolating
`from in vitro experiments. Zamore suggests
`that thousands might be disabled. Clearly, there‘
`fore. RNAi may have a major advantage over and
`sense in terms of drug potency.
`Apart
`from drug development, Ruth
`Ruprecht. an immunologist at the Dana Farber
`Cancer Institutes and professor at the Harvard
`Medical School (wwwmedfiaruordedu), sug-
`gests that RNAi will also have an immediate impact on functional
`genomics. Silencing genes quickly and easily could give scien-
`tists new windows into how genes participate in cellular processes.
`Scientists could simply turn the genes off and then see what baa
`pens. This is an important advance for biology because even
`
`RNAi’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_£s£p.com) to treat eye infections in AIDS
`patients—has won US. 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
`works. Antisense has perpet-
`ually frustrated researchers:
`The efficacy of new com-
`pounds can seem arbitrary.
`and gene-silencing effects are
`typically obtained only with
`considerable trial and error.
`
`
`
`Figure ‘l. Silence and antisonss. Gene expression can be silenced in many ways. In antisensa technology Hall]. on
`oligoribonucleotide binds with the mRNA molecule to prevent protein translation. With RNAi technology. however. the
`Dicar enzyme cleaves double—stranded ids] RNA molecules into short interference tsi) RNA molecules, which are shut-
`tled by the RISC protein complex to the mRNA molecule. Once bound, the complex digests the mHNA molecule, pre-
`
`III}-
`
`51:12am: “RNA
`
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`
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`enzyme
`
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`
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`
`RISC
`complex
`
`’lp Nucleus
`
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`mRNA
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`No translation
`
`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-
`
`

`

`though most of the human genome has been decoded, little is
`known about what vast regions of the genome actually 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 is criti
`cal to programmed cell death. The researchers then inhibited the
`functions of human versions of these genes in tumor cells and
`found that they could disrupt cell proliferation in at
`least two cell lines.
`
`
`
`
`
`THE soon To RNAI otscovenv
`The term RNAi was coined in 1998 by researchers
`Andrew Fire, then at the Carnegie Institution of
`Washington (www.cz‘w. edit), and Craig Mello from the
`University of Massachusetts Medical School upon their
`discovery that double-stranded RNA molecuies were
`remarkably potent inhibitors of a targeted gene in C. etc-
`guns (2). The technique was later shown to work in flies
`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.mpibpcgwdgde) and
`now at Rockefeller University (www.mckefellencom), overcame
`this problem by trimming the
`RNA molecules to 21—23
`
`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", Modem Drug Discovery, June 2003,
`p 26). It. for example. silencing a particular gene can
`cure a diseased animal, then the protein encoded by that
`gene is an attractive target for drug development. The
`standard technique in these experiments involves breed-
`ing transgenic animal species such as the p53 knockout mouse,
`a mutated strain 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 academic laboratory could
`knock those 100 kinases down
`in a month. That’s a whole lot
`faster than breeding 100 knockv
`outs." RNAi might also allow
`researchers to “interrogate"
`several genes simultaneously,
`Zamore adds. This is crucial
`
`Exploiting HNAi
`
`Therapeutics
`
`Company
`
`Website
`
`AGY Therapeutics,|nc. www.agyinc.com
`
`Alnylam
`Pharmaceuticals
`
`Cenix BioScience
`
`Devgen
`DSI Pharmaceuticals
`
`Nucleonics Inc.
`
`WWW.aInylam.com
`
`www.cenixv
`biosciencecom
`
`www.devgen.com
`
`www.osip.com
`wwwnucloonics
`inccom
`
`Target
`CNS disorders
`
`Diabetes,
`cancer, arthritis
`
`Cancer
`
`CNS disorders
`
`Cancer
`
`HIV. hepatitis
`
`typically
`because diseaSe
`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
`Exeiixis, Inc, (wwuiexelu'ismm)
`(1). Using RNAi to knock down
`expression of genes
`throughout the genomes of
`Drosophilo, Coenorhobdt'tis cle-
`goas, and zebralish—standard
`model organisms—an Exelixis
`team disrupted the function of
`more than 2700 druggabic gene
`targets in just 16 months.
`
`www.regsnaron.com
`
`Flibopharma AG
`
`wwwribopharmade Cancer
`
`Flibozvma
`Pharmaceuticals
`
`www.micom
`
`Hepanfis
`
`
`
`Screening
`
`Company
`
`Amgen Inc.
`Benitec
`
`Daltagen
`
`Eos Biotechnology
`Exelixis Pharmaceuticals
`
`lmmusol Inc.
`
`Website
`
`WWWBITIQBTHIDITI
`
`wwwbaniteccomau
`
`www.deltagen_com
`www.cosbiotechcom
`
`www.cxelixis.com
`
`wwwimmusolcom
`
`Millennium Pharmaceuticals
`
`www.mlnm.com
`
`Novartis Research Foundation
`
`http:/lwebgnfcrg
`
`Regeneron Pharmaceuticals
`
`tiny
`nucleotides. Tuschl’s
`siRNAs evaded the immune
`
`system to disable mRNAs in
`mammalian cells. Tuschl's dis-
`
`covery, announced at the RNA
`Society's annual meeting in
`Banif. 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 Shard-tar
`
`oi 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 siRNA injected
`directly into the bloodstreams
`of mice protected liver cells
`from the effects of chemically
`induced fulrninating hepatitis
`(6'). In this case, the siRNAs tar-
`
`

`

`the apoptosis of liver cells when over-
`expressed. Human FM is also involved
`in liver diseases.
`
`Recently. Francis Chisari and col-
`leagues at The Scripps Research
`Institute (wwwscrippsedn) tested the
`capability of RNAi to inhibit hepatitis
`C Virus (HCV) RNA replication (7).
`The researchers developed a series of
`siRNAs against various host and viral
`genes and transfected the RNA mole-
`cules into cultured HEY—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 nat4
`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 HCV infection."
`
`THe soap AHEAD
`Although promising, these findings also
`illustrate how far RNAi has to go before
`it yields human drugs. The injections in
`this case, given in three bolus doses,
`were equivalent to nearly half the ani-
`mal‘s total blood volume. When asked
`
`Table 2
`
`Some suppliers 0! HNAi tools and reagents
`Website
`
`Company
`
`Amaxa Biosystams
`Ambion
`
`Compugen
`Dharmacon
`
`Eurogantec
`
`Galapagos
`Genomics
`
`Gene Therapy
`Systems
`
`GanScript Corp.
`
`lmgenex Corp.
`
`lntradigm Corp.
`lnvivoGen
`
`www.an1axa.com
`
`www.cmbioncom
`
`www.cgoncom
`
`www.dharrnaoon.com
`
`marwsurogsnteccom
`
`www.galapagos
`genomicscom
`
`www.genetherapy
`svstemscom
`
`WWW.QBI'ISCI‘ipt.COfT‘l
`
`wwwimgenexcom
`
`www.intradigm.corn
`
`wmvinvivogoncom
`
`Invitrogan Corp.
`
`wwwjnvitrogencom
`
`(liegen
`
`wvmqiagencomlsiFlNA
`
`Mirus Corp.
`Molecula Research Labs
`
`httpjlganetransfenoom
`wwmolaculacom
`
`MWG Biotech Inc.
`
`New England
`Biolabs. inc.
`
`Novagen
`
`Uligosngina
`
`Proligo LLC
`
`Promage Corp.
`
`Sequitur, Inc.
`
`
`
`thesizing the required RNA molecules.
`It costs thousands of dollars to produce
`enough RNA for experiments in tissue
`cultures, he says. “In humans, every
`injection might run you SlthO.” He
`adds that the costs could conceivably be
`reduced by economies of scale: Should
`the technology prove therapeutically
`successful, pharmaceutical plants could
`make RNA in sufficient volume to bring
`down Costs.
`As its protagonists point out (in
`what seems to be a growing turf battle
`between the two approaches), antisense
`is farther along in terms of drug devel-
`opment than is RNAi. Frank Bennett,
`vice president for antisense research at
`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 approach is 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 other anti-
`sense mechanisms. Our conclusion
`
`from investigating these mechanisms is
`that there is no single mechanism that
`is vastly superior to the others. They all
`have potential utility."
`On this point, however, Alnylam's
`Maraganore begs to disagree. "RNAi
`is a catalytic process that uses the 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 we think
`RNAi therapeutics are going to be
`developed and made successful."
`
`www.mwghiotech.corn
`www.nab.com
`
`wuwnovagencom
`
`wwwoligoanginecom
`
`www.proligo.com
`
`www.cromepacom
`
`umwsequiturincnom
`
`wwwspringbiocom
`
`wunntstratagenecom
`
`what major challenges lie ahead, Zamora
`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—
`
`Spring Bioscience
`
`Stratagene
`
`ods are fine for cultured cells, says Zamore, buthe adds. "1 wou 1d-
`n't want anyone injecting 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-knockout species 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."
`
`Resonances
`(D Winkler, H.; Powell, 3. Targets 2003, 2, 42-44.
`(2) Fire, A: et al. Nature 1998, 391. 806-810.
`(3) Gerber, K. Technology Rec, December 2002, pp 42-48.
`(4) Novina, C. ll; et at. Not, Med. 2002. 8. 681-686.
`(5) jacque,l.-M.: et al. Nature 2002, #13. 435-438.
`(5} Fong, E.; et al. Nat. Med. 2003, 9, 347-351.
`(7) Kapadia. S. B.: et al. Proc. Natl. Acad. Sci. U.S.A. 2003. .100. 2014—
`EMS.
`
`CharlcsW. Schmidt is a freelance science writer from Portland. ME.
`Send your comments or questions about this article to mcld@acs.org
`or to the Editorial Office address on page 3. l
`
`KEY TERMS: cell biology, clinical, drug delivery, ganomics.
`
`high throughput. screening
`
`