Citation: Molecular Therapy–Nucleic Acids (2012) 2, e8; doi:10.1038/mtna.2011.9
`© 2012 American Society of Gene & Cell Therapy All rights reserved 2158-3188/11
`www.nature.com/mtna
`
`COMMENTARY
`The Business of RNAi Therapeutics in 2012
`
`Dirk Haussecker1
`
`Molecular Therapy–Nucleic Acids (2012) 2, e8; doi:10.1038/mtna.2011.9; published online 7 February 2012
`
`INTRODUCTION
`
`In its decade in existence, commercial RNA interference
`(RNAi) therapeutics development has seen great financial
`volatility. The causes of this volatility are broadly shared with
`what has been observed on other technology frontiers such
`as gene therapy in the case of drug development, with the
`amplitude of the volatility magnified or moderated by macro-
`economic factors. Volatility poses challenges especially for
`financially exposed small biotechnology companies, the core
`translational force of the industry, to establish the platform
`and develop drugs in a process that takes at least 15 years
`to bear fruits in the form of approved drugs and depends on
`the complex interactions between a diverse set of investors.
`Even small disruptions can have big repercussions leading
`to both euphoria and capitulation which can be equally dam-
`aging to the long-term health of a sector.
`This commentary is directed at companies already involved
`in RNAi therapeutics development or those interested in
`entering the space. By analyzing the forces that shape the
`business of RNAi therapeutics at the start of 2012 it aims
`to uncover key opportunities for value creation. It may also
`help investors identify related investment opportunities and
`inventors commercialize their intellectual property (IP). For a
`review of the fundamental business case for RNAi therapeu-
`tics, the reader is referred to an earlier article on the topic.1
`
`RNAi THERAPEUTICS BUSINESS TRENDS IN
`HISTORICAL PERSPECTIVE
`
`The business of RNAi therapeutics has just entered its fourth
`phase. The first, discovery phase (2002–05) was defined by
`the early adopters of RNAi as a therapeutic modality follow-
`ing the discovery of RNAi in human cells.2 These were small,
`risk-taking biotechnology companies such as Ribozyme
`Pharmaceuticals (aka Sirna Therapeutics), Atugen (aka
`Silence Therapeutics) and Protiva (aka Tekmira). As much
`as they may have believed in the potential of RNAi thera-
`peutics, their strategic reorientation was also a gamble on a
`technology with considerable technical uncertainties in order
`to turn around declining business fortunes by leveraging their
`nucleic acid therapeutics know-how to become leaders in a
`potentially disruptive technology. For example, exploration of
`in vivo gene knockdown had only just begun, not to speak
`
`of knockdown in larger animals following systemic delivery.
`This phase also saw the founding of Alnylam Pharmaceutical
`based on the idea of cornering the IP on the molecules that
`mediate RNAi (RNAi triggers) so that it may finance its own
`drug development by collecting a toll from all those engaged
`in RNAi therapeutics.
`(“Big
`Until
`then,
`larger pharmaceutical companies
`Pharma”) saw the value of RNAi largely as a research tool
`only. This, however, changed quickly when a few of them,
`including Medtronic, Novartis, and Merck, were seen by their
`peers to take an interest in RNAi as a therapeutic modality.
`The situation seemed reminiscent of monoclonal antibodies
`which had just established themselves as the major value
`creator in the pharmaceutical industry, but where Big Pharma
`was thought to be paying the price for having watched from
`the sidelines for too long. Another factor for Big Pharma’s
`surging RNAi therapeutics interest, the defining feature of
`the second, boom phase of RNAi therapeutics (2005–08),
`was the impending patent cliff and the hope that the technol-
`ogy would mature in time to soften its financial impact.
`A bidding war, largely for access to potentially gate-
`keeping RNAi trigger IP erupted. Most notably, Merck and
`Roche paid US$1.1B for acquiring Sirna Therapeutics and
`US$300M+ for a limited platform license from Alnylam,
`respectively. These deals were only rivaled in attention by
`the award of a Nobel Prize to Andrew Fire and Craig Mello
`for their seminal discovery of double-stranded RNA (dsRNA)
`as the trigger of RNAi. The industry naturally did not mind
`the attention and in some cases fanned the fire by raising
`unrealistic expectations. This atmosphere also gave rise to
`controversial publications in high-profile journals which lent
`credence to the mistaken notion that the technical barriers to
`exploiting the RNAi trigger IP would be low.3,4 Consequently,
`most Big Pharma companies had a stake in the technology.
`Yet, the US$2.5B–3.5B in investments largely failed to formu-
`late sound strategies for the real technical challenges such
`as delivery. Symptomatic for the times, the financial markets
`similarly failed to realize the value of truly enabling technolo-
`gies: in the 2 weeks following the publication of a seminal
`paper on systemic small interfering RNA (siRNA) delivery
`by Protiva (now Tekmira) and Alnylam on 26 March 2006,5
`Alnylam’s share price would decline by over 10%.
`It is therefore perhaps not surprising that this period of high
`expectations and blockbuster deals was followed by general
`
`1Department of Medical Biotechnology, College of Natural Sciences, Dongguk University, Seoul, Korea
`Correspondence: Dirk Haussecker, Department of Medical Biotechnology, College of Natural Sciences, Dongguk University, 26, Pil-dong 3, Jung-gu, Seoul 100-715,
`Korea.
`E-mail: dirk.haussecker@gmail.com
`
`PROTIVA - EXHIBIT 2015
`Moderna Therapeutics, Inc. v. Protiva Biotherapeautics, Inc.
`IPR2018-00739
`
`

`

`2
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`
`RNAi Therapeutics Business 2012
`
`backlash (2008–2011), the financial consequences of which
`were exacerbated by global economic turmoil and health-
`care rationing in the West. Big Pharma quickly realized their
`mistake of putting IP before enablement as they scrambled
`to scout for delivery technologies and found the majority of
`them not to live up to their claims.6 Roche, a year after their
`IP license from Alnylam, felt compelled to pay US$125M for
`Dynamic PolyConjugates from Mirus Bio, one of the more
`promising and differentiated delivery technologies, for which,
`however, significant risks related to translation into organ-
`isms beyond rodents and manufacturing/scale-up remained.
`Contributing to buyer’s remorse was the ageing and rapidly
`eroding gate-keeping potential of the RNAi trigger IP that
`had been the focus of their original investments.
`As much as delivery, it was the potential of certain RNAi for-
`mulations to stimulate innate immunity that caused much of
`the scientific angst that contributed to the deteriorating busi-
`ness sentiment in 2008.7,8 It almost came to be assumed that
`an in vivo RNAi efficacy claim was in fact an innate immuno-
`stimulatory artefact. Importantly, this suspicion extended to
`the preclinical data that formed the rationale for the industry’s
`lead clinical candidates in wet age-related macular edema
`(Acuity/Opko’s Cand5, Merck/Allergan’s Sirna-027/AGN-
`745, Quark/Pfizer’s PF-4523655)9,10 and respiratory viral
`infection (Alnylam’s ALN-RSV01),11 approaches which inci-
`dentally did not involve specific delivery chemistries. Mak-
`ing matters worse still, innate immune stimulation is a safety
`issue. Although today innate immunostimulatory potential is
`widely considered to be manageable through chemical modi-
`fication and choice of RNAi trigger structure, the reputational
`damage persists.
`Suffering from RNAi-specific scientific and credibility issues
`and with first drug approvals still years away, RNAi therapeu-
`tics was among the first to feel the cost-cutting axe at com-
`panies like Pfizer, Merck, Abbott Labs, and Roche which all
`started to suffer from patent expirations, drug approval and
`productivity issues, worsening drug reimbursement climates,
`and the general loss of confidence in their innovative abili-
`ties. Particularly the exit of Roche from in-house RNAi thera-
`peutics development sent shockwaves through the industry.
`Having invested heavily in the technology only 2–3 years ago
`and being considered an innovation bellwether within Big
`Pharma, Roche’s decision in late 2010 found a number of
`imitators among Big Pharma and has been functioning as a
`major barrier to new investments in RNAi therapeutics.
`The backlash, however, also had cleansing effects which
`form the basis for the 4th, recovery phase of RNAi therapeu-
`tics (2011–present). As a result of the financial restrictions
`and increased scientific scrutiny, there has been an overall
`increase in the quality of the science. RNAi therapeutics
`has also become less of a target for the quick-rich biotech
`schemes that constantly chase the next hot area in drug
`development. This quality shift is most evident in the evo-
`lution of the RNAi therapeutics clinical pipeline which has
`become more and more populated with candidates based
`on sound scientific rationales, especially in terms of delivery
`approaches and anti-immunostimulatory strategies. For the
`recovery, however, to firmly take root and for the long-term
`health of the industry, it is important for the current clinical
`dataflow to bring back investors.
`
`RNAi THERAPEUTICS ASSETS
`
`One measure for the health of an industry is in accounting
`its assets. These are also at the center of business activity.
`Because drugs are the ultimate objective of RNAi therapeu-
`tics and because of the significant de-risking that occurs dur-
`ing drug development, the clinical and late-stage preclinical
`pipeline weighs heavy. Equally important at this relatively
`early stage are the technologies that enable candidate devel-
`opment and drive platform efficiencies. These technologies
`need to be protected by patents or trade secrets for individ-
`ual companies to capture their full value.
`
`RNAi therapeutics development pipeline. As of the 2008
`review,1 there were eight candidates in clinical development
`(Table 1). What is noticeable is that most of them were local
`RNAi approaches that today would most likely not enter devel-
`opment due to uncertain scientific rationale or safety: naked
`delivery, in some cases with unmodified synthetic RNAi trig-
`gers (Cand5, Sirna-027, RTP-801i, ALN-RSV01, TD-101),
`liposomal delivery of a DNA-directed RNAi (ddRNAi) can-
`didate which could have been predicted to be inadequate
`for antiviral applications and was all but assured to cause
`immune stimulation (NucB1000),12 or first-generation ddRNAi
`expression systems subsequently13 found to frequently cause
`cellular toxicity (rHIV-shI-TAR-CCR5RZ; possibly NucB1000).
`Not surprisingly, many of these programs were either termi-
`nated, or their future development is doubtful. Among the
`latter, there is hope that Quark/Pfizer’s PF-4523655 and
`Alnylam’s ALN-RSV01 can still make it to market as long as
`they show appropriate safety and efficacy even though their
`value to RNAi therapeutics would be limited given the wide-
`spread skepticism about their mechanism of action.
`Since 2008, the development pipeline has not only grown
`in size (18 active clinical candidates today), but more impor-
`tantly it has improved in quality concomitant with a shift from
`local to systemic delivery: 7 of the 14 new clinical candidates
`since 2008 were delivered systemically, compared to only 1
`of the 8 before. This is largely the result of the clinical entry
`of the most advanced systemic delivery platforms, stable
`nucleic acid lipid particles (SNALP) and AtuPLEX. SNALP
`alone accounts for six clinical candidates (ALN-VSP02, TKM-
`ApoB, ALN-TTR01, TKM-PLK1, ALN-PCS02, TKM-EBOLA)
`and one more is expected to enter the clinic in the near future
`(ALN-TTR02).
`Given that the value of a given drug candidate is dynamic
`and can dramatically change with each new data point—such
`as a clinical trial result or even change in regulatory policy—
`it is beyond the scope of this commentary to determine the
`market value of the RNAi development pipeline. Some candi-
`dates, however, have been licensed which makes their market
`value easier to assess. Quark Pharmaceuticals for example
`has been quite successful in licensing its compounds. As of 31
`December 2010, Pfizer had invested $52.5M in PF-4523655
`which is in late phase II development for wet age-related mac-
`ular edema and diabetic macular edema. Quark moreover is
`eligible to receive substantial future milestones and royalties.14
`Still, the value of PF-4523655 has become highly uncertain
`after phase II study results suggested that PF-4523655 faces
`an uphill battle before it can be a commercially viable drug.
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`Table 1 RNAi therapeutics clinical pipeline
`
`Year of
`IND/CTA
`
`
`Candidate
`
`
`Indication
`
`2004
`2004
`2005
`
`2007
`
`2007
`2007
`
`2007
`
`2008
`2008
`
`2008
`
`2008
`
`2008
`
`2009
`
`2009
`
`2009
`2009
`
`2009
`
`2009
`
`2010
`
`2010
`
`2011
`
`2011
`
`2011
`
`Wet AMD, diabetic macular edema
`Cand5
`Sirna-027/AGN-745 Wet AMD
`ALN-RSV01
`RSV infection
`
`DGFi
`
`PF-4523655
`rHIV-shl-TAR-
`CCR5RZ
`NucB1000
`
`Acute kidney injury, delayed
`graft function
`Wet AMD, diabetic macular edema
`HIV infection
`
`Hepatitis B viral infection
`
`HBV RNAs
`
`TD101
`Therapeutic vaccine
`
`Pachyonychia congenita
`Metastatic melanoma
`
`Mutant keratin
`Immunoproteasome
`
`Excellair
`
`Asthma
`
`Syk kinase
`
`CALAA-01
`
`ALN-VSP02
`
`Atu027
`
`QPI-1007
`
`SYL040012
`TKM-ApoB
`
`bi-shRNAfurin/
`GMCSF
`ALN-TTR01
`
`siG12D LODER
`
`TKM-PLK1
`
`CEQ508
`
`ALN-PCS02
`
`Nonresectable or metastatic solid
`tumors
`Liver cancer, cancer with liver
`involvement
`Advanced solid tumors
`
`Chronic nerve atrophy, nonarteritic
`ischemic optic neuropathy
`Intraocular pressure and glaucoma
`Hypercholesterolemia
`
`M2 subunit of ribonucleotide
`reductase
`VEGF, KSP
`
`PKN3
`
`Caspase 2
`
`(cid:66)-Adrenergic receptor 2
`Apolipoprotein B
`
`Ovarian cancer, advanced
`melanoma
`Transthyretin amyloidosis
`
`Operable pancreatic ductal
`adenocarcinoma
`Solid cancers and lymphoma
`
`Furin
`
`Transthyretin
`
`Mutated KRAS
`
`Polo-like kinase 1
`
`Familial adenomatous polyposis/
`colon cancer prevention
`Hypercholesterolemia
`
`-Catenin
`
`PCSK9
`
`TKM-EBOLA
`
`Ebola infection (biodefense)
`
`Viral RNA
`
`
`RNAi Therapeutics Business 2012
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`3
`
`
`Target
`
`VEGF
`VEGF-R1
`Viral RNA
`
`p53
`
`RTP801/REDD1
`Viral RNA and host factors
`
`
`Delivery
`
`Intravitreal needle injection (retina; local)
`Intravitreal needle injection (retina; local)
`Inhalation of unformulated siRNAs
`(lung epithelium; local)
`Intravenous naked siRNA (proximal
`tubule cells; systemic)
`Intravitreal needle injection (retina; local)
`Lentiviral (hematopoietic stem cells;
`ex vivo)
`Liposomal plasmid (hepatocytes;
`systemic)
`Intradermal needle injection (skin; local)
`Electroporation (autologous monocytes;
`ex vivo)
`Inhalation of unformulated siRNAs
`(lung epithelium; local)
`RONDEL (solid tumor cells; systemic)
`
`SNALP liposome (hepatocytes;
`systemic)
`AtuPLEX lipoplex (vascular endothelial
`cells; systemic)
`Intravitreal needle injection
`
`Eye drop (ciliary epithelial cells; local)
`SNALP liposome (hepatocytes;
`systemic)
`Electroporation plasmid (autologous
`tumor samples; ex vivo)
`SNALP liposome (hepatocytes;
`systemic)
`LODER local drug elution
`
`SNALP liposomal (solid tumor cells;
`systemic)
`Bacterial (mucosal layer of small and
`large intestine; oral)
`SNALP liposome (hepatocytes;
`systemic)
`SNALP liposome (hepatocytes and
`phagocytes; systemic)
`
`Intradermal needle injection (skin; local)
`Lentiviral transduction transduction
`(hematopoietic stem cells; ex vivo)
`
`Select preclinical candidates
`
` 2012 (est.)
` 2012 (est.)
`
`RXI-109
`To be named
`
`Dermal scarring
`HIV infection
`
`CTGF
`CCR5
`
`Abbreviations: AMD, age-related macular edema; CTGF, connective tissue growth factor; GMCSF, granulocyte-macrophage colony-stimulating factor; HBV,
`hepatitis B virus; KSP, kinesin spindle protein; PKN3, protein kinase N3; RSV, respiratory syncytial virus; shRNA, small hairpin RNA; siRNA, small interfering
`RNA; SNALP, stable nucleic acid lipid particles; VEGF, vascular endothelial growth factor.
`
`Quark also sold an option for an exclusive license to its sec-
`ond-most advanced candidate, QPI-1002, then in phase I for
`acute kidney injury and delayed graft function, for a remark-
`able US$10M fee to Novartis. The market value of the only
`other partnered candidate in clinical development, ALN-
`RSV01, has decreased considerably as its target population
`has shrunk drastically and after Alnylam’s partners for this
`drug candidate, Kyowa Hakko and Cubist Pharmaceuticals,
`have distanced themselves from it despite having invested
`more than US$35M in upfront alone.
`The remaining value of the clinical pipeline largely rests on
`three oncology candidates (ALN-VSP, Atu027, TKM-PLK1)
`
`and the SNALP-enabled ALN-PCS02 for hypercholester-
`olemia and ALN-TTR01/02 for transthyretin amyloidosis.
`This judgment is based on delivery that has been de-risked
`to some extent for these candidates, almost nonexistent tar-
`get risks for three of them (TTR, PLK1, PCS02), and the fact
`that they all represent highly differentiated approaches for
`diseases of considerable unmet medical needs. Moreover,
`there exist early biomarker opportunities for two of them
`(TTR, PCS). Should these biomarker read-outs demonstrate
`effective target gene knockdown in their phase I studies, their
`value would increase considerably, possibly pegging their
`upfront partnering value in the high double-digit millions with
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`the potential to generate substantially more revenues down-
`stream. In the case of ALN-VSP and Atu027, early clinical
`data are already supportive of further development with the
`sponsors hoping to license these compounds in 2012.
`Although having only just entered clinical development,
`TKM-EBOLA may actually be the pipeline asset with the
`highest net present value in the industry. This is because
`the full development of this biodefense candidate is being
`funded under a US$140M contract from the US Department
`of Defense. This contract allows Tekmira to not only earn
`incentive fees and profit from eventual stockpiling contracts,
`but also to develop the candidate in a way that broadly ben-
`efits the platform on which it was built. Among the other pre-
`clinical pipeline candidates, RXi Pharmaceutical’s RXI-109
`for dermal scarring, and Calimmune’s ddRNAi candidate
`for HIV deserve special mention based on their promising
`preclinical results,15 differentiation, and potential to blaze the
`trail for their respective self-delivering rxRNA and lentiviral
`ddRNAi platforms.
`
`Enabling technologies. As indicated by the evolution of
`the RNAi therapeutics product pipeline, it is the underlying
`technologies, foremost delivery, that are the major value driv-
`ers. Other technologies, however, also add value by reducing
`adverse event risk, and in the case of RNAi trigger innova-
`tion by opening up new therapeutic frontiers.
`Delivery: one cell/tissue type, many indications. The pres-
`ent expansion of the SNALP-based pipeline reflects a fun-
`damental principle of RNAi therapeutics: once a delivery
`technology is found suitable for knocking down genes in a
`given cell/tissue type, any gene can be targeted in that cell/
`tissue type with the possible applications only limited by
`our exploding understanding of disease genetics (Table 2).
`SNALP, Tekmira’s PEG-stabilized monolamellar liposomes
`that encapsulate the RNAi trigger payload in its aqueous
`interior and which are neutrally charged at physiologic pH,
`is furthest developed for knocking down genes expressed in
`the liver, particularly hepatocytes.5 Solid tumor cells,16 sites of
`tissue inflammation, and phagocytic cells,17 however, are also
`suitable targets for SNALP due to their relative accessibility
`and/or natural propensity to take up nanosized particles.
`With the caveat that there is sequence-dependent variabil-
`ity, results from the SNALP-based trials with TKM-ApoB and
`ALN-VSP02 suggest that the SNALP formulations that were
`developed initially have potential for a few indications with
`
`Table 2 Tissues/cell types amenable to therapeutic RNAi today
`
`Tissue/Cell type
`
`Liver (hepatocytes, but also other cell types)
`Vascular endothelial cells
`Solid tumor cells
`Phagocytic cells, including antigen presenting cells
`Skin
`
`Hematopoietic stem cells
`CNS, eye
`
`Delivery
`
`SNALP
`AtuPLEX
`SNALP
`SNALP
`Self-delivering
`rxRNAs
`Lentivirus
`AAV, lentivirus
`
`Abbreviations: AAV, adeno-associated virus; CNS, central nervous
`system; RNAi, RNA interference; SNALP, stable nucleic acid lipid
`particles.
`
`less stringent tolerability and cost requirements. Improve-
`ments in the efficacy and tolerability of SNALP over the last
`5 years,18 however, have significantly widened applicability
`through an expected 100- to 1,000-fold improvement in the
`therapeutic index, and further enhanced the competitive pro-
`file of SNALP by reducing cost and treatment frequencies.
`Symbolizing the value shift from RNAi triggers to delivery,
`Alnylam, which once relied on its RNAi trigger IP for its indus-
`try-leading position, has been sued by Tekmira for scheming
`to unlawfully gain control and ownership over SNALP tech-
`nology and otherwise causing damage to Tekmira’s com-
`petitive position. Somewhat benefitting from this gridlock in
`SNALP is the industry’s second-most advanced systemic
`delivery technology, AtuPLEX by Silence Therapeutics. This
`multilamellar, positively charged, lipid-based formulation
`has proven useful for knocking down genes in the vascular
`endothelium in small and large animal models.19 The phar-
`macokinetic and safety data that emerges from the ongoing
`Atu027 trial (e.g., ASCO 2011 poster presentation) indicate
`this also likely to be the case in humans. With applications
`particularly in oncology (antiangiogenesis) and acute inflam-
`matory conditions (the vascular endothelium as a barrier to
`inflammatory cell infiltration), this technology has garnered
`increased partnership interest. Positively charged lipoplexes,
`in this case delivered by intravesical instillation, may also be
`useful for knocking down genes in the superficial layers of
`the bladder, including malignancies, as suggested by pre-
`clinical data from Marina Biotech.20
`Besides these and other lipid-based delivery technologies,
`there are a number of polymer and conjugate delivery tech-
`nologies in earlier development. What started with largely
`negatively charged RNAi triggers complexed to positively
`charged polymers, an approach frequently associated with
`toxicities,21 polymers appear to be more promising as neutrally
`charged polyconjugates.22 Especially the smaller conjugates
`may be suited for gene knockdown in tissues not accessible
`to the larger lipid-based formulations. Manufacturing chal-
`lenges and biodegradability issues, however, could be caus-
`ing delays in their clinical translation. This appears to be the
`case for the Dynamic PolyConjugates for which Roche paid
`US$125M in 2008, but which Arrowhead Research recently
`acquired for single-digit million US dollars.23
`Smaller than polyconjugates, simple conjugates such
`as the GalNAc-siRNAs (target organ: liver) developed by
`Alnylam may similarly reach a wider range of target cells/
`tissues and could also be amenable to subcutaneous admin-
`istration. Potency improvements, however, are required to
`render them competitive with the more complex formula-
`tions for systemic applications when the target cell/tissue is
`shared. It is in local/localized applications that similar small
`conjugates currently have most utility. A first such program is
`about to enter clinical development with RXi Pharmaceuti-
`cal’s intradermally injected self-delivering rx-RNA RXI-109 for
`dermal scarring. Ocular, central nervous system (intraparen-
`chymal, intrathecal) and respiratory (epithelial) applications
`may similarly benefit from simple conjugate solutions.
`The RNAi trigger versus delivery debate is more balanced
`in ddRNAi technology. This is because delivery technologies
`can be directly borrowed from the field of gene therapy, with
`particularly adeno-associated virus and lentiviral delivery
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`well suited for a number of central nervous system,24 ocular,25
`and hematopoietic stem cell-related applications.15 Con-
`versely, because ddRNAi is intended for gene silencing over
`extended periods of time following a single administration,
`and adverse reactions due to ddRNAi trigger activity cannot
`easily be reversed, ddRNAi trigger safety is paramount.13
`Some of the delivery technologies above can also be used
`for ex vivo delivery. Here, the delivery challenge is essentially
`reduced to a tissue culture problem by RNAi treating the tar-
`get cells outside the body using transfection, electroporation,
`or viral transduction, before (re-)introducing them into the
`patient. This approach holds particular promise for stem cell-
`based therapeutics15 and therapeutic cancer vaccines.26
`In summary, albeit delivery technologies of clinical and
`commercial maturity are still relatively few in number, today’s
`delivery capabilities already allow for a number of high-quality
`RNAi therapeutics opportunities. This is because each deliv-
`ery technology, once found to be suitable for gene knock-
`down in a given cell/tissue type, can be rapidly expanded
`to many target genes and applications. Control over and
`access to these technologies is critical for RNAi therapeutics
`platform success.
`RNAi triggers: potency matters, but value also in safety
`and new functionalities. One of the main developments in
`the RNAi trigger field has been the realization that many
`RNAs with dsRNA elements can induce RNAi gene silenc-
`ing at least to some degree.27 Together with the weaken-
`ing of Alnylam’s RNAi trigger IP estate in the course of
`the Kreutzer–Limmer (KL) and Tuschl patent prosecutions,
`choice and access to RNAi triggers has become less rate-
`limiting than it was once thought of. It also means that work-
`ing around somebody else’s IP estate alone does not easily
`compensate for deficiencies in scientific performance, espe-
`cially knockdown potency which normally determines both
`the maximal degree and duration of the knockdown. Conse-
`quently, non-Tuschl RNAi triggers should be at least equal
`in potency, if not superior, or offer additional advantages in
`safety and functionality.
`In terms of potency, a single asymmetric instead of sym-
`metrical 3(cid:97) overhangs on the guide strand has been found
`to improve on the knockdown efficacy of Tuschl siRNAs.28
`Potency can also be improved by applying thermodynamic
`design rules such as the Zamore rules to which Silence
`Therapeutics has an exclusive license.29 Although the Dicer-
`substrate RNAi triggers had once been proposed not only to
`fall outside of Alnylam’s RNAi trigger patent estate, but also
`to be more potent than Tuschl siRNAs,30 they may actually be
`a more appropriate example for the value of functional differ-
`entiation by facilitating certain delivery strategies31 and poten-
`tially also by extending the duration of gene silencing.32
`Synthetic small hairpin RNAs can either function as Dicer-
`substrate RNAs or also be smaller in size, yet still trigger RNAi
`(e.g., SomaGenics).32 These single-molecule RNAs have the
`benefit of increased thermodynamic stability which may be
`exploited for the manufacture of RNAi triggers with increased
`dsRNA yield than conventional two-stranded siRNAs33 as
`well as delivery approaches which require single-stranded
`phases during the delivery journey. Shorter small hairpin
`RNAs should also be less prone to induce innate immunity
`and interfere with endogenous small RNA processing. The
`
`latter attributes also apply to the first-generation asymmet-
`ric siRNAs (asiRNA) by Biomolecular Therapeutics which
`are characterized by shorter double-stranded elements
`than those in conventional siRNAs.34 RXi Pharmaceutical’s
`sd-rxRNAs have even shorter double-stranded elements, a
`feature the company claims to be critical for crossing hydro-
`phobic lipid bilayers during delivery. Nevertheless, because
`the success rate of finding potent RNAi triggers may drop
`noticeably for RNAi triggers with such short dsRNA ele-
`ments, these structures should be preferentially contem-
`plated in applications where they can add unique delivery or
`safety benefits.
`The structural flexibility of RNAi triggers has also been
`exploited for increased functionality by having them target
`more than one gene (“multitargeting”). This is particularly
`valuable for treating complex diseases or where resistance
`is an issue (cancer, viral infections). Multitargeting is already
`being pursued in ALN-VSP02 and Tekmira’s Ebola program
`which involve the inclusion of several conventional siRNAs in
`a given formulation,16 but it can also be achieved for exam-
`ple by using three- or four-stranded designs, both Dicer-
`substrate and non Dicer-substrate, in which the individual
`strands guide the cleavage of distinct targets.35 Tekmira has
`recently licensed a three-stranded RNAi trigger design from
`Halo-Bio.
`Although certainly adding to functionality, two RNAi trigger
`structures exploiting RNAi trigger structural diversity, immu-
`nostimulatory siRNAs (e.g., Alnylam)36 and single-stranded
`RNAi triggers (e.g., ISIS Pharmaceuticals)37 run counter to
`two core principles of RNAi in Man. First, it was the Nobel-
`Prize winning insight by Fire and Mello that dsRNA, and not
`for example single-stranded antisense RNA, is the trigger
`in RNAi. Second, the discovery of RNAi in mammals was
`based on the use of shorter dsRNAs that would not stimulate
`the nonspecific interferon response. It therefore remains to
`be seen whether the potency disadvantage (single-stranded
`RNAi) and safety liability (immunostimulatory siRNAs) of
`these triggers can be compensated for by their unique deliv-
`ery attributes (single-stranded RNAi) or any anticancer, anti-
`viral, or antiangiogenic effect of immunostimulatory siRNAs.
`Unlike in synthetic RNAi triggers, innovation in ddRNAi
`trigger design has somewhat stalled, particularly in the
`commercial and translational arenas, with most groups still
`employing first-generation minimal small hairpin RNAs driven
`by U6 and H1 Pol-III promoters. With safety remaining a con-
`cern for these systems,13 and the causes of toxicity still to be
`fully identified, there is considerable value to be created by
`establishing alternative ddRNAi expression systems.
`Tools to minimize RNAi-related adverse event risk. The
`challenges of drug development do not stop with hitting the
`target. In a risk-averse regulatory environment, even theo-
`rized or minor safety signals in preclinical studies can lead to
`substantial delays in the approval process. The value of tech-
`nologies that minimize adverse event risk is therefore not only
`in protecting patient safety, but also in avoiding regulatory
`surprises. In RNAi therapeutics, such technologies can be
`categorized into those that address acute toxicity and those
`that deal with the risks associated with their long-term use.
`Acute immune responses from activating innate immune
`receptors or the complement system is commonly considered
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`Molecular Therapy–Nucleic Acids
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`6
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`RNAi Therapeutics Business 2012
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`the next biggest scientific challenge besides delivery.8 Indeed,
`RNAi-related “flu-like symptoms” with inflammatory cytokine
`elevations have been observed in a number of clinical tri-
`als (e.g., TKM-ApoB, NucB1000, CALAA-01),12 and activa-
`tion of the alternative complement pathway was seen in the
`Atu027 trial. Although the clinical importance of such obser-
`vations can vary considerably, it is necessary to improve on
`that record. Fortunately, understanding and mitigating, if not
`abolishing acute immune responses has been one of the
`most fertile areas of RNAi therapeutics research in recent
`years. We now understand most of the relevant nucleic acid
`structural parameters (e.g., dsRNA length, GU-rich ele-
`ments) and chemical modifications (e.g., 5(cid:97)-triphosphates,
`2(cid:97)-O-methyl), as well as the innate immune receptors (TLR3,
`7, 8; RIG-I; PKR) and cell types by which such activation may
`occur. Consequently, it is possible to adjust the RNAi trigger
`structure and, in the case of synthetic RNAi triggers, apply
`chemical modifications to minimize acute immune stimula-
`tion risk, a prediction which can then be tested in predic-
`tive assays.38 While capitalizing on this progress by licensing
`related IP has proven difficult, companies like Tekmira have
`created brand value by establishing themselves as early
`adopters and experts in this area.8
`Adve