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`Library of Congre»s Cataloging-in-Publication Data
`
`Antisense drug technology : principles, strategies, and applications / editor Stanley T. Crooke. -- 2nd '
`ed.
`.
`. .
`.
`.
`,
`.
`
`p.;cm.
`Includes bibliographical references and index.
`ISBN-13: 978-0-8493-8796-8 (alk. paper)
`ISBN-10: 0-8493-8796-5 (alk. paper)
`1. Antisense nucleic acids--Therapeutic use. I. Crooke, Stanley T.
`[DNLM: 1. Oligonucleotides, Antisense--therapeutic use. QU 57 A6324 2006] I. Title.
`
`RM666.A564A567 2006
`615'.31--dc22
`
`Visit the Taylor & Francis Web site at
`http://www.taylorandfrancis.com
`
`and the CRC Press Web site at
`http:/ /www.crcpress.com
`
`2006101712
`
`

`

`Contents
`
`Pref ace ............................................... ............ • .............. ... ... ..... ...................................................... .. .ix
`
`Acknowledgments ................. ............ .. ,. ....................... , ..................................... .............................. .. xi
`
`The Editor ...................................................... ............. ........ . ,.,., ....... .. ........ ,, ..................... , .......... ........ xiii
`
`Contributors ...................................... ............................ ........... .......................... ............. ........... ... .. xv
`
`Part I
`Introduction .. , ..... ,, ....................... .. ... ,. ... .......................... ..... ............................................ ...................... 1
`
`Chapter 1
`Mechanisms of Antisense Drug Action, an Introduction ...... .. , ...... , ............................... ..................... 3
`Stanley T. Crooke, Timothy Vickers, Walt Lima, and Hongjiang Wu
`
`Chapter 2
`The RNase H Mechanism ............................. .,. ......... ,, ................. , ......... .. :.· ...... ,. .. ................................. 47
`Walt Lima, Hongjiang Wu, and Stanley T. Crooke
`
`Chapter 3
`Small RNA Silencing Pathways ....................................................................................................... ;75
`Alla Sigova and Phillip D. Zamore
`
`Chapter 4
`Splice Switching Oligonucleotides as Potential Therapeutics ....... ................................................... 89
`Peter Sazani, Maria A. Graziewicz, and Ryszard Kole
`
`Part II
`The Basics of Oligonucleotide-Based Therapeutics .......... ................................................ :.: ......... 115
`
`Chapter 5
`Basic Principles of Antisense Drug Discovery .................. ... ...................... .... .. , ............................. 117
`Susan M. Freier and Andrew T. Watt ·
`·
`
`'•'
`Chapter 6
`· The Medicinal Chemistry of Oligonucleotides .............. .... ............................................................ 143
`Eric E. Swayze and Balkrishen Bhat
`
`'·
`Chapter 7
`Basic Principles of the Pharmacokinetics of Antisense Oligonucleotide Drugs ........................... 183
`Arthur A. Levin, Rosie Z. Yu, and Richard S. Geary
`
`Chapter 8
`Routes and Formulations for Delivery of Antisense Oligonucleotides ........................................ ..217
`Gregory E. Hardee, Lloyd G. Tillman, and Richard S. Geary
`
`Chapter 9
`Liposomal Formulations for Nucleic Acid Delivery .. ................... .. ....... ........................................ 237
`Ian MacLachlan
`
`

`

`vi
`
`CONTENTS
`
`Part IIIA
`Hybridization-Based Drugs: Basic Properties 2' -O-Methoxyethyl Oligonucleotides .................. 271
`
`Chapter 10
`Pharmacological Properties of 2' -O-Methoxyethyl-Modified Oligonucleotides ................... ....... 273
`C. Frank Bennett
`
`Chapter 11
`Pharmacok.inetic/Pharmacodynamic Properties of Phosphorothioate 2' -O-(2-Methoxyethyl)-
`Modified Antisense Oligonucleotides in Animals and Man ................................................. ; ........ 305
`Richard S. Geary, Rosie Z. Yu, Andrew Siwkowski, and Arthur A. Levin
`
`Chapter· 12.
`Toxicologic Properties of 2' -O-Methoxyethyl Chimeric Aritisense Inhibitors
`in Animals and Man ............... ...................... .................................................... .... ........................... 327
`Scott P. Henry, Tae-Won Kim, Kimberly Kramer-Stickland,
`Thomas A. Zanardi, Robert A. Fey, and Arthur A. Levin
`
`Chapter 13
`An Overview of the Clinical Safety Experience of First- and Second-Generation
`Antisense Oligonucleotides ... '. ........... ., ............... , ............................................. ., ................................... 365
`T. Jesse K woh
`
`Chapter 14
`Manufacturing and Analytical Processes for 2' -O-(2-Methoxyethyl)~Modified
`Oligonucle.otides ........................................................... 1:· .................................. : .......•.••••••• : ..... .......... 401
`Daniel C. Capaldi and Anthony N. Scozzari
`
`Part Ill B
`Hybridization-Based Drugs: Basic Properties Duplex RNA Drugs ...... ........................................ .435
`
`Chapter 15
`Utilizing Chemistry to Harness RNA Interference Pathways for Therapeutics:
`Chemically Modified siRNAs and Antagomirs ................ ........................................................... ..437
`Muthiah Manoharan and Kallanthottathil G. Rajeev
`
`Chapter 16
`Discovery and Development of RNAi Therapeutics ...... ............................... ...................... ........... 465
`Antonin R. de Fougerolles and John M. Maraganore
`
`Part IV
`Other Chemical Classes of Drugs ....................... ........................................ .................................... 485
`
`Chapter 17
`Optimization of Second-Generation Antisense Drugs: Going. Beyond Generation 2.0 .............. ..487
`Brett P. Monia, Rosie Z. Yu, Walt Lima, and Andrew Siwkowski
`
`Chapter 18
`Modulating Gene Function with Peptide Nucleic Aci~s (PNA) .................................................... 507
`Peter E. Nielsen
`
`

`

`CONTENTS
`
`vii
`
`Chapter 19
`Locked Nucleic Acid ......... ........................................................... ............ ..................................... ... 5 I 9
`Troels Koch and Henrik 0rum
`
`Chapter 20
`Morpholinos .... .... .. .................... ........ ......................... ...... ... .............. ... .......................................... 565
`Patrick L. Iversen
`
`Part V
`Therapeutic Applications .. .. ................................. ......................................................................... .. 583
`
`Chapter 21
`Potential Therapeutic Applications of Antisense Oligonucleotides in Ophthalmology ................. 585
`Lisa R. Grillone and Scott P. Henry
`
`Chapter 22
`Cardiovascular Therapeutic Applications ................................... •-··········· ..................................... . 601
`Rosanne Crooke, Brenda Baker, and Ma.rk Wedel
`
`Chapter 23
`Developing Antisense Drugs for Metabolic Diseases: A Novel Therapeutic Approach ............... 641
`Sanjay Bhanot
`
`Chapter 24
`Inflarnrnatory Diseases ................................................................................................................... 665
`Susan A. Gregory and James G. Karras
`
`Chapter 25
`Antisense Oligonucleotides for the Treatment of Cancer .............................................................. 699
`Boris A. Hadaschik and Martin E. Gleave
`
`Chapter 26
`Targeting Neurological Disorders with Antisense Oligonucleotides ...................... , ...................... 721
`Richard A. Smith and Timothy M. Miller
`
`Chapter 27
`Mechanisms and Therapeutic Applications of Immune Modulatory Oligodeoxynucleotide
`and Oligoribonucleotide Ligands for Toll-Like Receptors ........................................................... . 747
`Jorg Vollmer and Arthur M. Krieg
`
`Chapter 28
`Aptamer Opportunities and Challenges .......................................... ···-··········· ................................ 773
`Charles Wilson
`
`Index ... ............................. .. ............................................................................................ ................. 80 l
`
`

`

`•
`
`1 •
`
`,
`
`•
`
`,
`
`•
`
`•
`
`·
`
`\
`
`•
`
`.
`
`I
`
`\
`
`·• .
`
`.
`
`..
`
`.
`
`• . . • ,
`
`.. .
`
`'
`
`'
`
`,i
`
`16.3
`
`I
`
`•
`
`,
`
`.,
`
`•
`
`,""4 l •
`
`'
`
`'
`
`CHAPTER 16
`
`·Discovery and Development of ·RNAi Therapeutics
`.
`.
`
`A.ntonin R. de Fougerolles and John M. Mar~ganQ~e
`
`CpNTENTS
`
`.
`.
`16.1 · Introduction ................ , .................................................................. ......................................... 466
`.
`'
`.
`In Vitro ~ele~tion of Le~d C~ndiqates, ........... ; .. ..................... ..... ................... ? .. ... . .. .. , ... .... .466
`16.,2
`16 .. 2.1
`. Potency ............ _. ................................. ~···:··· ........ ................................. 1 .... . .. .... .. . .. .. .. 467
`16.2.2 Specificity ································•··•.•!••··_-·············· .. ···i········:········ .............. , ................. 4.68
`16,2.1 St&pility :., ........... , ... _. ................... ,.'.'." : .... , ........... ...... , .... , .............. : ... .' ... ,,. ... :~--•·:····· .. ·•···468
`162.4
`.Therapeutic .Considerations, ..... r . . . .. ... ..... ... .... , .... . ... , . .. .... .. ... ......... ... , .. .. , ••••. . ••.•.•••• .469
`In Vivp q~livery ·r,•··--·······--·.··.···:··· .. ···· ...... . "'.":"'•···· .................................... ................ ,, .. , ......... '..471
`16.3.1 Naked siRNA .............................................. .... ....................................................... 471
`16.3.1.1 Ocular ......................................... ... .......................................................... 471
`16.3.1 ,2· . . ::Respiratory ................. J.: :.: .. : .. _. .............. .l •• • :. '. •• : ...... :: . • .1 . ......... ....... . ., ... ..... 472
`16.3.1.3 Nervous System .... ,. .... ,..,. ...... .,.,. ..... ........... .......... .................................... 474
`·16.3 . .2. Conjugation., ............. :: .. .-...... :; ... • ... ;::.,. ............ ,. ..................... ..r. .... .... : ... ................ ~ ..... 474
`,
`16.3.2.1
`. Cholesterol .. ; .......... ....... ,,.1.,: ... ............. : ........... : ......... , ........ .. .... • .... .... ... :; .. 474
`'
`: 16.3.2.2 Other Natural. Ligands ......... · ........................ ., ............. ............................ .415
`16.3.2.3 Aptamers ................. ............................... ; ......... : ..................................... 475
`16.3.2.4 Small Molecules ................................................................................... 475
`16.3.3 Liposomes and Lipopl~!(.es ..................................... ..................... ............................. 475
`16.3.4 Peptides and .Polymers ....... _. .......................... : ....... :,. ........... ........ ...... ............... ......... 477
`16.3.5 Antibodies ............................................................... ,. ................ ........ ., ................ .... 478
`16.4 Clinical Trials .. : ...... : ................................................ ..... .......... ........ : ........ ............................... 478
`16.4.1 Ocular ............................ .......... .............. ,. ..... ... : .... \ .,; ............................... .,., .......... .... 478
`16.4.Ll · v"EoF ................................................. .,.,.,.,. ... , ........................................... 480
`16.4.L2 · VEGF Receptor ....... ~ ........... :: ............................................................... 480
`16.4.2 Respiratory .................. .................................. ............................. ............................. 480
`16.4.2.:1
`: RSV ........................................................................ , .... ............................. 480
`16.5 Summary : .................. ................................................. .......... .. ................. ................................ 480
`References .......................... ... .......... ............. ,, ....................................... ................. ,. ............................. 481
`
`465
`
`

`

`466
`
`ANTISENSE DRUG TECHNOLOGY, SECOND EDITION
`
`16.1
`
`INTRODUCTION
`
`•
`
`•
`
`.
`
`•
`
`:>
`
`\
`
`In less than a decade since its discovery, RNA interference (RNAi) as a novel mechanism to
`selectively silence messenger RNA (mRNA) expression has revolutionized the biological sciences
`in the postgenomic era. With RNAi, the target rriRNA is enzymatically cleaved, leading to decreased
`levels of the corresponding protein. The specificity of this mRNA silencing is controlled very
`precisely at the nqcleotide level. Given the identification and s;quencing of the entire human
`genome, RNAi is a fundamental cellular mechanism that can also be harnessed to rapidly develop
`novel drugs against any disease target. The reduction in expression of pathological proteins through
`RNAi is applicable to all classes of molecular targets, including those that have be.en traditionally
`difficult to target with either small molecules· or proteins, including monoclonal antibodies.
`Numerous proof-of-concept studies in animal models of human disease have demonstrated the
`broad potential applicability of RNAi-based therapeutics. Further, RNAi therapeutics are now under
`clinical investigation for age-related macu.llfr'a.egeneration (AMD) anci respiratory syncytial vfrus
`(RSV) infection, with numerous other drug candidates poised to advance into clinical development
`in the years to come.
`In this review, we will outline and discuss the various considerations that go into developing
`RNAi-based therapeutics starting from in vitro lead design and identification, to in vivo preclinical
`drug delivery and testing, and lastly, to a review of clinical experiences to date With RNAi thera(cid:173)
`peutics. While both nonviral delivery of small interfering.RNAs (siRNA) and viral delivery of short
`hairpin RNA (sliRNA) are being advanced as potential therapeutic approaches based on RNAi, this
`review will focus solely on development of synthetic siRNA as drugs. Synthetic siRNAs can har(cid:173)
`ness the cellular RNAi pathway in a consistent and predictable manner with regard to the extent and
`duration of action, thus making them particularly attractive· as drugs. As a consequence, siRNAs are
`the class of RN Ai therapeutics that is most advanced in preclinical and clinical studies. 1
`
`•.
`
`16.2
`
`IN VITRO SELECTION OF LEAD CANDIDATES
`
`This section highlights the various steps required. to identify potent lead siRNA can.dictates
`starting from bioinformatics design through to in vitro characterization. The overall scheme for
`turning siRNA into.drugs is summarized in Figure 16.1. The three inost important attributes to take
`into account when designing and selecting siRNA are potency, specificity, and nuclease stability.
`
`• Select siRNA
`In si/ico design
`»
`In vitro assays
`
`»
`
`• Stabilize siRNA
`» _Chemistry
`
`• Select Delivery
`» Naked .
`» Conjugation
`» Liposomes
`» Peptides/Polymers
`» Antibodies
`
`-~·
`
`Figure 16.1 Turning siRNA into drugs. Outline of steps involved in development of an RNAi therapeutic. This
`three-step process begins with in silico design and in vitro screening of target siRNA, followed by
`incorporation of stabilizing chemical modifications on lead siRNA as required, and ending with
`selection and in vivo evaluation of delivery technologies appropriate for the target cell type/organ
`and the disease setting.
`
`

`

`DISCOVERY AND DEVELOPMENT OF RNAi THERAPEUTICS
`
`467
`
`With regard to specificity of siRNA, the two issues of "off-targeting" due to the silencing of genes
`sharing partial homology to the siRNA and "immune stimulation" due to recognition of certain
`siRNAs by the·innate immune system have been of special concern: With an increased under(cid:173)
`standing of the molecular and structural mechanism of RNAi, all issues around lead siRNA
`selection are better understood and also are now generally resolvable through the use of bioin(cid:173)
`formatics, chemical modifications, and empirical testing. Thus, it is now possible to very rapidly,
`in the s.pan of several months, identify potent, specific, and stable in vitro active lead siRNA
`candidates to any target of interest.
`
`16.2.1 Patency
`
`Work by Tuschl and colleagues [ 1] represented the first published study to demonstrate that
`RNAi could be mediated in 'mammalian cells through the introduction of small'fragments of double(cid:173)
`stranded RNA (dsRNA), termed small interfering RNA, and that siRNAs had a specific architecture
`comprised of. 21 nucleotides in a staggered 19-nucleotide duplex with a 2-nucleotide 3' overhang on
`each strand (Figures 16.1 and 16.2), Further elaboration and dissection of the RNAi pathway,
`including· insights from X-ray crystallographic structures, have· revealed that long dsRNAs are
`naturally processed into siRNAs via a cytoplasmic RNaseIIMike enzyme calledDicer. A multienzyme
`complex known. as the RNA-induced silencing complex (RISC) then unwinds the siRNA ·duplex.
`The siRNA- RISC complex functions enzymatically to recognize -and cleave· mRNA strands com(cid:173)
`plementary to the "antisense'·' or "guide" strand of the siRNA. The target mRNA is then cleaved
`between nucleotides 10 and 11 (relative to the 5' end of the siRNA guide strand). Loading of RISC
`with respect to the sense and antisense siRNA strands is not symmetrical.- The-efficiency with which
`the antisense or · guide strand is incorporated into the RISC machinery ( versus incorporation of' the
`sense strand) is the most important determinant of siRNA potency. Through analysis of strand-specific
`
`. , ; ,
`
`· Therapeutic
`gene silencing
`
`~ siRNAs
`
`Natural
`process of
`RNAi
`
`J C. omplementary pairing ·
`
`l ,
`
`·
`
`I
`
`mRNA
`degradation
`
`'-./"-
`.
`
`•
`V
`
`_
`
`1
`
`Cleaved mRNA
`
`,/"'../'\../'\. f'-.F (A)n
`I
`,
`.mRNA
`_ f .. Cleavage
`
`'
`
`(A)n
`
`/
`
`Figure 16.2
`
`Harnessing the natural. RNAi process with synthetic; siRNA. Long double-stranded RNA (dsRNA)
`·is cleaved into short stretches of dsRNA called siRNA. The siRNA interact with the RISC to selec(cid:173)
`tively siience target mRNA. siRNA against any mRNA target can be chetnicaily synthesized and
`introduced into cells, resulting in specific therapeutic gene silencing.
`
`

`

`1
`
`468
`
`ANTISENSE DRUG TECHNOLOGY, SECOND EDITION
`
`reporter constructs [2] aftd large sets of siRNA of varying potency [3,4], it was found that RISC
`preferentially associates with the siRNA duplex strand whose 5' end is bound less tightly with the
`other strand. A detailed description of RNAi-mediated silencing as it relates to siRNA and other
`small RN As by Sigova and Zamore can be found in Chapter 3 of the' book
`
`16.2.2 Specificity
`
`RNAi-mediated silencing of gene expression has been shown to be exquisitely specific as evi(cid:173)
`denced by silencing fusion mRNA without affecting an unfused allele [5,6] and by studies show(cid:173)
`ing ability to silence point-mutated genes over wild-type sequence [7]. Nevertheless, .along -with
`on-target mRNA silencing, siRNA might have the potential to recognize nontarget mRNA, other(cid:173)
`wise known as "off-target" silencing. On the basis of in vitro transcriptional profiling studies,
`siRNA duplexes have been reported to silence multiple genes in addition to the intended target
`gene under certain conditions. Not surprisingly, many of these observed off-target genes contain
`regions that are complementary to one of the two strands in the siRNA duplex [8,...10]. More
`detailed bioinformatic analysis revealed that complementarity between the 5' end of. the guide
`strand and the mRNA was the key to off~target silencing, w.ith the critical nucleotides being in
`positions 2-8 (from the 5' end of the guide strand) [11,12]. Accordingly, careful bioinformatics
`design of siRNA can reduce potential off-target effects. Further, published work has shown that.the
`incorporation of 2'-O-Me ribose modifications into nucleotides can suppress most off-target
`effects while maintaining tf1[get rhRNA silencing [13,14]. In fact, incorporation of.a: single 2' -O~Me
`modification at nucleotide 2 was sufficient to suppress most off-target sileJ1.cing of partiall;y
`complementary mRNA transcripts by all siRNAs tested. Thus; in summary, bioinformatics design
`and position-specific, sequence~independent chemical moc;Ufications can be ·incorporated into
`siRNAthat reduce off-target effects while maintairung target silencing.
`A second mechanism whereby siRNA can induce potentially unwanted effects is through stim(cid:173)
`ulation of the innate immune system in certain specialized immune cell types. It has been demon~
`strated that siRNA duplexes contain distinct sequence motifs that can engage Toll-l~ke receptors
`(TLRs) in plasmac)(_toid dendritic cells and leac;l to increased interferon-alpha production [15]. In
`much the same way•that.~eJtajn _CpG motifs i~ antisens~ oligonµcle.ot1des are responsible for TLR-
`9-mediated im~u-nostimulation, the interferon· induction seen with discrete · siRNA nucleotide
`motifs was found to occur largely via TLR-:7. Much additional 'wo:rk remai~s to;beidone in identi(cid:173)
`fying the full spectrum of immunostimulatory motifs. and whether other receptors might also be
`involved (reviewed in [16]). Severaj approaches exist to circumvent the immunosttm$tory prop(cid:173)
`erties of certain siRNA duplexes. First, in vitro assays exist to rank-order duplexes for their abil(cid:173)
`ity to induce interferon when traiisfected into plasmacyioid dendritic cells [15]. Second, several
`groups have shown that introduction of chemical modifications, such as 2' -O-Me modifications,
`are capable of abolishing immunqstimulatory activity [15,17,18]. Third, siRNA delivery strategies
`can be employed that would ·a\:0id the cell types responsible for immune stimulation.
`
`16.2.3 Stability
`
`Not surprisingly, numerous studies h·av~\ shown that the chemical modification of siRNA
`duplexes, including chemistries already in use ·with antisense oligonucleotide q11d aptamer thera(cid:173)
`peutics, cap protect against nuclease degradation with no effect or intermediat~ effects on activity
`(19-21]. For instance, introduction ofa ,phospborothioate (P=S) backbone linkage at the 3' end is
`used to protect against exonuclease degradation and 2' sugar modification (2'-O-Me, 2'-F, others}
`is used for endonuclease resistance. With respect to maintenance of RNAi silencing activity,
`exonucleas'e-stabilizing modifications are all very well tolerated. Introduction of internal ·sugar
`modifications to protect against endo'nucleases is also generally tolerated but can be more
`dependent on the location of the modification within the duplex, with the sense strand being more
`
`

`

`DISCOVERY AND DEVELOPMENT OF RNAi THERAP EUTICS
`
`469
`
`amenable to modification than the antisense strand. Nevertheless, using simple, well-described
`modifications such as P = S, 2' -O-Me, and 2' -F, it is possible in most instances to fully nucle(cid:173)
`ase-stabilize an siRNA duplex and maintain mRNA silencing activity. Importantly, the degree of
`modifications required to fully stabilize the siRNA duplex can generally be limited in extent,
`thereby avoiding the toxicities associated with certain. oligonucleotide chemistries.
`Improved nuclease stability is especially important in vivo for siRNA duplexes that are exposed
`to nuclease-rich environments (such as blood) and are formulated using excipients that do not them(cid:173)
`selves confer additional nuclease protection on the duplex. As might be expected in these situations,
`nuclease-stabilized siRNA show improved pharmacokinetic properties in vivo (Alnylam, unpublished
`results). In other situations, when delivering siRNA directly to more nuclease-amenable sites such
`as the lung or when delivering in conjunction with delivery agents such as liposomes, the degree of
`nuclease stabilization that is required can be reduced significantly. While the ability ofan siRNA
`duplex to reach ibl target cell type intact is vitally important, whether nuclease protection confers a
`measurable benefit once an siRNA is inside the cell remains to be determined. While in vitro
`comparisons of naked siRNA versus fully stabilized siRNA do not reveal significant differences in
`longevity of mRNA silencing [22], these studies have typically been performed using rapidly divid(cid:173)
`ing cells, where dilution due to cell division, and not intracellular siRNA half-life governs the dura(cid:173)
`tion of gene silencing [23]. With the recent advent of fluorescence resonance energy transfer studies
`using siRNA [24], it should be possible in the near future to understand more completely the intra(cid:173)
`cellular benefit of nuclease stabilization on the longevity of RNAi-mediated silencing., .
`
`16.2.4 Therapeutic Considerations
`
`With the identification of a_ctive siRNA, a set of rules were initially ptop'osed for selecting
`potent siRNA duplex sequences (I]. Subsequently, a number of groups 'have developed more
`sophisticated algorithms based on empiric testing to identify multiple criteria that can contribute to
`defining an active siRNA [25~27]. Using current algorit~ s, sub-nM IC50 in vitro active siRNA
`can be routinely identified ll\:/i quarter to a half of the de,~iig-~ed siRNA with a subset of_ siRNA often
`demonstrating low pM activ_in,.
`·
`..,,
`-~
`·
`In designing :siitN'As f~
`erapeutic puri -?Ses, ot¥fconsiderations beyond an .active target
`sequence exist. Where possf~~' it is desJrable J'o fdentifft~rget_sequences that.have identity across
`all the relevant species use~
`:safety,a~d effi¢acy stuq~es; thus enabling devel(cid:144)prnent of a single
`drug candidate from resear~ stage _,all the \Y.af throu:g~ clinical trials. Other considerations in
`selecting a target sequence foV.olve; the presence of single,..nuclemide polymoj_-p?isms and general
`ease of chemical synthesi~,
`Predicting the nucleotide sequence and chemical modifications required to yield an ideal RNAi
`therapeutic still remains ·a work fa p;6gress. While rtmch progress has been made in understanding what
`atajbut~s a.(e requi{~d. to identify an {IJ. vitro active a11d stable si_RNA, much foss, is ~own agout how
`well those attributes translate into identifying in vivo active siRNAs;- For example, many of the issues
`arnuiid' -specificity are based orf · in v'itro data and their · in vivo relevance remain~· to be determined.
`For ex~ple, the. i:ange of off-target genes, identified in tissue culture cart 4iffer dramatically depend(cid:173)
`ing upon the transf~ction method. used -to introduce siRNAs into cells {28]~ Likewise, induction of
`innate immune responses by certairt siRNAs has beeri shown to be cell-fype'specific [29]. At present,
`in order to, identify ,robusUn vit~o. activy,'iead candidate siRNAs ~ui(able for subsequent in vivo study,
`the practical and prudent approach is to synthesize and screen a library of siRNA duplexes for potency,
`specificity; nuclease sta~ity, and i:rn;munostimulatory · activity. A good example of such an empiric
`approach was a screen that we conducted for siRNA tc\[geting all vascular endothelial growth factor
`(VEGF)-A spliced isoforms to treat AMO. Over 200 siRNA with different sequences and chemistries
`were evaluated from which an optimized clinical candidate, ALN-VEG0 1, was selected. This opti(cid:173)
`mization procedure resulted in an siRNA with picomolar in vitro activity and sustained silencing in the
`relevant ocular cell type than was superior to other published VEGF siRNA compounds (Figure 16.3).
`
`\
`
`

`

`I
`
`I : !
`
`j
`I
`
`I
`I
`
`470
`
`ANTISENSE DRUG TECHNOLOGY, SECOND EDITION
`
`(a)
`
`Hela Cells
`
`0
`
`Transfect
`siRNA
`
`100
`
`24h
`
`48h
`
`Ch ange supernatant Quantitate VEGF
`by ELISA
`
`80
`
`-~
`00
`
`t~ 60 ·
`
`(9 -'
`W 0
`>"'"°'.
`• ai 40
`'cl-.:=,
`
`20
`
`0 +-~ ~ ~~-
`0.01
`0.11
`
`~ T-rTTT,..,.,-_,.......,:'-,-,-.:,....::,::;,,r
`10
`
`siRNA (nM)
`
`(cid:127)
`
`L2000
`
`...,._ ALN-VEG01 MM
`-+- Luciferase siRNA
`-0- Cand5 VEGF siRNA
`
`-<;-- ALN-VEG01
`
`(b)
`
`Human ARPE-19 Cells
`
`0
`
`1 day
`
`5days
`
`10 days
`
`Transfec:t RPE
`with VEGF siRNA
`
`Change supernatant & quantitate VEGF by ELISA
`
`C
`
`20 oo
`
`' -0
`0.. (\J
`LL -'
`(9 0
`w "'-:
`> ai
`. ,._
`o~
`~
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`(cid:127) 30 nM
`(cid:127) 3.33 nM
`(cid:127) 0.37 nM
`
`00.04 nM
`
`ALN-VEG01
`
`ALN -VEG01 MM ,
`
`_ Cand5
`
`Figure 16.3
`
`Identification of highly potent VEGF siRNA, ALN-VEG01 . Hela cells or ARPE-19 human retinal
`pigment epithelial cell line were plated in 96 well plates and transfected 24 h later with the indicated
`concen.tration of siRNA in Lipofectamine 2000. A Lipofectamine-alone control (l.2000) is also indi(cid:173)
`cated. At 24 h post-transfe,ction, culture medium was completely removed and 100 µI of fresh 10%
`FBS in DMEM added. Fbllowing this medium change, cultured supernatants from Hela and
`ARPE-19 cells were collected 24 h (48 h post-transfection) and 96 h (5-day post-transfectibn) later,
`respectively. Fresh culture supernatant was added on day 5 to the confluent ARPE~19 cells and
`supernatants coll~cted again 5 days later (10-day post-transfection). Thus, the effect of siRNA in,hi(cid:173)
`bition on VEGF protein production was measured over different time periods post-transfection
`(Hela, 24-48 h; ARPE-19, days 1~5, days 6~10). Quantitation of human VEGF protein in the cul(cid:173)
`tured supernatants was by ELISA. Positive control is Cand5 hVEGF siRNA [32) and negative con(cid:173)
`trols include an irrelevant siRNA (luciferase) and an ALN-VEG01 siRNA containing four inverted
`nucleotide mismatches (ALN -VEG01 MM).
`
`

`

`DISCOVERY AND DEVELOPMENT OF RNAi THERAPEUTICS
`
`471
`
`16.3
`
`IN VIVO DELIVERY
`
`Effective delivery is the most challenging remaining consideration in the development of RNAi
`as a broad therapeutic platfonn. To date, animal studies using siRNA either have not employed
`additional formulation (i.e., "naked siRNA") or have delivered · siRNA formulated as conjugates,
`as liposome/_lipopfexe_s, or as complexes (peptides, polymers, or antibodies). The route of admini(cid:173)
`stration of siRNA has also ranged from-local, direct delivery to systemic administration. Local
`delivery or "direct RNAi" has particular advantages for a developing technology in that as with any
`pharmacol?gic approach, doses of siRNA required for efficacy are substantially lower when
`siRNA are injected into or administered at or near the target tissue. Direct delivery also allows for
`a more foctJsed delivery of siRNA that might circumvent -any theoretical undesired side effects
`resulting from' ·systemic delivery. Systemic d