Alnylam Exh. (cid:20)(cid:19)(cid:24)(cid:19)
`
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`Library of Congress Cataloging-in-Publication Data
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`Antisense drug technology : principles. strategies. and applications I editor Stanley T. Crooke. -- 2nd
`ed.
`
`p. ; cm.
`
`Includes bibliographical references and index.
`iSBN-IS: 978n0~8493~8796-8 (alk. paper)
`[SEN—10:0-8493-3796-5Ilalk. paper)
`i. Crooks. Stanley T.
`1. Antisense nucleic acids-Therapeutic use.
`[DNLM: I. Oligonucleotides. Antisenseutherapeutic use. QU 57 A6324- 2006] I. Title.
`
`RM666.A564A56’Z 2006
`615’.31--dc22
`
`Visit the Taylor St Francis Web site at
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`and the CRC Press Weir site at
`hitpdfwwwxrcpresscam
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`2006101712
`
`

`

`Preface ‘.‘__‘....n-u~t..lx
`
`Contents
`
`The Editor ............. . ........................................................................................................................ xiii
`
`Contributors .................................................................................................................................... XV
`
`Part I
`
`Introduction ..................................................................... . .................................................................. 1
`
`Chapter 1
`Mechanisms ofAntisense Drug Action, an Introduction. 3
`Stanley T. Crooke, Timothy Viekers, Walt Lima, and HongjiangWu
`
`Chapter 2
`The RNase H Mechanism ...................................................................... . ......................................... 4?
`
`Walt Lima, Hongjiang Wu, and Stanley T. Crookc
`
`Chapter 3
`Small RNA SilencingPathways75
`Alla Sigova and Phillip D. Zamore
`
`Chapter 4
`Splice Switching Oligonucleotides as Potential Therapeutic589
`Peter Sazani, Maria A. Graziewicz, and Ryszard Kole
`
`Part H
`
`The Basics of Oligonucleotide—Based Therapeutics ...................................................................... l 15
`
`Chapter 5
`Basic Principles ofAntisense Drug Discovery1 1?
`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
`[an MacLachlan
`
`

`

`vi
`
`Pa rt III A
`
`CONTENTS
`
`Hybridization-Based Drugs: Basic Properties 2’-0-Methoxyethyl Oligonucleotides 27]
`
`Chapter 10
`Pharmacological Properties of 2’—0-Methoxyethyl-Modified Oligonucleotides .......................... 273
`C. Frank Bennett
`
`Chapter 11
`PharmacokineticfPharmacodynamic Properties of Phosphorothioate 2'-0-(2-Methoxyethyl)—
`Modified Antisense Oligonucleotides111 Animals and Ma11..
`... 305
`Richard S Geary, Rosie Z. Yu, Andrew Siwkowski, and Arthur A Levin
`
`Chapter 12
`Toxicologic Properties of 2'-OuMethoxyethyl Chimeric Antisense Inhibitors
`in Animals and Man”
`327
`Scott P. Henry, Tae-Won Kiiii,Kimberly Kramer-Strekland
`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 ....... '..................................................................................................... 36.1
`T. Jesse Kwoh
`
`Chapter 14
`Manufacturing and Analytical Processes for 2’~0(2-MethoxyethyI)—Mcdified
`
`Oligonucleotides ..............................................................................................................................401
`Daniel C. Capaldi and Anthony N. Scozzari
`
`Part. III 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 Drugs485
`
`Chapter 1?
`Optimization of Second-Generation Antisense Drugs: Going. Beyond Generation 2.0 W48?
`Brett P. Monia, Rosie Z. Yu, Walt Lima, and Andrew Siwkowski
`
`Chapter 18
`Mnrlnlatincr Gene Function with pentirlp Nucleic Acids: {DNA}
`
`an?
`
`

`

`CONTENTS
`
`vii
`
`Chapter 19
`Locked Nucleic Acid... 519
`Tmels Koch and Henrik 9min
`
`Chapter 20
`Morpholinos
`Patrick L. Iversen
`
`PartV
`Therapeutic Applications
`
`Chapter 21
`Potential Therapeutic Applications of Antisense Oligonucleotidcs in Ophthalmology
`Lisa R. Grillone and Scott P. Henry
`
`...565
`
`583
`
`.585
`
`Chapter 22
`Cardiovascular Therapeutic Applications .......................................................
`Rosanne Crookc, Brenda Baker, and Mark Wade]
`
`............................
`
`601
`
`Chapter 23
`Developing Antisense Drugs for Metabolic Diseases: A Novel Therapeutic Approach ...............
`Sanjay Bhanot
`
`641
`
`Chapter 24
`inflammatory Diseases ...................................................................................................................
`Susan A. Gregory and James G» Karras
`
`665
`
`Chapter 25
`Antisense Oligonucleotides for the Treatment of Cancer ..............................................................
`Boris A. Hadaschik and Martin E. Gleave
`
`69‘}
`
`Chapter 26
`Targeting Neurological Disorders with Antisense Oligonucleotides .............................................
`Richard A. Smith and Timothy M. Miller
`
`”321
`
`Chapter 2?
`Mechanisms and Therapeutic Applications of Immune Modulatory Oligodeoxynucleotide
`and Oligoribonucleotide Ligands for Toll-Like Receptors ............................................................
`Jtirg Vollmer and Arthur M. Krieg
`
`74'?
`
`Chapter 28
`Aptamer Opportunities and Challenges .........................................................................................
`Charles Wilson
`
`773
`
`Index
`
`Sill
`
`

`

`Discovery and Development of RNAi Therapeutics
`
`CHAPTER 16
`
`Antonin R. de Fougerolles and John M. Maraganore
`
`CONTENTS
`
`16.1
`162
`
`Introduction. 466
`In Vim; Selection of Lead Candldates 466
`16.21 Potency... 46'?
`
`16.23 Stability... 468
`
`16.3
`
`16.2.4 Therapeutic CODSIdBI’atIOI'IS 469
`In Vivo Delivery... 471
`16.3.1 NakedSIRNA4'11
`
`16.3.1.2 Respuatory472
`16.3.1.3 Nervous 331516111424
`16.3.2 Conjugation... 474
`16.3.21 Cholesterol 1474
`
`'16. 3 2. 2 Other Natural ngands 475
`16.3.2. 3 Aptamers.. 475
`16.3.2.4 Small Molecules...................................................................................475
`16.3.3 Liposomes and Lipoplexes .................................................................................... 475
`16.3.4 Peptides and Polymers .......................................................................................... 47'?
`16.3.5 Antibodies ............................................................................................................. 478
`
`16.4 Clinical Trials 4'18
`
`16.4.1.2 VEGF Receptor 480
`16.4.2 ReSpiratory ............................................................................................................480
`16.4.2.1 RSV ......................................................................................................480
`
`Summary .-............................................................................................................................480
`16.5
`References481
`
`465
`
`

`

`466
`
`ANTISENSE DRUG TECHNOLOGY, SECOND EDlTION
`
`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 mRNA is enzymatically cleaved. leading to decreased
`levels of the corresponding protein. The specificity of this mRNA silencing is controlled very
`precisely at the nucleotide level. Given the identification and sequencing 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 been 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 maeular degeneration (AMD) and respiratory syncytial virus
`(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 viva preclinical
`drug delivery and testing, and lastly, to a review of clinical experiences to date with RNAi thera-
`peutics. While both nonviral delivery of small interfering RNAs (siRNA) and viral delivery of short
`hairpin RNA (shRNA) 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-
`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 RNAi therapeutics that is most advanced in preclinical and clinical studies.
`
`16.2
`
`IN WTRO SELECTION OF LEAD CANDIDATES
`
`This section highlights the various steps required to identify potent lead siRNA candidates
`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 most important attributes to take
`into account when designing and selecting siRNA are potency, specificity, and nuclease stability.
`
`S‘WE‘ ' """"
`Anleense
`
`
`
`°Se|90t siRNA
`.
`.
`I:
`in srllco dealgn
`»
`in wire assays
`
`. Stabilize siFlNA
`n Chemistry
`
`' Select Delivery
`» Naked
`
`» Conjugation
`a Llposomes
`» Peptides/Polymers
`WW » Antibodies
`mm
`
`Figure 16.1 Turning isNA Into drugs. Outline 0! steps involved in development of an RNAi therapeutic. This
`three-step process begins with in silico design and in vllro screening of target lelNA, followed by
`incorporation of stabilizing chemical modifications on lead siFlNA as required, and ending with
`selection and in viva evaluation of delivery technologies appropriate lor the target cell Iypeforgan
`and the disease setting.
`
`

`

`DISCOVERY AND DEVELOPMENT OF FlNAi 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—
`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-
`formatics, chemical modifications, and empirical testing. Thus. it is now possible to very rapidly,
`in the span of several months, identify potent, specific, and stable in vitro active lead siRNA
`candidates to any target of interest.
`
`16.2.1 Potency
`
`Work by Thschl 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-
`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 Z-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 cytOplasrnic RNaseIH-like enzyme called Dicer. A multienzyrne
`complicit known as the RNA-induced silencing complex (RISC) then unwinds the siRNA duplex.
`The siRNA—RJSC complex functions enzymatically to recognize and cleave mRNA strands corn-
`plernentary 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 respectto 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
`
`”Synthetic
`isNAs M
`
`
`
`
`
`Dicer
`
`
`m siFlNAs
`
`Strand separation
`
`Therapeutic
`
`R'SC
`
`Natural
`process of
`FINN
`
`Jr Complementary pairing
`
`
`
`Cleavage
`
`
`gene silencing
`I
`mFINA
`degradation
`
`L/\
`i-xnl'vxn/ - (Aln
`
`mRNA
`
`
`l Cleavage \
`\[\l
`\J\_l
`
`{Aln
`
`Cleaved mFlNA
`¢___
`
`(
`
`/l
`
`Figure 16.2 Harnessing the natural FINN process with synthetic siFtNA. Long double-stranded FINA (dsFlNA)
`is cleaved into shod stretches of dsFtNA called siFiNA. The siFlNA interact with the FIISC to selec-
`tively silence target mFlNA. siFlNA againsi any mFlNA large! can be chemically synthesized and
`introduced into cells. resulting in specific therapeutic gene silencing.
`
`

`

`468
`
`ANTISENSE DRUG TECHNOLOGY. SECOND EDITION
`
`reporter constructs {2] and 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 RNAs 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—
`denced by silencing fusion mRNA without affecting an unfused allele [5.6] and by studies show-
`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-
`wise known as “off«target" silencing. On the basis of in nine 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 cemplernentarity between the 5' end of the guide
`strand and the mRNA was the key to off-target silencing, with the critical nucleotides being in
`positions 2—8 (from the 5’ end of the guide strand) [11,12]. Accordingly, careful bioinforrnatics
`design of siRN A can reduce potential off-target effects. Further, published work has shown thatthe
`incorporation of 2’-0-Me ribose modifications into nucleotides can suppress most off-target
`effects while maintaining target mRNA silencing [13,14]. In fact, incorporation of a single 2’-0-Me
`modification at nucleotide 2 was sufficient to suppress most off-target silencing of partially
`complementary mRNA transcripts by all siRNAs tested. Thus, in sununary, bioinformatics design
`and position-specific, sequence-independent chemical modifications can be incorporated into
`siRNA that reduce off-target effects while maintaining target silencing.
`A second mechanism whereby siRNA can induce potentially unwanted effects is through stim-
`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-like receptors
`(TLRs) in plasmacytoid dendritic cells and lead to increased interferon-alpha production [15]. In
`much the same way 'that certain CpG motifs in antisense oligonucleotides are responsible for TLR-
`9—rnediated immunostimulation, the interferon induction seen with discrete siRNA nucleotide
`motifs was found to occur largely via T'LR-"i. Much additional work remains to be done in identi-
`fying the full spectrum of immunostimulatory motifs and whether other receptors might also be
`involved (reviewed in 116]). Several approaches exist to circumvent the immunostimulatory prop-
`erties of certain siRNA duplexes. First, in. vitm aSSays exist to rank-order duplexes for their abil-
`ity to induce interferon when transfected into plasmacytoid dendritic cells [15]. Second, several
`groups have showu that introduction of chemical modifications, such as 2’—0~Me modifications,
`are capable of abolishing inununostimulatory activity [15,17,18]. Third. siRNA delivery strategies
`can be employed that would avoid the cell types responsible for immune stimulatiou.
`
`16.2.3 Stability
`
`the chemical modification of siRNA
`Not surprisingly, numerous studies have shown that
`duplexes, including chemistries already in use with antisense oligonucleotide and aptamer thera-
`peutics. can protect against nuclease degradation with no effect or intermediate effecm on activity
`[19—21]. For instance, introduction of a phosphorothioate (P=S) backbone linkage at the 3’ end is
`used to protect against exonuclease degradation and 2’ sugar modification (2’-0—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 endonucleases is also generally tolerated but can be more
`dependent on the locatiou of the modification within the duplex, with the sense strand being more
`
`

`

`DlSCOVEFiY AND DEVELOPMENT OF FiNAi THERAPEUTFCS
`
`469
`
`amenable to modificatiOn than the antisense strand. Nevertheless, using simple, well-described
`modifications such as P ' S, 2’-0-Me, and 2’-F, it is possible in most instances to fully nucle—
`ass-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—
`seIVes 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 liposornes, the degree of
`nuclease stabilization that is required can be reduced significantly. While the ability of an SiRNA
`duplex to reach its 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—
`ing cells, where dilution due to cell division, and not intracellular siRNA half-life governs the dura-
`
`tion cf 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-
`cellular benefit of nuclease stabilization on the longevity of RNAi-mediated silencing.
`
`16.2.4 Therapeutic Considerations
`
`With the identification of active siRNA, a set of rules were initially proposed for selecting
`potent siRNA duplex sequences [1]. 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 algorithms, sub—11M ICSO in vino active siRNA
`can be routinely identified in a quarter to a half of the designed siRNA with a subset of siRNA often
`demonstrating low pM activity.
`In designing siRNAs forqtherapeutic purposes, other considerations beyond an active target
`sequence exist. Where possible, it is desirable to identify target sequences that have identity across
`all the relevant species used in safety and efficacy studies, thus enabling development of a single
`drug candidate from research stage all the way through clinical trials. Other cousiderations in
`selecting a target sequence involve the presence of single-nucleotide polymorphisms and general
`case of chemical synthesis.
`
`Predicting the nucleotide sequence and chemical modifications required to yield an ideal RNAi
`therapeutic still remains a work'in progress. While much progress has been made in understanding what
`attributes are required to identify an in vine active and stable siRNA, much less is known about how
`well those attributes translate into identifying in viva active siR-NAs. For example. many of the issues
`around specificity are based on in tram data and their in viva relevance remains to be determined.
`For example, the range of off—target genes identified in tissue culture can differ dramatically depend-
`ing upon the transfeetion method used to introduce siRNAs into cells [28]. Likewise, induction of
`innate imr'nune responSes by certain siRNAs has been shown to be cell-type'specific [29]. At present,
`in Order to identify robust in were active lead candidate siRNAs suitable for subsequent in vivo study,
`the practical and prudent approach is to synthesize and screen a library of siRNA duplexes for potency,
`specificity, nuclease stability, and inununostimulatory activity. A good example of such an empiric
`approach was a screen that we conducted for siRNA targeting all vascular endothelial growth factor
`(VEGB-A splicad isoforrns to treat AMI). Over 200 siRNA with different sequences and chemistries
`Were evaluated from which an optimized clinical candidate, ALN-VEGOI, was selected. This opti-
`mization procedure resulted in an siRNA with picomolar in vino activity and sustained silencing in the
`relevant ocular cell type than was superior to other published VEGF siRNA compounds (Figure 16.3).
`
`

`

`470
`
`ANTISENSE DRUG TECHNOLOGY. SECOND EDITION
`
`{a}
`
`HeLa Cells
`
`
`0
`24h
`48h
`I
`|
`I
`Transfect
`Change supernatant
`Quantitate VEGF
`siHNA
`by ELISA
`
`
`
`
`
`
`
`
`°/o,VEGFprotein(rel.toL2000)
`
`—O-' ALN—VEGD1
`
`+ Luciferase siRNA
`
`‘0'- CandS VEGF siFiNA
`
`I L2000
`
`+ ALN-VEG01 MM
`
`0.01
`
`0.11
`
`1
`
`10
`
`siFtNA (nM)
`
`(b)
`
`Human AFtF'E-lQ Cells
`
`
`0
`1 day
`5 days
`10 days
`r
`l
`I
`l
`
`‘~
`v
`J \
`Y
`1
`Change supernatant Sr quantitate VEGF by ELISA
`
`Transfeot RPE
`with VEGF siFiNA
`
`120
`
`E, 8 100
`0 O
`31% 30
`l1.
`(5 o
`60
`w I
`>_ e
`39
`
`4e
`20
`
`0
`
`I] 0.04 nM
`
`I so nM
`
`l 3.33 nM
`
`I 0.37 mm
`
`Figure 16.3 Identification of highly potent VEGF siFiNA, ALN-VEGDl. HeLa cells or AFtPE-19 human retinal
`pigment epithelial cell Ilne‘were plated in 96 well plates and transfected 24 h later with the indicated
`concentration of siFtNA in Lipofectamine 2000. A Lipofectamine—alone control (L2000) is also indi'
`cated. At 24 h post-transtection. culture medium was completely removed and 100 pl of fresh 10%
`FBS in DMEM added. Following this medium change, cultured supernatants from HeLa and
`ARPE-19 cells were collected 24 h (48 h post-transtection) and 96 h (5-day post-transfection) later,
`respectively. Fresh culture supernatant was added on day 5 to the confluent ARPE-tQ cells and
`supernatants collected again 5 days later (1 O-day post-transfeotion). Thus, the affect of siRNA inhi—
`bition on VEGF protein production was measured over different time periods post—transfection
`(HeLa, 24—48 h: AFtPE-19, days 1—5. days 6—»10). Quantitation cl human VEGF protein in the cul-
`tured supernatants was by ELISA. Positive control is Cand5 hVEGF siFt NA [32] and negative con—
`trols include an irrelevant siFtNA (lucil‘erase) and an ALN-VEGOl siFtNA containing four inverted
`nucleotide mismatches (ALN-VEGOt MM).
`
`

`

`DISCOVERY AND DEVELOPMENT OF FiNAi THERAPEUTICS
`
`471
`
`15.3
`
`”V Vii/O DELIVERY
`
`Effective delivery is the most challenging remaining consideration in the development of RNAi
`as a broad therapeutic platform. To date, animal studies using siRNA either have not employed
`additional formulation (i.e., “naked siRN ”) or have delivered siRNA formulated as conjugates,
`as liposomellipOplexes, or as complexes (peptides, polymers, or antibodies). The route of admini-
`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
`pharmacologic 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 focused delivery of siRNA that might circumvent any theoretical undesired side effects
`resulting from systemic delivery. Systemic delivery of siRNA especially with cholesterol conju-
`gates and liposome formulations have also been widely explored with considerable success. While
`this section will provide a review of the different delivery approaches utilized with siRNA, it is not
`an exhaustive description of all in viva experimentation. Several recent publications offer such a
`review [20,30,31].
`
`16.3.1 Naked siFlNA
`
`Many reports describing success with RNAi in viva involve direct delivery of “naked" siRNA to
`tissues such as eye, lung, and central nervous system. As used here. the term “nak " siRNA refers
`to the delivery of siRNA (unmodified or modified) in saline or other simple excipients such as 5%
`dextrose (DSW). The ease of formulation and administration using direct delivery of “naked” siRNA
`to tissues-make this an attractive therapeutic approach. Not surprisingly, the initial development of
`RNAi therapeutics has focused on disease targets and clinical indications (AM!) and RSV infection)
`that allow for direct administration of siRNA to the diseased organ.
`
`16.3. 1. 1 Ocular
`
`Multiple examples of efficacious local delivery of siRNA in the eye exist. where proof of con-
`cept has been attained in animal models of ocular neovascularization and scarring using both
`saline and lipidnbased formulations [32—36]. Much evidence suggests that direct administration
`of “naked" isNA is able to target cell types in the back of the eye and have profound disease-
`modifying effects. Using the optimized VEGF targeting siRNA ALN~VEGOI described above,
`we have demonstrated robust specific inhibition of pathologic retinal neovascularization in a rat
`oxygen-induced model of retinOpathy (Figure 16.4). Following a single intravitreal injection of
`saline~forrnulated ALN—VEGOI, we achieved over 75% inhibition of pathological neovascular-
`ization with no effect on the normal retinal vasculature. The inhibition seen with ALN—VEGOI
`
`was both dose-dependent and specific as a mismatched si‘RNA showed no inhibition.
`Interestingly, the degree of inhibition seen with ALN—VEGOI was dramatically more profound
`than that seen with either a VEGFlfis—specific aptamer (pegaptanih, approved for intravitreai use
`in AMD patients) or a VEGF-receptor immunoglobulin fusion protein (Figure 16.4). Separate ear—
`lier studies using lipid-formulated VEGF siRNA had shown a reduction of laser-induced
`choroidal neovascularization (CNV) in a mouse model of AMD [32]; this initial study was fol-
`lowed by nonhuman primate laser-induced CNV study where it was reported that intravitreal
`injection of a saline-formulated VEGF siRNA was well tolerated and efficacious [33]. Lastly,
`intravitreal injection of saline-formulated siRNA targeting VEGF receptor—l was effective in
`reducing the area of ocular neovascularization by U3 to 2:3 in two mouse models [34]. These
`encouraging proof-of-concept studies in animal models have lead to clinical trials of siRNA tar-
`geting the VEGF pathway in AMI).
`
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`Figure 16.4 (See color insert following page 270.) ALN-VEGU-1 specifically Inhibits retinal neovasculariza—
`tion in a rat oxygenénduced retlnopathy model.- Newborn rats were exposed to alternating high
`oxygen concentrations from days 0—14 as outlined previously [101]. On day 14. therapeutic
`agents were given once intravitreally (5 pl volume) at the amounts Indicated and rats placed in
`room air tor the following 6 days (days 14-20); On day 20. rats were sacrificed and llat'mount reti-
`nal preparations stained with ADPase was used to quantitate (a) pathologic neovascularization
`and (b) normal vascular development; representative ADPase flat mount preparations are shown
`following administration of (c) irrelevant control siHNA or (d) ALNNEGOL Experimental groups:
`no injection (No lni), saline '(PBS), high- and low-does ALN-VEGm siFtNA (sNEGF), high—dose
`ALN-VEGOt mismatch siRNA (siMM), clinical-grade VEGF aptamer (Pegaptanib), and research-
`grade VEGF receptor immunoglobulin fusion protein from R&D Systems (VEGF Ftc lg). All groups
`were scored blinded; N = 10 per groUp. Neovascularization data are expressed as mean neo-
`vascular area (mm?) 2': SE (A) and normal'retinal vasculature data are expressed as percentage
`vascular area (13E). Scheife’s post-hot: analysis was employed to identify significant differences
`in both neovascular area and normal vascular area. One of three representative experiments.
`
`16.3. “t .2 Respiratory
`
`A number of studies have demonstrated that intranasal and orotracheal administration of
`
`formulated siRNA can result in a significant target gene silencing in the lung leading to distinct
`disease~modifying phenotypes (Table 16.1.). Typically, siRNAs were administered in concentrate
`tions of ~100 pg per mouse and were directed against viral or- endogenous disease-related
`targets. Most of the examples of successful direct delivery of