R E V I E W
`
`The chemical evolution of oligonucleotide
`therapies of clinical utility
`
`Anastasia Khvorova1,2 & Jonathan K Watts1,3
`
`After nearly 40 years of development, oligonucleotide therapeutics are nearing meaningful clinical productivity. One of the
`key advantages of oligonucleotide drugs is that their delivery and potency are derived primarily from the chemical structure of
`the oligonucleotide whereas their target is defined by the base sequence. Thus, as oligonucleotides with a particular chemical
`design show appropriate distribution and safety profiles for clinical gene silencing in a particular tissue, this will open the door
`to the rapid development of additional drugs targeting other disease-associated genes in the same tissue. To achieve clinical
`productivity, the chemical architecture of the oligonucleotide needs to be optimized with a combination of sugar, backbone,
`nucleobase, and 3(cid:96)- and 5(cid:96)-terminal modifications. A portfolio of chemistries can be used to confer drug-like properties onto
`the oligonucleotide as a whole, with minor chemical changes often translating into major improvements in clinical efficacy. One
`outstanding challenge in oligonucleotide chemical development is the optimization of chemical architectures to ensure long-term
`safety. There are multiple designs that enable effective targeting of the liver, but a second challenge is to develop designs that
`enable robust clinical efficacy in additional tissues.
`
`The informational nature of oligonucleotide drugs1 (i.e., drugs
`designed on the basis on sequence information) promised to lend
`itself well to the postgenomic era of medicine. Researchers were
`drawn by the promise of rapid and rational design of drugs against
`virtually any genetic target. However, it has taken more than three
`decades for these therapies to reach clinical maturity.
`As with any therapeutic modality, the success of an oligonucleotide
`drug is defined both by its ability to affect a target and its pharma-
`cokinetic behavior, including absorption, distribution, metabolism,
`and excretion (ADME). Oligonucleotide therapeutics comprise a
`diverse class of drugs, including small interfering RNAs (siRNAs)2,
`antisense oligonucleotides (ASOs)3, microRNAs4, aptamers5, and
`others6. As these all work by different mechanisms, the activity and
`pharmacokinetic properties can to some extent7 be optimized inde-
`pendently (Fig. 1). In contrast, these are inseparable for traditional
`small molecule drugs, necessitating a unique, iterative process of
`optimization for each.
`A drug’s pharmacokinetic properties depend on a set of molecular
`features we refer to as the dianophore, from the Greek dianomi, which
`means distribution or delivery. For oligonucleotide drugs, the diano-
`phore is defined largely by chemical and structural architecture, such
`as chemical modifications of sugars, bases, and phosphate backbone,
`single strand or duplex structure, and the presence or absence of a
`targeting ligand. In contrast, the pharmacophore (the ensemble of
`
`1RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester,
`Massachusetts, USA. 2Program in Molecular Medicine, University of Massachusetts
`Medical School, Worcester, Massachusetts, USA. 3Department of Biochemistry
`and Molecular Pharmacology, University of Massachusetts Medical School,
`Worcester, Massachusetts, USA. Correspondence should be addressed to A.K.
`(anastasia.khvorova@umassmed.edu) or J.K.W. (jonathan.watts@umassmed.edu).
`
`Received 18 August 2016; accepted 12 December 2016; published online
`27 February 2017; doi:10.1038/nbt.3765
`
`molecular features that determine target regulation) is defined by its
`nucleotide sequence.
`Although base sequence and the precise pattern of chemical modi-
`fications can affect the global properties of an oligonucleotide and its
`trafficking, cellular uptake, and other behaviors7, the ability to sepa-
`rately optimize the pharmacophore and dianophore, at least to some
`extent, is a key advantage of oligonucleotide drugs. Development of
`an optimized dianophore, a chemical architecture enabling effec-
`tive delivery to a certain tissue, enables rapid progression of drugs
`with predictable ADME profiles for multiple indications, as long
`as the same tissue and cell type are involved in disease progression
`(for example, siRNAs formulated in lipid nanoparticles for the liver
`or N-acetylgalactosamine (GalNAc)-conjugated ASOs and siRNAs
`for hepatocytes).
`Early on, unmodified or minimally modified compounds
`were rushed to the clinic without conjugates or delivery vehicles.
`Massive dose requirements and limited clinical efficacy created a dra-
`matically negative view of the technology, damaging the reputation
`of the field of oligonucleotide therapeutics for years. A consequent
`decrease in available funding delayed progress. But advances in oli-
`gonucleotide chemistry and an understanding of fundamental princi-
`ples that define the in vivo behavior of oligonucleotides have enabled
`oligonucleotide therapeutics to approach clinical productivity (at least
`in some tissues).
`As a result, the current pipeline of oligonucleotide drugs is broad
`and includes a variety of molecules with different mechanisms of
`action. In hepatitis B virus (HBV) treatment, for example, four oli-
`gonucleotide drugs are currently undergoing human testing. Two are
`siRNAs (Arbutus is using a lipid nanoparticle (LNP) and Alnylam
`a GalNAc conjugate) whereas Ionis is developing both naked and
`GalNAc-conjugated ASOs. The fact that four platforms are being
`tested simultaneously allows several shots on goal, and the clinical
`comparison of these four platforms for the same tissue and disease
`
`238
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`VOLUME 35 NUMBER 3 MARCH 2017 NATURE BIOTECHNOLOGY
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`© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
`
`

`

`R E V I E W
`
`can also be increased with 2(cid:96)-fluoro (2(cid:96)-F) modification of RNA (2(cid:96)-F-
`RNA, (cid:36)Tm ~2.5 °C per modified nucleotide).
`Reducing the conformational flexibility of nucleotides can increase
`their binding affinity29,30. Locked nucleic acid (LNA), which links the
`2(cid:96)-oxygen and 4(cid:96)-carbon of ribose, shows unprecedented increases
`in binding affinity ((cid:36)Tm 4 °C to 8 °C per modification when binding
`RNA)31–33. The very high binding affinity of LNA and its methyl-
`ated analog, known as ‘constrained ethyl’ (cEt) (Fig. 2), has opened
`new doors in nucleic acid chemical biology and therapeutics34
`(Fig. 3). Tricyclo-DNA (tcDNA) is another constrained nucle-
`otide based on a very different three-ring scaffold35. Its binding
`affinity ((cid:36)Tm ~2 °C) is smaller than that of LNA, but it has shown
`much promise in splice-switching applications, for reasons that are
`not fully understood36.
`This variety of sugar modifications can be used to make chimeric
`oligonucleotides with very high binding affinities or help offset nega-
`tive effects caused by another modification. For example, fully LNA-
`modified oligomers longer than ~8 nucleotides tend to aggregate,
`so LNA and cEt modifications are often used in chimeric oligo-
`nucleotides containing multiple types of modified nucleotides
`(for example, mixtures of LNA and DNA or LNA, 2(cid:96)-OMe, and
`MOE-RNA). Although MOE and tcDNA have lower binding affinities
`per modification than LNA, they can both be used to make longer,
`fully modified oligomers.
`An ASO that simply binds and blocks its RNA target requires rela-
`tively few constraints on chemistry besides nuclease resistance and
`high binding affinity. If an enzyme such as RNase H or Argonaute is
`required, the constraints on chemical modification are more complex.
`Below, we describe the two most common categories of ASO.
`
`RNase H–dependent ASOs
`RNase H cleaves the RNA strand of a DNA–RNA hybrid; as such,
`the sugar-modified RNA-like nucleotides described above do not
`elicit RNase H cleavage of complementary RNA. The most common
`solution, called a ‘gapmer’ ASO, consists of a central window (i.e.,
`a gap) of PS DNA, which recruits RNase H, flanked by modified
`RNA-like nucleotides (Fig. 2).
`There are no hard-and-fast rules about gapmer symmetry.
`Asymmetric ASOs with high-affinity modifications on one end of the
`oligonucleotide can also be used, sometimes with a cap or ligand on
`the other end to help prevent nucleolytic decay37. The overall affinity
`of an oligomer for its target needs to be high enough to displace RNA
`secondary structure or compete with RNA-binding proteins. But
`cleaved target RNA fragments must be released before an ASO can
`find, bind, and cleave the next target, so overly high binding affinity
`can actually reduce potency in vivo38.
`Short (12–15 nt) gapmer ASOs built with LNA and cEt nucleotides
`tend to be more potent than longer oligonucleotides built with lower-
`affinity chemistry39,40. Thus, the high binding affinity of the con-
`strained ribose allows shorter oligomers to bind their RNA targets
`with sufficient affinity to be functional. The improved potency means
`that a wider range of tissues can be accessed by systemic administra-
`tion of naked ASOs17.
`LNA and cEt ASOs have been associated with liver toxicity41.
`The risk of toxicity seems to apply equally to LNA and cEt, despite
`previous reports to the contrary, and is sequence dependent. In
`the past year, three groups independently demonstrated that LNA
`and cEt gapmer ASOs induce liver toxicity by directing off-target
`RNase H cleavage of mismatched transcripts, particularly within
`introns42–44. Armed with this information, computational methods
`
`Traditional, small-molecule drug
`
`Informational drug
`
`Chemical structure
`
`Chemistry of the
`backbone and ligand
`
`Sequence
`
`Dianophore
`
`Pharmacophore
`
`Dianophore
`
`Pharmacophore
`
`Figure 1 The key advantage of an informational drug is that the
`pharmacophore (molecular features that determine target specificity) and
`the dianophore (molecular features that determine tissue distribution
`and metabolism) can be optimized separately. When a dianophore for a
`particular tissue or cell type is defined, it can be applied to a range of
`pharmacophores that are rationally designed based on sequence information.
`
`will surely inform the direction of future clinical development of
`oligonucleotide drugs in the liver.
`In this review we describe current aspects of the evolution of the
`chemistry of both antisense oligonucleotides and siRNAs that have
`opened the way for clinical utility. We place particular emphasis on
`ASO and siRNA conjugates currently in human testing. Advances in
`nucleic acid chemistry that are earlier in the preclinical pipeline have
`been reviewed elsewhere8–11.
`
`Chemical evolution of ASOs
`In 1978, Zamecnik and Stephenson demonstrated that an oligonu-
`cleotide that is antisense (i.e., complementary) to a viral RNA could
`reduce protein translation and viral replication12,13. It is now clear that
`ASOs can make use of multiple mechanisms to reduce or modulate
`gene expression14. Nonetheless, all ASOs require chemical modifica-
`tion to be sufficiently active in vivo.
`The first chemical modification applied to antisense technology
`is still the most widely used—the phosphorothioate backbone15
`(Fig. 2). Although originally incorporated to provide nuclease stabil-
`ity, the major impact of phosphorothioate modification has been on
`oligonucleotide trafficking and uptake15–18. ASOs bearing phospho-
`rothioate linkages are compatible with recruitment of RNase H, which
`cleaves the targets of ASOs.
`Although they improve oligonucleotide stability, phosphorothio-
`ates alone do not fully protect ASOs from nucleases and the in vivo
`efficacy of first-generation ASOs (which comprised phosphorothioate
`(PS) DNA; Fig. 3) required repeated administration at high doses.
`Moreover, phosphorothioates reduce the binding affinity of an oli-
`gonucleotide toward its RNA target. Improved stability and increased
`affinity have been achieved through the use of nucleotides with sugar
`modifications, including 2(cid:96)-modified and conformationally con-
`strained nucleotides (Fig. 2).
`The 2(cid:96)-O-methyl (2(cid:96)-OMe) modification of RNA (2(cid:96)-OMe-RNA),
`which occurs in nature, improves binding affinity and nuclease resist-
`ance19–21 and reduces immune stimulation22. Using 2(cid:96)-O-methyl as a
`starting point, medicinal chemists worked to find an ideal 2(cid:96)-O-alkyl
`substituent23–27. Among dozens of variants tested, 2(cid:96)-O-methoxyethyl
`(2(cid:96)-MOE)28 emerged as one of the most useful analogs, providing a
`further increase in nuclease resistance and a jump in binding affinity
`((cid:36)Tm) 0.9 °C to 1.7 °C per modified nucleotide. The approved anti-
`sense drug Kynamro, as well as numerous oligonucleotide drugs cur-
`rently in clinical trials, carry the 2(cid:96)-MOE modification. ASO affinity
`
`© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
`
`NATURE BIOTECHNOLOGY VOLUME 35 NUMBER 3 MARCH 2017
`
`239
`
`

`

`R E V I E W
`
`can be used to select ASOs with minimal complementarity to
`off-target transcripts (including introns).
`Chemistry can be used to improve ASO specificity. Gapmer ASOs
`that are highly selective for single-nucleotide polymorphisms (SNPs)
`have been developed using combinations of modifications—including
`
`2-thiothymidine, 3(cid:96)-fluorohexitol nucleic acid (FHNA), cEt, a 5-modified
`pyrimidine base, and an analog called (cid:65),(cid:66)-constrained nucleic acid
`((cid:65),(cid:66)−CNA) in which the phosphate is included in a ring structure
`(Fig. 2)—in combination with shorter gaps45,46. These gapmers mini-
`mize the region that can be cleaved by RNase H without reducing
`
`Base
`
`Base
`
`O
`
`O
`
`N
`
`OP
`
`O
`
`Me2N
`
`O
`
`N
`
`P
`
`O
`
`O
`
`Me2N
`
`PMO
`
`(cid:37)(cid:68)(cid:86)(cid:72)
`
`Base
`
`O
`
`O
`
`O
`
`O
`
`O
`
`P
`
`OO
`
`O
`
`(S
`
`C5(cid:1) R
`
`p)-(cid:3),(cid:2)-CNA
`
`O
`
`O
`
`Base
`
`O
`
`O
`
`Base
`
`O
`PO
`–S
`
`O
`
`O
`PO
`–S
`O
`
`Base
`
`O
`
`Base
`
`O
`
`R
`
`p isomer
`
`O
`
`S
`
`p isomer
`
`O
`
`Phosphorothioate (PS)
`(shown for DNA)
`
`Phosphate backbone modification
`
`O
`
`O
`
`Base
`
`O
`
`O
`
`Base
`
`O
`
`O
`
`Base
`
`O
`
`F
`
`O
`
`Base
`
`O
`
`OCH3
`
`O
`
`O
`
`O
`
`F
`
`O
`
`2(cid:1)-OMe-RNA
`
`MeO
`
`2(cid:1)-O-MOE-RNA
`
`2(cid:1)-F-RNA
`
`2(cid:1)-F-ANA
`
`2(cid:1)-modifications
`
`O
`
`NH
`
`O
`
`N
`
`S
`
`O
`
`O
`
`2-thio-dT
`
`Steric blockers
`RNase H
`RNAi
`
`Base modification
`
`O
`
`O
`
`Base
`
`O
`
`O
`
`Base
`
`O
`
`OO
`
`LNA
`
`OO
`
`(S)-cEt
`
`O
`
`Base
`
`H
`
`O
`
`tcDNA
`
`O
`
`Base
`
`O
`
`O
`
`F
`
`FHNA
`
`O
`
`O
`
`Base
`
`O
`
`O
`
`Base
`
`O
`
`X
`
`(S)-5(cid:1)-C-methyl
`
`O
`
`OH
`
`UNA
`
`Base
`
`O
`
`P O–
`O
`
`O–
`
`O
`
`Base
`
`O
`
`P S–
`O
`
`O–
`
`O
`
`Base
`
`O
`
`P O–
`
`O–
`
`O
`
`Base
`
`O
`
`P O–
`
`O
`
`O–
`
`RO
`
`E-VP
`
`RO
`
`O
`
`R
`
`RO
`
`Methyl phosphonate
`
`5(cid:1)-phosphorothioate
`
`(S)-5(cid:1)-C-methyl with phosphate
`
`Constrained nucleotides
`
`Other modified sugars
`
`Sugar modification
`
`5(cid:1)-phosphate stabilization
`
`Figure 2 Structures of chemical modifications discussed in this review. Combining modifications of the oligonucleotide backbone, sugars, bases, and
`the 5(cid:96)-phosphate are necessary to develop compounds with optimal activity. Some modifications are used for oligonucleotides that work by different
`mechanisms (indicated by colored lines): steric blockers, green; RNase H, blue; RNAi, orange.
`
`240
`
`VOLUME 35 NUMBER 3 MARCH 2017 NATURE BIOTECHNOLOGY
`
`© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
`
`

`

`R E V I E W
`
`ASO technology maturation
`
`5(cid:1)
`
`3(cid:1)
`
`5(cid:1)
`
`3(cid:1)
`
`5(cid:1)
`
`3(cid:1)
`
`C o n j u g a t e s
`
`PS-DNA
`
`‘Gen 2 gapmers’
`
`LNA and cEt gapmers
`
`Stereoselective PS
`
`5(cid:1)
`
`Chemistry for specificity
`
`5(cid:1)
`
`O
`
`O
`
`Base
`
`O
`
`O
`
`Base
`
`-(GalNAC)3
`
`3(cid:1)
`
`3(cid:1)
`
`Y
`
`O
`
`O–
`P
`O
`
`oligonucleotide
`
`O
`
`NH
`
`3
`
`O
`
`O
`NHAc
`
`X
`
`OH
`
`HO
`
`Base
`
`HO
`
`O
`
`P O–
`
`O–
`
`O
`
`OO
`
`LNA
`
`O
`
`F
`
`2(cid:1)-F-RNA
`
`Base
`
`Base
`
`O
`
`O
`
`Base
`
`O
`PO
`–S
`
`O
`
`GalNAc conjugates
`OH
`O
`
`oligonucleotide
`
`PUFA conjugates
`
`O
`
`NH
`
`O
`
`O
`
`Base
`
`OO
`
`(S)-cEt
`
`RO
`
`E-VP
`
`3(cid:1)
`
`5(cid:1)
`
`i n g c o n j u g a t e s
`
`T a r g e t
`
`Lipophilic conjugates
`
`Partly or fully single stranded
`
`5(cid:1)-E-VP
`
`3(cid:1)-(GalNAC)3
`
`5(cid:1)-E-VP
`
`3(cid:1)-PUFA
`
`5(cid:1)-E-VP
`
`5(cid:1)
`
`3(cid:1)
`
`5(cid:1)
`
`3(cid:1)
`
`3(cid:1)
`
`O
`
`O
`
`PS DNA
`
`O
`
`O
`
`O
`
`OCH3
`
`2(cid:1)-OMe-RNA
`
`siRNA technology maturation
`
`5(cid:1)
`3(cid:1)
`
`3(cid:1)
`
`5(cid:1)
`
`5(cid:1)-E-VP
`
`3(cid:1)
`
`5(cid:1)
`3(cid:1)
`
`3(cid:1)
`5(cid:1)
`
`Native
`siRNA
`
`Partially
`modified
`siRNA
`
`Fully chemically
`stabilized siRNA
`
`Figure 3 The evolution of RNase H antisense and RNAi technologies, including key chemical modifications and structural configurations that have
`enabled major advances toward clinical efficacy. White circles, 2(cid:96)-OH (RNA) or 2(cid:96)-H (DNA); gray, 2(cid:96)-F; black, 2(cid:96)-OMe or 2(cid:96)-MOE; blue, LNA or cEt;
`green, specificity-enhancing modification; red, phosphorothioate backbone modification (direction of the bond indicates positional stereopurity
`Rp or Sp). PUFA, polyunsaturated fatty acids; gen 2, second generation.
`
`cleavage of the desired site (for example, a disease allele), but a
`mismatch near the desired cleavage site (i.e., normal allele) incurs
`a major loss of cleavage activity47. SNP-selective ASOs to treat
`Huntington’s disease are expected to be the first to enter the clinic. It
`remains to be seen how readily the principles used for SNP selectivity
`can be applied to the more general problem of target selectivity.
`As an alternative to the gapmer approach, modifications that adopt
`a DNA-like conformation can also be used to improve the affinity
`and stability of RNase H–compatible ASOs. Fluoroarabinonucleic acid
`(2(cid:96)-F-ANA) is the paradigmatic example of this approach48,49.
`Although 2(cid:96)-F-ANA modification at every position of an ASO
`increases stability and affinity, the RNase H cleavage rate drops sub-
`stantially. But rapid kinetics of cleavage can be restored by combining
`2(cid:96)-F-ANA with DNA50,51. 2(cid:96)-F-ANA and other DNA mimics are thus
`valuable tools for tuning the thermodynamic properties of RNase
`H–dependent ASOs.
`
`Steric blocker ASOs
`The second major class of ASOs does not seek to recruit RNase H, and
`therefore a DNA-like gap in the oligonucleotide is unnecessary. This
`class of ASOs has seen two major clinical uses to date: splice switching
`and microRNA (miRNA) inhibition.
`
`In the past year, two splice-switching oligonucleotides have achieved
`clinical success. Last August, the US Food and Drug Administration
`(FDA) approved Exondys 51 (eteplirsen, made by Sarepta
`Therapeutics), a 30-mer phosphorodiamidate morpholino oligomer
`(PMO; Fig. 2) for treatment of Duchenne muscular dystrophy52.
`The molecule was approved, despite controversy over the levels of
`Exondys 51 that actually reached muscle tissue and the degree of splice
`switching attained. Four months later, Spinraza (nusinersen), a fully
`MOE-modified 18-mer ASO that redirects the splicing of the SMN2
`gene53, was approved for treatment of spinal muscular atrophy54.
`Several chemical approaches have been used for oligomer-mediated
`miRNA inhibition55. A direct comparison of anti-miRNAs (anti-
`miRs) showed that chimeric LNA–2(cid:96)-OMe-RNA oligomers with
`phosphorothioate backbones are the most potent56. Researchers gen-
`erally design anti-miRs to be complementary to the mature miRNA
`sequence and thereby inhibit them directly, but in some cases, anti-
`miRs can also target or disrupt the precursor miRNA structures and
`inhibit miRNA maturation57. A family of miRNAs that shares a com-
`mon seed sequence can be inhibited by a single, short (8-nt) oligomer
`that is fully modified with LNA58. These ultra-short oligomers some-
`times show enhanced distribution in some tissues compared with
`longer anti-miRs.
`
`NATURE BIOTECHNOLOGY VOLUME 35 NUMBER 3 MARCH 2017
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`© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
`
`

`

`Antisense technology
`
`Importance of RNase H
`
`Discovery of the
`antisense principle
`
`Discovery of
`splice-switching
`
`Momentum
`shifts to RNAi
`
`Invention of
`automated
`oligonucleotide
`synthesis
`
`Foundational
`chemistry: PS
`
`Discovery of LNA,
`MOE, PMO, 2(cid:1)F-ANA
`
`Short BNA gapmers show
`increased potency
`
`Fomivirsen
`approval
`
`Mipomersen approved
`by FDA but not EMA
`
`Oblimersen
`fails in
`phase 3
`
`Development of GalNAc ASOs
`
`Increasing understanding
`of toxicity mechanisms
`
`1980
`
`1990
`
`2000
`
`2010
`
`2020
`
`Foundations of 1st
`generation approaches
`
`Foundations of 2nd
`generation approaches
`
`New designs improve
`potency and specificity
`
`RNAi
`
`Discovery of
`RNAi
`
`Big pharma
`enters
`
`Development of
`stabilized
`GalNAc siRNAs
`
`Nobel Prize to Fire
`and Mello
`
`Several advanced
`clinical trials fail
`
`Early clinical trials
`
`First product expected
`~2018
`
`Big pharma
`leaves
`
`LNPs continue to
`show immune
`effects
`
`Market size, optimism, visibility
`
`Market size, optimism, visibility
`
`2000
`
`2005
`
`2010
`
`2015
`
`2020
`
`Foundational
`biology
`
`Focus: minimal
`modification
`and LNP delivery
`
`Focus: extensive
`modification
`and conjugate delivery
`
`Figure 4 Key events in antisense and RNAi therapeutics mapped to the
`Technology Curve. Both antisense (a) and RNAi (b) approaches have
`passed through the stages of novel technology trigger, peak of inflated
`expectations, and trough of disillusionment and are now approaching the
`plateau of productivity.
`
`Metabolic stabilization
`When injected into the bloodstream, naked siRNAs are degraded
`within minutes71. Studies quickly revealed, however, that relatively
`few chemical modifications are sufficient to increase stability, prevent
`innate immune activation72 and reduce off-target effects73. Extensive
`modification of siRNAs (to ~50% of nucleotides) does not signifi-
`cantly increase the duration of silencing in vivo when siRNAs are
`delivered by lipid nanoparticles or hydrodynamic injection71,74.
`Moreover, the RNAi machinery can efficiently bind heavily modi-
`fied siRNAs (i.e., those with most or all ribose content removed)75–78,
`but extensive modification can negatively affect efficacy. Consequently,
`the idea that a minimal number of modifications could improve stabil-
`ity and activity in vivo was viewed as a key advantage of RNAi technol-
`ogy over antisense technology for years. (This minimal modification
`was more recently shown to be inadequate for conjugate-mediated
`delivery, as discussed below.)
`
`R E V I E W
`
`Other ASO developments
`The length of an ASO contributes to its dianophore, affecting distribu-
`tion and tissue uptake. Shorter ASOs tend to distribute more to the
`kidney, and longer oligomers to the liver59. Shorter ASOs bind plasma
`protein poorly and consequently have a short half-life in plasma, but
`they can be assembled into multimers using cleavable linkers60.
`Idera Pharmaceuticals has found that connecting two first-generation
`phosphorothioate-modified ASOs by their 5(cid:96) ends (leaving the
`3(cid:96) ends exposed) substantially increases the potency of gene silencing
`and reduces innate immune activation61. This approach may provide
`an independent way to increase potency and specificity.
`The phosphorothioate linkage introduces a stereocenter at phos-
`phorus, and oligonucleotides are normally a mixture of 2n–1 diaster-
`eomers (for example, an 18-mer phosphorothioate oligonucleotide
`has 217 diastereomers). The Sp and Rp diastereomeric linkages (Fig. 2)
`have different properties: the Rp diastereomer is less resistant to nucle-
`ases than the Sp diastereomer, but it binds with higher affinity and
`elicits RNase H more effectively62–64. Overall, uniformly stereopure
`phosphorothioate ASOs (i.e., all Sp or Rp) are inferior to the ster-
`eorandom phosphorothioate ASOs. Precise patterns of alternating
`stereochemistry at phosphorus (for example, RpRpSp and SpSpRp) may
`improve mismatch discrimination and RNase H activity compared
`with stereorandom or stereopure oligonucletides65. On the basis of
`this principle, WaVe Life Sciences is planning to advance a stereo-
`defined SNP-selective ASO drug to treat Huntington(cid:96)s disease to
`clinical trials. Because specificity and mismatch discrimination are
`becoming increasingly important in ASO therapeutics, the increased
`specificity of stereoselective phosphorothioate ASOs may find wide
`application in improving other drug candidates.
`
`Chemical evolution of siRNAs
`RNAi was discovered in 1998 (ref. 66), and RNAi silencing of
`gene expression in mammalian cells was first described in 2001
`(ref. 67), roughly coincident with the completion of the human
`genome sequence. This resulted in an explosion of interest in, and
`funding for, RNAi. The original hope was that siRNAs (the double-
`stranded oligonucleotide triggers of RNAi) could be used to silence
`any gene in any cell. Several biotech companies, including the flag-
`ship RNAi company Alynlam, and many major pharmaceutical
`companies entered the fray (Fig. 4). Confident in the power of
`RNAi, in which an siRNA becomes associated with Argonaute and
`other proteins to form the RNA-induced silencing complex (RISC)
`and cleave complementary RNA, programs moved rapidly
`toward the clinic, most using local delivery by eye injection or
`intranasal spray68,69.
`In many of these early programs, completely unmodified or slightly
`modified compounds were administered in the hope that a small
`but sufficient amount of oligonucleotide would be taken up by the
`appropriate cells and silence the target. Ultimately, most of these
`attempts showed limited clinical efficacy and unacceptable toxicity,
`primarily from induction of the innate immune response by non-
`modified duplex RNAs. Thus, chemical modification of siRNA is
`absolutely necessary to achieve clinical utility.
`The significant legacy of nucleic acid chemistry developed for
`ASO therapeutics sped up the evolution of RNAi technology tre-
`mendously70. Nevertheless, the molecular requirements for effective
`recruitment of the RNAi enzymatic machinery and the double-
`stranded nature of RNAi imposed a unique set of limitations on the
`chemical modification of siRNAs, which took years of investigation
`to overcome.
`
`© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
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`

`

`Table 1 Clinical programs based on GalNAc conjugates
`Drug
`Company
`Mechanism and chemistry Target
`
`Disease
`
`Development
`
`R E V I E W
`
`Revusiran
`Fitusiran
`Inclisiran
`IONIS-APO(a)-LRx
`IONIS-ANGPTL3-lRx
`RG-101
`ALN-CC5
`ALN-AS1
`
`IONIS-HBV-LRx
`RG-125
`
`Alnylam
`Alnylam
`Alnylam
`Ionis
`Ionis
`Regulus
`Alnylam
`Alnylam
`
`Ionis
`Regulus
`
`siRNAa
`siRNAb
`siRNAb
`ASOc
`ASOc
`anti-miRd
`siRNAb
`siRNAb
`
`ASOc
`anti-miRd
`
`Initial siRNA compounds were therefore modified at only a few
`positions. Many different chemical configurations have been used
`to stabilize siRNAs, particularly combinations of 2(cid:96)-OMe, 2(cid:96)-F, and
`phosphorothioate72,79,80. Modifications that increase or decrease
`sugar flexibility have also been explored, including LNA and unlocked
`nucleic acid (UNA)81, but they are used mainly to introduce chemical
`asymmetry into duplex siRNAs. That is, they block passenger strand
`entry and promote RISC loading of the guide strand, which can also
`be easily achieved by 2(cid:96)-OMe modification of the two nucleotides at
`the 5(cid:96) end of the passenger strand73.
`The most common configurations include modification of ter-
`minal nucleotides82, of every second sugar with 2(cid:96)-OMe83, or of all
`pyrimidines. The popularity of the last stemmed from the high cost
`and low availability of 2(cid:96)-F-modified purines, which only recently
`became widely accessible. The guide strand must bind efficiently
`to the RNAi machinery and is therefore more sensitive to chemical
`modification. 2(cid:96)-F, which is the best mimic of the 2(cid:96)-OH group by size
`and charge, is generally well tolerated and has been used extensively
`as a primary guide strand modification84. Often, the guide strand is
`modified with 2(cid:96)-F and the sense strand with 2(cid:96)-OMe85.
`Modifications typically interfere with silencing activity by making
`the duplex too stable, which prevents removal of the passenger strand
`and interferes with proper loading of guide strand, or by forcing the
`nucleic acid into a suboptimal geometry86. The 2-F and 2-OMe modi-
`fications favor the C3(cid:96)-endo ribose conformation and support the
`A-form helical structure of the guide strand, which positions the
`target mRNA into the cleavage center of RISC87. But both modifi-
`cations introduce slight structural distortions. 2(cid:96)-F-RNA slightly
`overwinds the duplex (leading to more stacking and higher Tm),
`and 2(cid:96)-OMe-RNA slightly underwinds the duplex (less stacking).
`Either modification is tolerated in any individual position of an
`siRNA76, but a fully modified 2(cid:96)-OMe guide strand is completely
`inactive, and a fully modified 2(cid:96)-F guide strand often has substan-
`tially reduced activity78. When 2(cid:96)-OMe and 2(cid:96)-F modifications are
`alternated, however, the combination creates a compound ideally
`suited for RISC assembly and function75.
`Thermodynamic or structural tuning88 may further enhance the
`efficacy of modified siRNAs. Many of the advanced clinical com-
`pounds carry additional stretches of 2(cid:96)-OMe and 2(cid:96)-F (for example,
`three of either modification in a row, or sometimes longer stretches of
`2(cid:96)-OMe)89 in the context of the alternating 2(cid:96)-F–2(cid:96)-OMe-RNA pattern
`(Fig. 3). The pattern was designed to chemically mimic the sinusoidal
`thermodynamic stability described for highly functional siRNAs90.
`An ideal guide strand has a more flexible 5(cid:96) end, which can be easily
`introduced by structural and chemical modifications73; a high-affinity
`
`‘seed’ region, which drives the initial base pairing between the guide
`strand and target; and a lower-affinity 3(cid:96) region required for product
`release. This profile was initially derived by comparing active and
`inactive siRNAs90, but recent single-molecule RISC studies provide a
`clear mechanistic explanation91. Structures of fully modified siRNAs
`bound to the Argonaute protein Ago2 will also enable more precise
`tuning of modification patterns to optimize RISC binding and activ-
`ity92.
`Additional nuclease stability is conferred by backbone modi-
`fications14. Limited phosphorothioates are tolerated by Ago2, and
`phosphorothioate modifications at both ends of both strands of
`an siRNA duplex are incorporated into many of the leading clini-
`cal candidates. This simple combination of backbone and sugar
`modification provides additional resistance to exonucleases—the
`primary effectors of RNA degradation—and an order-of-magnitude
`increase in oligonucleotide accumulation in vivo. Methylation of
`the 5(cid:96)-carbon to give (S)-5(cid:96)-C-methyl-RNA93 has also been used to
`enhance 3(cid:96) exonuclease resistance.
`
`5(cid:96)-phosphate stabilization
`The 5(cid:96)-phosphate of a siRNA guide strand is essential for recognition
`by RISC94–96. siRNAs with a 5(cid:96)-hydroxyl are efficiently phosphor-
`ylated and loaded onto Ago2 inside cells97. Blocking phosphorylation
`of the 5(cid:96)-hydroxyl in siRNA prevents RISC loading and activity98.
`Chemical modification (e.g., 2(cid:96)-OMe or 2(cid:96)-F) of the 5(cid:96)-ribose of the
`guide strand can interfere with intracellular phosphorylation but the
`activity of these 5(cid:96)-modified guide strands can be restored if a 5(cid:96)-phosphate
`is introduced chemically75,99. Chemical phosphorylation does not
`significantly increase the cost or complexity of chemical synthesis,
`and most commercial sources of modified siRNAs add a 5(cid:96)-phosphate
`chemically. However, when dosed systemically, the 5(cid:96)-phosphate is
`quickly removed by phosphatases, resulting in an accumulation of
`biologically inactive siRNAs. Within 2 h after intravenous administra-
`tion, at least 90% of fully modified siRNAs are dephosphorylated, and
`within 24 h the phosphorylated guide strand is essentially undetect-
`able (R. Haraszti, L. Roux, and A. Khvorova, unpublished data).
`Phosphatase-resistant analogs of the 5(cid:96)-phosphate can improve
`in vivo efficacy100. Ionis modified the 5(cid:96) end of single-stranded siRNA
`(ss-siRNA) with E-vinyl phosphonate (5(cid:96)-E-VP), which substitutes the
`bridging oxygen with carbon in the context of a double bond101,102
`(Fig. 2). The 5(cid:96)-E-VP is in a suitable conformation for RISC binding,
`whereas the other stereoisomer (5(cid:96)-Z-VP) shows reduced activity due
`to inappropriate positioning of the phosphonate92,103. In this con-
`text, 5(cid:96) chemical stabilization was absolutely essential for the in vivo
`efficacy of ss-siRNAs101,102.
`
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