General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.

Users may download and print one copy of any publication from the public portal for the purpose of private study or research.

You may not further distribute the material or use it for any profit-making activity or commercial gain

You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from orbit.dtu.dk on: Jul 07, 2020

Locked nucleic acid: modality, diversity, and drug discovery

Hagedorn, Peter H.; Persson, Robert; Funder, Erik D.; Albæk, Nanna; Diemer, Sanna L.; Hansen, DennisJ.; Møller, Marianne R; Papargyri, Natalia; Christiansen, Helle; Hansen, Bo R.Total number of authors:13

Published in:Drug Discovery Today

Link to article, DOI:10.1016/j.drudis.2017.09.018

Publication date:2018

Document VersionPublisher's PDF, also known as Version of record

Link back to DTU Orbit

Citation (APA):Hagedorn, P. H., Persson, R., Funder, E. D., Albæk, N., Diemer, S. L., Hansen, D. J., Møller, M. R., Papargyri,N., Christiansen, H., Hansen, B. R., Hansen, H. F., Jensen, M. A., & Koch, T. (2018). Locked nucleic acid:modality, diversity, and drug discovery. Drug Discovery Today, 23(1), 101-114.https://doi.org/10.1016/j.drudis.2017.09.018

Review

s� K

EYNOTE

REV

IEW

Drug Discovery Today �Volume 23, Number 1 � January 2018 REVIEWS

Teaser LNA-modified antisense oligonucleotides (LNAs) are widely used in RNA therapeu-tics. The structural diversity of LNAs affects most drug properties. Exploiting this diversity

offers new opportunities for discovering LNA-based drugs.

Locked nucleic acid: modality,diversity, and drug discoveryPeter H. Hagedorn1, Robert Persson2, Erik D. Funder1,Nanna Albæk1, Sanna L. Diemer1, Dennis J. Hansen1,Marianne R. Møller1, Natalia Papargyri3,Helle Christiansen1, Bo R. Hansen1, Henrik F. Hansen1,Mads A. Jensen1 and Troels Koch1

1 Therapeutic Modalities, Roche Pharma Research and Early Development, Roche Innovation Center Copenhagen,2970 Hørsholm, Denmark2 Pharmaceutical Sciences, Roche Pharma Research and Early Development, Roche Innovation CenterCopenhagen, 2970 Hørsholm, Denmark3Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark

Over the past 20 years, the field of RNA-targeted therapeutics has

advanced based on discoveries of modified oligonucleotide chemistries,

and an ever-increasing understanding of how to apply cellular assays to

identify oligonucleotides with improved pharmacological properties

in vivo. Locked nucleic acid (LNA), which exhibits high binding affinity

and potency, is widely used for this purpose. Our understanding of RNA

biology has also expanded tremendously, resulting in new approaches to

engage RNA as a therapeutic target. Recent observations indicate that each

oligonucleotide is a unique entity, and small structural differences

between oligonucleotides can often lead to substantial differences in their

pharmacological properties. Here, we outline new principles for drug

discovery exploiting oligonucleotide diversity to identify rare molecules

with unique pharmacological properties.

IntroductionThe flow of genetic information is a highly complex process. Of the approximately 43 000

human genes currently annotated by the Ensembl project (release 89), approximately 47%

encodes proteins, and the rest are all transcribed into different types of noncoding RNA (ncRNA)

[1]. It is clear that these ncRNAs represent a long and growing list of different types of RNA with

various functional roles and regulatory interactions [2,3]. This knowledge has provided a

stronger basis for the specific annotation of the relationships between structure, type, and

function of RNAs and human disease [4]. This is one reason for the increased interest in RNA

Peter Hagedorn has aMSc in theoretical physicsand biophysics from theNiels Bohr Institute at theUniversity of Copenhagen,and a PhD in molecularbiology and bioinformaticsfrom Ris½ NationalLaboratory, Denmark. Forthe past 6 years, Peter has been exploring thestructural diversity of LNA-modified antisenseoligonucleotides and how this impacts measuredproperties, with a particular focus on developingpredictive mathematical models that capture thisdiversity.

Robert Persson has aPhD in microbiology andbiochemistry from theUniversity of Lund,Sweden. He has workedwith the intracellulartransport of secretoryproteins and viralmembrane proteins andwith the pharmacokinetics of proteins andoligonucleotides for more than 25 years. During thepast 8 years, Robert has focused on the bioanalysis,biodistribution, and pharmacokinetics of LNAoligonucleotides.

Troels Koch has a PhD inbioorganic chemistry fromthe University ofCopenhagen, Denmark. Hehas worked in the area ofnucleic acid chemistry andbiology for 20 years. He co-founded Santaris PharmaA/S, which pioneered thelocked nucleic acid (LNA) platform and LNAtherapeutics. Santaris was acquired by Roche inAugust 2014, and he is presently vice president andhead of research at the Roche Innovation Centre inCopenhagen. His main responsibilities are to developthe chemical and biological properties of LNA,improve and upgrade the LNA platform, refine LNAantisense drug discovery processes, and establish aRNA therapeutics drug pipeline in Roche.

Corresponding author: Koch, T. (troels.koch@roche.com)

1359-6446/ã 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).https://doi.org/10.1016/j.drudis.2017.09.018 www.drugdiscoverytoday.com 101

Reviews�K

EYNOTE

REV

IEW

REVIEWS Drug Discovery Today �Volume 23, Number 1 � January 2018

therapeutics, which focuses not only on classic protein-coding

mRNA intervention, but also on how the manipulation of non-

coding regulatory RNA can provide novel drugs for previously

untreatable diseases.

A rational strategy to target RNA is by synthetic oligonucleo-

tides. Although RNA exists in a complex biological matrix, en-

dogenous enzymatic RNA-processing mechanisms can be

exploited and recruited by the heteroduplex formation between

RNA and synthetic oligonucleotides. RNA exhibits high turnover

rates and is more labile than DNA [5]; thus, inhibitory effects

induced by the oligonucleotide binding complement can be

executed rapidly and effectively. Zamecnik and Stevenson [6]

were the first to show that a single-stranded synthetic DNA

oligonucleotide could inhibit the expression of RNA, resulting

in the stimulation of substantial research and development ac-

tivity [7–13].

The past four decades of research and development in oligo-

nucleotide-based RNA therapeutics has produced groundbreak-

ing results. The recent successes are based on improvements in

three essential parameters for antisense technologies: (i) medici-

nal chemistry efforts have devised much-improved nucleic acid

modifications for antisense oligonucleotides (AONs), of which

the high-affinity LNA [14–17] is among the most widely used; (ii)

the knowledge revolution of the functions and biological molec-

ular mechanisms of RNA; and (iii) the translational drug discov-

ery part (i.e., discovery processes and models used in drug

discovery). The latter relates to bioinformatics drug design, pre-

diction algorithms, in vitro/vivo assays, better pharmacokinetics/

pharmacodynamics (PK/PD) models, absorption, distribution,

metabolism, and excretion (ADME) models, and toxicology study

designs.

Recent developments have also provided insights into the fun-

damental properties of the chemical modality. Small chemical

modifications of AONs have been shown to produce large property

differences even among AONs belonging to the same chemical or

structural class. The old concept of the ‘portability of chemistry’

for AONs, whereby nearly all properties are shared for a given

chemical class, has turned out not to be true for many important

drug properties (e.g., activity and toxicity). In other words, we now

know that the structure–activity relationships (SARs) for AONs are

more subtle. The fact that AONs belonging to a given structural

class only on a few parameters share the same properties and act

more as individual unique compounds diversifies and complicates

drug discovery [13,18–20]. However, the fact that small chemical

modifications, even the configuration of a single bond, can have

profound property impacts creates a basis for SAR studies compa-

rable to classic small-molecule drug discovery. Therefore, during

the discovery phase, it is now possible to produce more unique

compounds for nearly any RNA target with larger therapeutic

indexes (TI).

In this review, we focus on three aspects of LNA: (i) state of the

art of the modality, including structure, fundamental properties,

preferred designs, mechanisms of action, and cellular uptake; (ii)

illustrate how subtle structural modifications have profound

impacts and, in some cases, produce ‘all or none’ phenotypic

effects; and (iii) how the insight or ‘Erkenntnis’ of property diver-

sity can be used to transform and improve the classic oligonucle-

otide drug discovery process and lead generation.

102 www.drugdiscoverytoday.com

Chemistry sets boundaries for successIn the first generation of single-stranded therapeutic oligonucleo-

tides, the phosphorothioate (PS) internucleoside linkage was the

only chemical modification [8,21–24]. The PS modification

replaces one of the nonbridging oxygen atoms with sulfur in

the internucleoside phosphate (Fig. 1ai). This modification creates

a chiral center at phosphorous producing two isomers: Rp and Sp.

Thus, for every PS linkage introduced in an oligonucleotide, two

diastereoisomers are formed. Given that conventional solid-phase

PS synthesis is not stereoselective, an n-mer PS oligonucleotide

contains random mixtures of 2n–1 diastereoisomers [25–30]. How-

ever, recent developments have now made it possible to synthesize

individual stereoisomers with high stereoselectivity [31–34], and

with good yields [35–37]. Diastereoisomers are different chemical

compounds and the diversity of properties for a given PS oligonu-

cleotide will be a gradient spanning from a little to a very large

difference in property. Generally, oligonucleotides with a Sp

configuration provided better exonuclease resistance compared

with oligonucleotides with a Rp configuration, whereas Rp iso-

mers were better substrates for DNA-dependent RNA poly-

merases, RNase H, and stimulated immune responses [38–40].

Compared with the random mixtures, Rp oligonucleotides exhib-

ited higher melting temperature (Tm) against RNA, whereas Sp

analogs had lower Tm. In the first-generation oligonucleotide

drugs, the PS modification was normally not stereocontrolled.

Except for rare examples [41], this class of drugs has not provided

an adequate TI for AONs [42]. Two reasons for this were that the

PS modification decreases the Tm of the hybrid duplex and,

although it protects against nuclease degradation to some extent,

this protection was still not adequate on its own for most thera-

peutic purposes [43]. Still, the PS modification, stereocontrolled

or not, turned out to be a central modification for later genera-

tions. Combined with the increased nuclease stability, PSs also

contribute to improved PK and cellular uptake properties of

AONs. Unless otherwise stipulated, all antisense data described

here come from fully PS-modified AONs.

A preferred strategy to improve the binding affinity of AONs to

RNA was to introduce electronegative substituents in the 20-posi-tion of the furanose (Fig. 1aii) [7,8,44–47]. For nucleic acid analogs

of this structural class, melting temperatures increased by approx-

imately 0.5–1.5 �C/modification against RNA. The most signifi-

cant members of this class are the 20-F, 20-O-CH3 and 20-O-

CH2CH2-O-CH3 (MOE) substituent groups [44,46]. This second

generation of drugs shows clear improvements compared with

first-generation PS drugs, and two drugs of this type, mipomersen

[48] and nusinersen [49], have now been approved by the US Food

and Drug Administration (FDA).

A high point for synthetic nucleic acid analogs was reached in

1997 with LNA [15,50] (Fig. 1aiii). LNA exhibited unprecedented

high RNA-binding affinity [51], providing melting temperature

increases of 3–9 �C/modification depending on the oligonucleo-

tide position and design complements [52]. The affinity increase

per LNA nucleoside substitution is a reproducible (‘portable’)

property [14,51–55] and, combined with the increased nuclease

stability, the driver behind the many therapeutic and diagnostic

life-science applications that have been demonstrated with, and

published on, LNAs [56–63]. Given these significant improve-

ments, LNA is now widely used and acknowledged by many to

Drug Discovery Today �Volume 23, Number 1 � January 2018 REVIEWS

5'-

5'-

- 3'

- 3'

LNA DNA PS

(a)

I II III IV V

Rp Sp

(b)Gapmer

Mixmer

(c)

AAAAA

Nucleus

Transcription

Post-transcriptionalmodification

Mature mRNA

5' cap 3' poly(A) tai l

AAAAA

AAAAA

AAAAA

AAAAA

AAAAA

AAAAA

Translation

Target protein

60S ribosomalsubunit

40S ribosomal subunit

4 Inhibition of 5' cap formation

5 Inhibition of RN A splicing

2 Activation of RNase H

1 Normal protein translation

2 Activation of RNase H 3 Steric hindrance ofribosomal subuni t binding

Hybridization

Formation of AON-mRN Aheteroduplex

40S

60S

RNase H

Transcriptionand cropping

miRNA gene

Mature miRNA

mRNA2

mRNA1

7 Derepression of proteintranslation

6 Repression of proteintranslation

DNA

pre-mRNA

Gapmer

Mixmer

Pre-miRNA

Mixmer

Export, AGO loading,and unwinding

AAAAA

AAAAA

AAAAAAAAAA

AAAAA

AGO

Cytoplasm

⇔

Drug Discovery Today

FIGURE 1

Chemistry of antisense oligonucleotides and mechanisms of action on RNA. (a) First-generation phosphorothioates (PS) (i), second-generation 20-O-substitutedribofuranoses (ii), third-generation locked nucleic acid (LNA) illustrated as non-stereodefined phosphorothioates (iii), the bicyclic structure of LNA (iv), and in (v)the two stereodefined diastereoisomers of the internucleoside phosphorothioate (Rp and Sp). (b) Gapmer design (top) in which consecutive LNA segments (darkorange) are flanking a central DNA segment (yellow), and mixmer design (bottom), where the nucleotides are ‘mixed’ throughout the compound. Nearly alldesigns contain PS internucleoside linkages (red line). The gray line below each nucleoside indicates the potential for forming hydrogen bonds withcomplementary nucleobases in other nucleosides via Watson–Crick base-pairing. (c) Mechanisms of action by which LNA oligonucleotides can intervene withand inhibit RNA function. Adapted from Ref. [141].

Review

s� K

EYNOTE

REV

IEW

belong to a third generation of chemical modifications used in

RNA therapeutics.

‘LNA’ refers to the bicyclic structure in which the flexibility of

the parent furanose has been locked [15] (Fig. 1aiv). The bicyclic

structure of LNA nucleosides is the key to the improved binding

properties, and has served as scaffold for many other bicyclic

analogs also exhibiting improved binding properties [64–68].

The 20-O-CH2-40 linkage is transformational and converts a flexible

furanose into a rigid bicycle [69–74]. One thermodynamic conse-

quence of the ‘locking’ compared with native deoxyribose is that

the free energy of solvation is 1–2 kcal/mol lower and, thus, more

favorable because of the lipophilic nature of the 20-O-CH2-40

linkage [75].

The difference between LNA and 20-OMe RNA, for example,

can be observed in the helical structures that they form. Recent

structural data indicate that LNA/RNA duplexes perturb the

A-type helix, whereas 20-OMe RNA/RNA attain a more classic

A-type structure [76]. LNA/LNA duplexes result in an extreme

‘underwinded’ helix in which the major grove is increased by a

dramatic decrease of the slide-and-twist values and a pitch value of

39 A compared with 29 A for RNA/RNA. There is a consecutive

order of structural change of the classical A-type helix from

RNA/RNA, RNA/LNA to LNA/LNA [76,77].

Transforming a flexible furanose moiety into a rigid bicycle

reduces the number of conformational degrees of freedom,

including rotations around internal bonds [75]. Structurally, this

www.drugdiscoverytoday.com 103

REVIEWS Drug Discovery Today �Volume 23, Number 1 � January 2018

Reviews�K

EYNOTE

REV

IEW

preorganizes the molecule and reduces the hybridization entropy

penalty. Although a net reduction in the entropy change for

hybridization (DDS) has been reported as a main driver for the

high affinity [78], the situation is more complex. Given that LNA

distorts the classic A-form helix, enthalpic contributions, such as

hydrophobic interactions and P–P-stacking between the bases,

are also increased [77].

LNA designs and RNA intervention mechanismsLNA designs can be divided in two main categories: mixmers and

gapmers (Fig. 1b). In a mixmer, LNA and DNA nucleosides (dark-

orange and yellow boxes, respectively in Fig. 1b) are interspersed

throughout the sequence of the oligonucleotide, whereas, in a

gapmer, two LNA segments at both ends of the oligonucleotide are

separated by a central segment or gap of DNA nucleosides. At

present, LNA designs are usually made with fully modified non-

stereodefined PS internucleoside linkages (red lines in Fig. 1b).

Unless otherwise specified, all data mentioned here are obtained

from such LNA PS random mixtures.

To inhibit mRNA expression and protein translation (Fig. 1c,

mechanism 1), gapmer designs are the most potent. This is because

the central DNA/PS segment, which is longer than 7–8 DNA

nucleotides (nt), recruits the RNA-cleaving enzyme RNase H when

the gapmer is hybridized to the mRNA [79–84]. Gapmers targeting

both exons and introns work equally efficiently and, thus, all

stages of mRNA and also ncRNA processing can be addressed.

Nuclease recruitment by gapmers occurs in both the cytoplasm

and the nucleus [85,86], although predominantly in the nucleus

[80] (Fig. 1c, mechanism 2). Mixmers can also be used to block

translation, but must be designed to bind close to, at, or upstream

of the translational start site to do so [83,84] (Fig. 1c, mechanism

3). The mixmer will inhibit translation by blocking the binding of

the ribosomal subunits to the mRNA. Alternatively, mixmers

binding close to the 50 end of the pre-mRNA can prevent 50-capformation and thereby inhibit translation (Fig. 1c, mechanism 4).

There is sometimes the need to intervene with RNA processing

and information flow without cleaving or degrading the target, or

inhibiting protein translation. For instance, in the case of splice

switching/redirection, or blocking natural antisense or other kinds

of ncRNA, mixmers are often the preferred option [87–93] (Fig. 1c,

mechanism 5). A mixmer cannot support RNA cleavage but acts as

an efficient steric block to mediate a phenotype without destroy-

ing the target RNA. This intervention changes the expression

profile, creating potentially both ‘loss-of-function’ and ‘gain-of

function’ phenotypes [91–93].

Inhibition, or sequestering, of miRNA is a gain of function that

has attracted a special interest [94–97]. miRNAs are an important

class of regulatory ncRNAs that can bind to partially complemen-

tary sites located in the 30 untranslated regions (UTRs) of target

mRNAs, promoting translational repression or deadenylation

and degradation (Fig. 1c, mechanism 6). Sequestering miRNAs

by LNA mixmer hybridization has wide therapeutic potential

[98] (Fig. 1c, mechanism 7). The miR-122-antagonizing LNA mix-

mer ‘miravirsen’ was progressed to Phase 2 clinical trials and

exhibited safe and potent HCV viral titer reductions [97]. The

high binding constant of LNA mixmers produces a slow off-rate,

which is the critical parameter for efficient miRNA antagonism.

Combined with the long tissue half-life of LNA, the slow off-rate

104 www.drugdiscoverytoday.com

produces a long half-life of the LNA/miRNA heteroduplex, leading

to long lasting derepression of the proteins that are under the

control of the miRNA.

Affinity, potency, and specificity of LNA oligonucleotidesIt is well documented that the high RNA-binding affinity of LNA is

linked to its potency [58,57,99]. The term ‘potency’ is used here for

the activity per administered dose and is not necessarily related to

the concentration in cells or in tissues. Although high target

affinity is a driver behind higher potency [14,18], many other

factors are also determinants, and higher affinity can be balanced

out by other potency-limiting parameters. Examples of such lim-

iting factors are: design, RNase H recruitment [100], tissue/cellular

uptake, and/or tissue distribution. For instance, it has been shown

that a fully LNA-modified 14-mer targeting the coding region of a

transcript did not inhibit protein synthesis [84,101], because the

ribosome ‘read through’ the binding site of the LNA, despite the

high RNA affinity. When some of the central LNA nt were substi-

tuted with DNA-nt so that the 14-mer became a LNA gapmer, the

gapmer recruited RNase H and mediated cleavage and degradation

of the message, despite the lower affinity [84].

It has also been shown that affinity increase by length

expansion of gapmers only improved the potency to a certain

compound-specific point/threshold, after which the potency de-

creased as the length, and affinity, increased [99]. This ‘threshold

affinity’ was studied further by Pedersen et al. using a kinetic model

to simulate LNA gapmer recruitment by RNase H [100]. The model

showed very fast LNA–RNA hybridization on-rate and recruitment

of RNase H. IC50 curves were generated as a function of LNA

concentrations and, when these were plotted as a function of

LNA–RNA affinity, a parabola emerged. This illustrated in affinity

terms that potency increased as the free energy of hybridization

decreased to approximately �DG� = 20–25 kcal/mol, but below

this point, and for compounds with even higher affinity, the effect

reversed and potency decreased. The existence of such ‘optimal’

affinity was explained to be a tradeoff of initial efficient target

binding coupled with inefficient deassociation of the cleaved

LNA–RNA duplex. Nonspecific sequestration of LNAs through

protein binding might also be length dependent because of

the negative charge contributed by each PS linkage, and might

also contribute to the observed length-dependent potency of LNAs

[99].

It is a cornerstone in RNA-based drug discovery to ensure that

oligonucleotides interact specifically with their intended RNA

targets. For LNA gapmers that bind with high affinity to fully

complementary RNA target regions, extra care must be taken to

ensure that they do not bind efficiently to partially mismatched,

unintended RNA targets [18]. Although the mismatched base pairs

in the unintended RNAs reduce the binding affinity compared

with the fully matched target, it has been reported that, in some

instances, affinity of LNAs can be designed so high that duplexes

between mismatched unintended RNAs and gapmers persist long

enough, and in sufficient amounts, to allow effective RNase H-

cleavage [18,102–104]. However, character- and thermodynamics-

based bioinformatics algorithms allow for computational gapmer

sequence analysis where the likelihood of binding to mismatched

unintended RNA can be predicted. Thus, bioinformatics enable

the selection of sequence-specific oligonucleotides designed to

Drug Discovery Today �Volume 23, Number 1 � January 2018 REVIEWS

Review

s� K

EYNOTE

REV

IEW

have either no or only a few mismatched hybridizations. The

sequence specificity of gapmers can also be evaluated experimen-

tally using transcriptome-wide profiling approaches, such as

microarrays or RNA-seq [18]. Taken together, these computational

and experimental methods are important tools to identify com-

pounds with high sequence specificity and low off-target potential

[105].

Cellular uptakeEntry of oligonucleotides into cells is a complex process and a

comprehensive report is beyond the scope of this review. Here, we

focus on central elements that are relevant for LNAs, but the

interested reader may consult recent reviews for more information

[106–109].

When oligonucleotides are transfected with cationic lipids, the

lipids fuse with the plasma membrane and deliver oligonucleo-

tides directly into the cytoplasm. However, the oligonucleotides

concentrate quickly in the nucleus, which is advantageous for

antisense because RNase H is predominantly found in the nucleus

[80]. When oligonucleotides are incubated without added trans-

fection agents, ‘naked’ uptake also occurs [106,110–112]. It was

illustrated that ‘naked’ LNAs were taken up and exhibited potent

activity in cell cultures [113]. This process was named ‘Gymnosis’

after the Greek ‘gymnos’ or ‘naked’, and activity was found for

nearly all cell types, including ‘hard-to-transfect’ cells, such as

suspension cell lines and T cells. Gymnotic silencing provides

more AON-specific phenotypes. Given that transfection/delivery

lipids are not used in gymnotic assays, a cellular response is only

related to, or is a consequence of, the added AON [113–115].

Cellular uptake of naked oligonucleotides and LNA is predomi-

nantly driven by endocytosis. Many different pathways and cellu-

lar proteins are engaged in the uptake, such as clathrin, dynamin,

and caveolar-dependent endocytosis [116,117]. However, cla-

thrin-independent endocytosis also exists [118], and it has been

shown that antisense activity in hepatocytes can be blocked by

adaptor protein AP2M1 but not by clathrin and caveolin [119].

It was recently shown that LNA gapmers were taken up in

gymnotic assays in human primary T cells via macropinocytosis

(MP) [120]. MP is a fluid-phase endocytosis pathway driven by

actin–myosin cell protrusions creating larger vesicles. The fate of

macropinosomes differs from that of other endosomes and they

are directed toward the late-stage endosomes/lysosomes. Once

taken up, LNAs were released from MPs/late endosomes and

showed potent and specific RNA target knockdown.

The mechanisms by which oligonucleotides are released from

endosomes/macropinosomes are still unclear, but maturation of

the endosomes from early to late endosomes (multivesicular bod-

ies) is important [85]. It was also shown the LNAs co-localize with

various cellular bodies (GW and P-bodies) and that proteins, such

as Ago2, TPC-, Hsc-70, and PKC-a proteins, which bind to endo-

somes, are involved in the endosomal maturation process and

probably also in their release [121]. Once oligonucleotides have

escaped from the endosomes, activity occurs in the cytoplasm [85]

or, after rapid trafficking, also in the nucleus [122,123].

One of the most successful uptake enhancers is the triantenn-

ary N-acetylgalactosamine (GalNAc) ligand, which targets the

high-density asialoglycoprotein receptor (ASGR) expressed on

hepatocytes [108,124]. When oligonucleotides are conjugated

to GalNAc, the high turnover of ASGR will internalize recep-

tor-GalNAc-oligonucleotide efficiently via clathrin receptor-me-

diated endocytosis. GalNAc-conjugated oligonucleotides

typically provide 10–20-fold reductions in the dose where half

maximum responses were seen, and have revolutionized hepatic

RNA targeting [108].

It is possible that the major uptake observed from the endoso-

mal/MP pathways operates together with other uptake mecha-

nisms. In this way, high uptake/low productivity pathways

(endocytosis) might operate in parallel with low uptake/high

productivity pathways. For example, it has been shown that

nucleic acid channels exist in rat kidney and brain cells [125],

although it has not been demonstrated that this is a general uptake

mechanism. Cellular uptake is a highly diverse and differentiated

process that is dependent on many parameters, such as cell type,

proliferation/cell cycle state, extracellular composition (both in

vitro and in vivo), oligonucleotide design/substitutions pattern,

concentration, and phosphorothioate configuration (see below).

Diversity: an inherent attribute for oligonucleotidesStructural diversityIt is normal practice in the field of small-molecule drug discovery

to produce collections or libraries of chemical entities. It is gener-

ally appreciated that library size is not a goal in itself, but rather the

generation of structural, and thereby functional, diversity, and

property refinement [126]. For oligonucleotides, structural differ-

ences also define functional diversities, but because of the partic-

ular compound class, the diversity framework is derived from

different property determinants. The diversity framework can be

divided in five components or determinants for structural diversi-

ty: (i) nucleobase or sequence diversity: the variation in nucleo-

base composition that constitutes oligonucleotide sequence; (ii)

nucleoside diversity: the variation in chemical structure of the

nucleoside building blocks used to produce a given oligonucleo-

tide; (iii) backbone diversity: the variation in backbone internu-

cleoside linkage structure, including variations in the chiral

orientation of any phosphorothioate linkages; (iv) architectural

diversity: the variation in design and combinatorial nucleotide

make-up of an oligonucleotide; and (v) macromolecular structure

diversity: the variation in structure and topology of the entire

molecule.

The first four diversity determinants are classic oligonucleotide

parameters. They represent the ‘digital’ Watson–Crick hybridiza-

tion properties. The antisense literature has covered these in great

detail over the past 30 years, but it was only recently appreciated

that the nucleobase sequence is not the only determinant, but that

the global properties are derived from the position of each struc-

tural unit in the context of the entire molecule. The term

‘sequence-related properties’ has been used frequently and some

properties can be related to a nucleobase sequence motif alone.

However, this description is at best inadequate, and can be

meaningless without reference to the specific composition of

the molecule.

Macromolecular structure diversity is related to the variation in

overall structure and topology of the oligonucleotide. This param-

eter relates more to the ‘analog’ or aptameric binding properties.

When the first four determinants are fixed, a resulting single

molecule will be able to attain many different shapes and overall

www.drugdiscoverytoday.com 105

REVIEWS Drug Discovery Today �Volume 23, Number 1 � January 2018

Gapmers with same nucleobase sequence but different LNA patte rns

HIF

1A m

RN

A (

% c

ontr

ol)

0

20

40

60

80

100

Drug Discovery Today

FIGURE 2

Architectural diversity of locked nucleic acid (LNA) gapmers affects targetknockdown in vitro. LNA gapmers with the same nucleobase sequence butdifferent LNA design patterns were screened in a gymnotic HeLa cell assay at5 mM, and knockdown of target mRNA was measured after 3 days by qRT-PCR. The gapmers were targeted to the same 13-nucleotide (nt) region onthe human HIF1A mRNA. In all, 32 design patterns were evaluated, written asLxxxDDDDDDxxL (L: LNA, D: DNA, x: either LNA or DNA). All values are shownas averages across four independent biological replicates. Error bars indicatethe SD.

Reviews�K

EYNOTE

REV

IEW

structures each representing different energy levels. This diversity

parameter can be illustrated by modeling, and quantum mechani-

cal (QM) calculations have demonstrated the 3D relations in

molecular structure, topology, and electrostatics of LNA PS oligo-

nucleotides [127,128]. Among all possible macromolecular struc-

tures, QM or molecular dynamics calculations will be able to

identify a specific minimized structure that is most representative

for the molecule [75,127]. Below, we first describe functional

diversity for the classic PS random mixtures, and then diversities

for stereo-defined PS LNAs.

Functional diversity in vitroHuman pre-mRNA sequences are typically between 10 000 and 65

000 nt in length with a median length of approximately 25 000 nt

[1]. Therefore, tens of thousands of possible sites on a pre-mRNA

can be targeted by gapmers. Libraries of gapmers that are posi-

tioned at unique and nonoverlapping sites on a RNA target mani-

fest diversity with respect to their nucleobase composition. It is

widely recognized that gapmer potency is affected by the target

nucleobase sequence. This is usually attributed to the structure of

the RNA in the target region, where less-structured RNA leads to

more-potent gapmer responses [129]. However, other factors that

affect potency can also be attributed to the nucleobase sequence,

such as sequence-specific intracellular localization [109].

Nucleobase diversity was illustrated in a study of 57 and 71 fully

phosporothioated gapmers (20mers, mixtures of 219 diastereoi-

somers). The gapmers were designed with five 20-O-methoxyethyl

(MOE) modifications in each flank (5–10–5 design). A range of

activities was seen when screened in mouse primary hepatocytes

and human T-24 cells [130]. Some gapmers were not active at all

and showed no knockdown of the target. Most compounds

showed some activity and a few in both libraries were judged to

be potent.

In another study, more than 200-fold differences in potency in

vitro were reported for a library of 47 LNA-phosphorothioate

gapmers targeted to various regions of the HCV RNA genome

[131]. Most of these nucleobase sequence-diverse gapmers were

16mers with four LNAs in each flank (a 4–8–4 design, mixtures of

215 diastereoisomers). Interestingly, when screening for general

cytotoxic effects in a cell proliferation assay, the gapmers were

observed to also demonstrate more than 200-fold differences in

the concentrations at which half-maximal cytotoxicity was ob-

served. These properties were not closely correlated, and the

authors were able to select seven gapmers that were both well

tolerated and highly potent in vitro. Out of the seven ‘hits’, the

gapmer with the highest potency reduced antiviral activity to

<5%. This target region was explored in detail by designing 21

additional LNA gapmers with a 4–8–4 design closely tiled across

this region. Upon screening of this library, a range of activities was

again observed. Some compounds did not affect HCV activity at

all, but three gapmers, consecutively spaced only 1 nt apart, were

even more potent than the original ‘lead’. The authors also briefly

explored architectural diversity by changing the LNA-modifica-

tion pattern of the gapmer with highest potency and designed an

additional 47 gapmers. They observed that 4–8–4 and 3–10–3

designs had similar potency, whereas 5–6–5 and 2–12–2 designs

were three and five times less potent, respectively. Notably, al-

though the 4–8–4 and 3–10–3 designs were of similar potency in

106 www.drugdiscoverytoday.com

vitro, the 3–10–3 design had a twofold longer half-life in kidney

after intravenous (i.v.) administration to mice [131].

An evaluation of both architectural and nucleoside diversity was

reported by Stanton et al. [132]. An initial screening campaign

identified three potent PS LNA-gapmers targeted to the Glucocor-

ticoid Receptor mRNA. Each of the three gapmers was of the 3–8–3

design but with completely different nucleobase sequences. The

authors designed a library of 109 gapmers where they varied

lengths between 14 and 20 nt, and modified flanks with various

numbers of LNA, MOE, 20-OMe, and 20F nt. Some gapmers

contained both LNA and 20-OMe, or LNA and 20-F in the flanks.

In vitro and in vivo potency were observed to be highly sensitive

to small changes in length, nucleoside composition, and number

of modifications in the flanks. Thus, the study demonstrated

an apparently unpredictable and wide property diversity of

oligonucleotides.

Another example of how architectural diversity can impact

potency in vitro is shown in Fig. 2. Here, 32 iso-sequential LNA-

gapmers targeting HIF1A, were screened for target knockdown at

5 mM in HeLa cells. The LNA design for these gapmers was

LxxxDDDDDDxxL, where L stands for LNA, D for DNA, and x

for either LNA or DNA, giving 32 different combinations. Mea-

sured knockdowns ranged from no effect to 80%, achieved by the

compound with the design LLLDDDDDDDDDL. The predicted Tm

[100] of the designs ranged from 40 �C to 60 �C, and did not

correlate with potency.

Functional diversity in vivoAdministered oligonucleotides will interact differentially at each

of the steps they encounter on their way to the site of action. These

steps include absorption at the site of administration and plasma

protein binding in the circulation; the uptake in different tissues

and protein binding at the cell surface; the uptake into cells; and

their intracellular transport to the site of action. Under in vivo

conditions, the five diversity parameters described above will also

collectively govern the properties of the specific molecule. Func-

tional diversity is very high for some properties and smaller for

others.

Drug Discovery Today �Volume 23, Number 1 � January 2018 REVIEWS

Review

s� K

EYNOTE

REV

IEW

Until recently, the least diversity had been observed in the

systemic exposure to LNAs after subcutaneous administration.

In circulation, LNAs are bound to plasma proteins to a high degree,

primarily serum albumin, although the exposure is species depen-

dent. Mice need doses ten times higher than humans per kg to

reach the same exposure in plasma [133]. Such comparisons have

been discussed for 20-O-(2-methoxyethyl)-modified oligonucleo-

tides [133] and are important for our understanding of species-

specific effects. At therapeutically relevant plasma concentrations

(up to 10 mg/ml), 95–99% of LNAs are bound to plasma proteins in

the rat. In monkey and humans, a lower fraction is bound to

plasma proteins (90–95%) and, in the mouse, even a lower frac-

tions [134]. The free (unbound) fraction of oligonucleotide is

secreted via the urine. Within the first 24 h post dose, the plasma

concentration is reduced by two to three orders of magnitude. This

reduction results from the rapid redistribution of the oligonucleo-

tide to different tissues, predominantly liver and kidney, followed

by skin, bone marrow, spleen, uterus, lymph node, and lung

[56,101].

Using a dosing schedule of 15 mg/kg on days 0, 3, 7, 10, and 14

of nine LNA oligonucleotides in mice (dosed, i.v.), a different

uptake and accumulation was revealed in the liver (green bars in

Fig. 3). The 16mer gapmer (3–10–3) oligonucleotides with differ-

ent nucleobase sequences were designed against the same RNA

target. At sacrifice on day 16, the liver concentration of some of

these oligonucleotides was as low as 15 mg/g liver tissue, whereas

others showed values of almost 80 mg/g liver tissue. These results

are by no means unique. Other studies showed differences in the

liver uptake of up to 25-fold. Uptake of LNA oligonucleotides in

kidney and other tissues show similar diversities. A kidney:liver

disposition ratio spanning over more than two orders of magni-

tude has been observed in mice (H.F. Hansen, pers. commun.).

Saline GA C D EB F H I0

50

100

0

700

1400

2100

2800

Oligonucleotide

Olig

onuc

leot

ide

conc

entr

atio

nor

mR

NA

leve

l

ALT

concentration25

75

Drug Discovery Today

FIGURE 3

Nucleobase diversity affects uptake, target knockdown, and hepatotoxicpotential in vivo. Nine LNA gapmers (labeled A–I) of the design 3–10–3, withdifferent nucleobase sequences but all targeted to the same target RNA,were administered intravenously at 15 mg/kg on days 0, 3, 7, 10, and 14 inmice (n = 5 mice per treatment group). On day 16, the livers were isolatedand homogenized, and the oligonucleotide concentration (green bars, mg/gtissue) and target mRNA levels (blue bars, % saline control) were determinedby hybridization-ELISA and qPCR, respectively. The alanine aminotransferase(ALT) level (red bars, IU/L) was measured in corresponding serum samples byELISA. For comparison, mRNA and ALT levels for mice in the studyadministered with saline are shown to the left. All values are shown as groupaverages. Error bars indicate the SD.

Taken together, tissue uptake of LNAs shows a high degree of

diversity. The question is whether the differences in tissue uptake

among a series of compounds are correlated with target activity

and adverse effect of the oligonucleotides. The short answer is no:

there is no direct correlation between the tissue uptake across a

panel of different compounds and activity. In the study illustrated

in Fig. 3, the target knockdown (blue bars) and tissue uptake (green

bars) varied fivefold. Again, there was no direct correlation be-

tween the liver uptake and target knockdown across the com-

pounds. Thus, an oligonucleotide with a very low liver uptake

could be very active.

The higher affinity of LNAs and LNA analogs has been reported

to result in hepatotoxicity [135]. By contrast, it has also been

reported that hepatotoxicity was significantly reduced by using

shorter version of the LNAs (from 20mer to 14mer) [135]. One

should be careful to calculate any property correlations in relation

to a single determinant, such as length. In other words, does a

property difference result from a length change or because differ-

ent compounds are being compared? For instance, it was shown

that a 13mer LNA was toxic, but, by adding just one LNA nt, the

corresponding 14mer was nontoxic [19]. The specific composition

and the nucleotide positioning (including LNA nt) is also a deter-

minant because LNAs with the same nucleobase sequence and

with different positions of the LNA in the flanks can have different

hepatotoxic profiles [19,136]. These studies indicate that some

sequence motifs influence the hepatotoxic potential. Recent

experiments in mice suggest that the number of unintended

RNA targets that are effectively reduced in the liver after systemic

administration of high-affinity gapmers, such as LNA gapmers, are

also correlated with the hepatotoxic potential of the gapmers [96–

98]. High-affinity gapmers can also affect unintended partially

mismatched RNA targets [137]. These results are consistent with a

model relating increased unintended targeting with increased risk

of some of these targets being involved in critical cellular functions

leading to hepatotoxicity. Alternatively, it could be that the

concentration of cleaved RNA in the hepatocytes can reach a level

that is in itself toxic to the cells. That hepatotoxicity can be a

consequence of off-target effects also helps explain why structural

diversity affects the hepatotoxic potential. As discussed above,

structural diversity clearly affects activity on the intended target

and, therefore, it is reasonable to expect that structural diversity

also can affect activity on off-targets.

Using the dosing schedule described above, five administrations

over 15 days (every third to fourth day) of 15 mg/kg, and measur-

ing alanine aminotransferase (ALT) in plasma/serum as a measure

for hepatotoxicity, high hepatotoxicity was found for both oligo-

nucleotides taken up at high and very low tissue concentrations

(red bars in Fig. 3). Likewise, there are oligonucleotides that are

taken up in high concentrations without being toxic. Taken

together, these results suggest that the liver uptake for different

oligonucleotides does not necessarily relate to activity or toxicity

even for oligonucleotides that are seen as ‘similar’ (i.e., with same

length, design, and chemistry).

It has been reported that some LNAs are toxic [19,102–

104,131,132,135]. The toxicity cannot be caused by the LNA

nucleosides per se, because there are also many reports of LNAs

that are not toxic [19,97,99,102,103,131]. It has also been clearly

demonstrated that toxicity can be mitigated by either increasing or

www.drugdiscoverytoday.com 107

REVIEWS Drug Discovery Today �Volume 23, Number 1 � January 2018

Diastereoisomeric gapmers with dif ferent backbone configu rations

HIF

1A m

RN

A (

% c

ontr

ol)

020406080

100120

HIF1A mRNA (% control)

Num

ber

of d

iast

ereo

isom

ers

0 40 80 120

0

10

20

30

40

50

(a) (b)

Drug Discovery Today

FIGURE 4

Backbone diversity of diastereoisomers affects target knockdown in vitro. (a) Selected stereoisomers (n = 236) of a 13mer human HIF1A targeting LNA gapmer,screened in HeLa cells at 5 mM by gymnosis. The phosphorothioates (PS) configuration (Rp/Sp) for at least one phosphate is varied in each stereoisomer. After 3days, knockdown of the target mRNA was measured by qPCR (and compared with a saline control). (b) The measured target knockdowns of the diastereoisomersapproximately follow a normal distribution (P = 0.32 by the Shapiro–Wilk test of normality).

0

20

40

60

80

100

Oligon ucleotide

Olig

onuc

leot

ide

conc

entr

atio

nor

mR

NA

leve

l

Sal

ine

RT

R38

33

Ste

reo

A

Ste

reo

B

Ste

reo

C

Ste

reo

D

Ste

reo

E

Ste

reo

F

0

100

200

300

400

ALT

concentration

500

120

140

600

700

Drug Discovery Today

FIGURE 5

Backbone diversity of diastereoisomers affects target knockdown in vivo. Arandom-mixture locked nucleic acid (LNA) gapmer, RTR3833, targeted to themouse APOB mRNA, as well as six different fully stereodefined gapmers withthe same nucleobase sequence as RTR3833, labeled Stereo A–F, wereadministered at 1 mg/kg in mice (n = 5 mice per treatment group) on day 0.On day 3, the livers were isolated and homogenized, and the oligonucleotideconcentration (green bars, mg/g tissue) and target mRNA levels (blue bars, %saline control) were determined by hybridization-ELISA and qPCR,respectively. The alanine aminotransferase (ALT) level (red bars, IU/L) wasmeasured in corresponding serum samples by ELISA. All values are shown asgroup averages. Error bars indicate the SD.

Reviews�K

EYNOTE

REV

IEW

decreasing the number of LNA nucleotides or, for iso-sequential

AONs, by changing the nucleoside composition (architectural

diversity), or, as observed in Fig. 3, by changing the nucleobase

sequence (nucleobase diversity). Irrespective of the underlying

mechanism affected by these structural changes, it is apparent

that diversity can be exploited to mitigate toxicity as well as

optimizing other properties.

Stereodefined LNA oligonucleotidesNearly all AONs are synthesized without stereodefinition of the PS

internucleoside linkage. Thus, for the LNA PSs described above,

each ‘compound’ exists as a random mixture of diastereoisomers.

Fundamentally, diastereoisomers are not identical and do have

different properties. Moving away from the random mixtures and

making single stereodefined PS AONs offers a new dimension to

RNA therapeutics [32]. One aspect of this is that macromolecular

diversity can now be studied, such as by computer modeling (see

above). An interesting outcome of this was QM calculations illus-

trating that changing a single PS bond configuration can produce a

macromolecular structure diversity to the same extent as, for

example, nucleobase or nucleotide changes [127,128].

In an in vitro experiment, 236 stereodefined LNAs, randomly

selected from 4096 possible isomers of a 13mer targeting human

HIF1A, were tested. A difference in knockdown ranging from 0% to

77% was observed (Fig. 4a). The distribution of HIF1A mRNA levels

after treatment with gapmer is represented in a histogram in

Fig. 4b (gray bars). The solid line in Fig. 4b represents a normal

distribution with mean and standard deviation estimated as the

median (70%) and the median absolute deviation (22%) of the

knockdown data. From this, it can be seen that the distribution of

the knockdown data as represented in the histogram is well

approximated by the normal distribution (Fig. 4b). The original

random-mixture LNA gapmer reduced HIF1A mRNA to approxi-

mately 50%. If it can be assumed that the extent of the knockdown

achieved by each of the 4096 isomers follows a normal distribu-

tion, as suggested from the 236 isomers measured and represented

in Fig. 4b, we can infer that approximately 20% of all 4096 isomers

will reduce HIF1A mRNA to a larger extent than the original

random-mixture LNA gapmer.

108 www.drugdiscoverytoday.com

In another experiment, six 13mer stereodefined ApoB-targeting

LNAs were tested in mice. The LNAs were dosed i.v. at 1.0 mg/kg

(Fig. 5). The stereodefined LNAs (A–F) downregulated ApoB

mRNA from 87% to 20%, with the random-mixture LNA,

RTR3833, reducing mRNA to approximately 50% (blue bars in

Fig. 5). The uptake in liver spanned one order of magnitude (1.34–

0.135 mg/g, green bars in Fig. 5). It was interesting to note that

high uptake did not relate to the highest activity for the six

stereodefined compounds (Pearson’s correlation r = 0.35 and

P = 0.44; Fig. 5). Only small differences in ALT concentrations

Drug Discovery Today �Volume 23, Number 1 � January 2018 REVIEWS

Review

s� K

EYNOTE

REV

IEW

were seen for the stereodefined LNAs (A–F) and none of them

were above twofold of the ALT concentration observed in saline-

treated controls (red bars in Fig. 5). Consequently, none of the

stereodefined LNAs displayed markedly increased hepatotoxicity.

Thus, in line with the QM observations, in vitro knockdown and

in vivo uptake and knockdown are highly diverse among diaster-

eoisomers.

The observation that individual stereoisomers can have sub-

stantially improved pharmacological properties compared with a

random mixture was also recently reported for the 5-10-5 MOE-

gapmer mipomersen [33]. Mipomersen comprises 524 288 stereo-

isomers and targets APOB RNA. The authors demonstrated for a set

of six different fully stereodefined versions of mipomersen that

three were more stable than the random mixture in both rat liver

homogenate and in rat serum, and two were cleaved faster by

RNase H in vitro [33]. The stereoisomer that was both more stable

and cleaved significantly faster was also tested in mice, and

showed significantly longer-lasting lowering of serum ApoB pro-

tein levels compared with the random mixture when dosed intra-

peritoneally eight times at 10 mg/kg [33]. Interestingly, this

stereoisomer had a triplet stereochemical motif, SSR, within the

gap, which the authors argued could be particularly suited to

promote target RNA cleavage by RNase H. The authors tested

the code in stereodefined gapmers targeted to FOXO1 RNA and

APOC3 RNA, and demonstrated an improvement compared with

the random mixtures. However, this finding is in contrast to an

earlier study, where, among a set of 31 stereoisomers, no clear

differences in in vitro potency was observed between the stereo-

isomers containing the SSR code and those that did not [34]. More-

comprehensive analyses with larger sets of stereoisomers are need-

ed to establish whether such a preferred triplet code is a general

phenomenon.

The diversity data observed for stereodefined AONs also point

the way to a novel understanding of how AON chemistry affects

function. In charged AONs, the PS linkage is required for optimal

antisense activity [23]. This is also true for AONs in which the PS

linkage is not required for making them nuclease resistant. Mech-

anistically, the importance of the PS linkage is often assigned to

the heavy atom effect of sulfur, which alters the sphere of hydra-

tion surrounding the molecule relative to the PO linkage. This,

plus the increased polarizability of the PS linkage and its ability to

form salt bridges, alters protein binding in addition to increasing

AON lipophilicity. However, because all possible combinations of

Rp and Sp diastereomers are present in the classic nonstereode-

fined AON random mixtures (see above), the effects of each

individual Rp and Sp are so diminutive that the PSs collectively

appears ‘pseudosymmetric’, essentially neutralizing the chiral

contributions of a PS linkage. However, the nature of a classic

random mixture, essentially a library of diastereoisomers, might in

itself have a positive role. Given that random mixtures exhibit all

possible isomers, including isomers with extreme and/or desired

properties, such as high potency, they might outbalance less

active, or inactive, isomers [34,138]. It is possible that such a

‘library effect’ and the heavy atom effect work together to promote

AON PS activity. By contrast, advantages with AON technology

might occur when both the heavy atom effects of sulfur combined

with pure phosphorus chirality are used (see below). Naturally, this

can only occur for stereodefined PSs.

Diversity offers new opportunities for RNAtherapeuticsThe standard, target-centric, model of RNA drug discovery begins

with the identification of a target known to be causally involved in

a particular disease (Fig. 6a). From the sequence of the RNA target,

libraries of complementary AONs can then be designed and syn-

thesized. The functional properties of the synthesized AONs are

evaluated in a screening cascade of cell-based assays and animal

models, to identify active and well-tolerated compounds (Fig. 6b).

In this approach, medicinal chemists are confronted with a

large chemical space to explore. For gapmers designed to knock

down the target RNA, as well as mixmers inhibiting miRNAs or

modulating splice switching on pre-mRNA, the number of chemi-

cally feasible compounds against any target is on the order of 1020

(Box 1). Despite great advances in high-throughput synthesis and

screening technology, such a large space prohibits exhaustive

evaluation.

The current target-centric discovery approach relies on the

principle of ‘evaluation in-parallel and local optimization’. That

is, libraries of AONs that predominantly explore one parameter (

for example potency) are designed, synthesized, and screened in

parallel, and, from these, sets of AON hits are selected that are

optimal with respect to that parameter. Given that synthesis and

screening of AONs takes on the order of weeks for the in vitro part of

the screening cascade, and on the order of months for the in vivo

part, parallel evaluation of libraries of AONs is necessary to keep

the timespan of the discovery project on the order of 1–2 years.

Using the analogy of chemical space, the map guiding the search

for active and tolerated AONs is as such constructed ‘along the

way’, and the path forward determined only by what is known in

the local regions explored up until that point. This approach does

not necessarily lead to the identification of the globally most-

active and best-tolerated AON, but historically have nevertheless

performed well and produced numerous drug candidates that have

entered clinical testing in human trials (see above).

To make these concepts more concrete, we illustrate in Fig. 6c,d

a recent drug discovery project in which local regions of chemical

space were explored with respect to nucleobase as well as archi-

tectural diversity (Fig. 6c). The gapmers from the first scanning

library (green dots in Fig. 6c,d) were screened for activity on the

intended target as well as cellular toxicity. As seen in Fig. 6d, some

were active and well tolerated, but many were not. The scanning

library primarily explores the nucleobase determinant, a classic

‘gene-walk’ (Fig. 6c). Next, the nucleobase sequences for a handful

of interesting compounds were identified and used as a basis for

designing optimization libraries exploring nucleoside and/or ar-

chitectural diversity (blue dots in Fig. 6c,d). As seen in Fig. 6d, the

optimization libraries identified a higher fraction of leads that

were potent and well tolerated compared with the hits in first

scanning libraries (upper right quadrant in Fig. 6d).

Furthermore, having identified leads where both nucleobase

sequence and architectural design had been locally optimized,

additional improvements in functional properties might be possi-

ble by synthesizing new libraries of fully stereodefined versions of

these gapmers (Fig. 6a). One such example was presented above for

fully stereodefined gapmers targeting HIF1A mRNA (Fig. 4). The

high property diversity observed for PS diastereoisomers offers a

new macromolecular diversity parameter for AON optimization.

www.drugdiscoverytoday.com 109

REVIEWS Drug Discovery Today �Volume 23, Number 1 � January 2018

Nucleobase diversity(start position on RNA)

Arc

hite

ctur

al d

iver

sity

(ran

ked

by p

reva

lenc

e)

(d)

(b)(a)

Diseaseknowledge

RNA targetidentification

Scanning library (n = ~1000)spread out across target

using a few standard designsto identify hits

Optimization libraries (n = ~500)tiled closely in a narrow regionusing many different designs

to identify leads/candidate drugs

Optimization libraries ( n = ~500)of fully stereodefined gapmers

to identify candidate drugs

Cellular activity assaysevaluating target RNA and protein

after treatment with gapmer

Cellular toxicity assaysfor relevant tissues where the

gapmers will accumulate

Animal modelsto evaluate safety and PK/PD

Clinical development in humans

(c)

ScanningOptimization

HighLow or no

Cellular activity assay

Cel

lula

r to

xici

ty a

ssay

Hig

hLo

w o

r no

1 140 000

Drug Discovery Today

FIGURE 6

RNA drug discovery and diversity. (a) The standard, target-centric, model of RNA drug discovery is a linear process in which new RNA targets are identifiedthrough knowledge of a particular disease, and libraries of gapmers complementary to the RNA target are synthesized. The hits from one library are used as abasis for designing gapmers in the next library. (b) Simplified screening cascade starting with high-throughput screening in cellular assays and ending with morelow-throughput evaluation in animal models. (c) An example where a region of chemical space was explored using 3661 LNA gapmers based on the principle of‘evaluation in-parallel and local optimization’. Coordinates for nucleobase diversity were approximated by the start position on the RNA target (1706 uniquepositions evaluated). For architectural diversity, all 1186 different designs used in the discovery project were ranked by the number of gapmers in which theywere used, and this ranking was used as the coordinate. The most often-used design is at the top of the graph. (d) Measured activity and toxicity in vitro for thesame gapmers as in (c). Based on the measured values, gapmers were divided into four regions, and the relative fraction of gapmers from scanning andoptimization libraries in each region are shown using pie charts. Gapmers selected for evaluation in animal models came from the upper-right region with highactivity and low or no toxicity. Abbreviation: PK/PD, pharmacokinetics and pharmacodynamics.

Reviews�K

EYNOTE

REV

IEW

Also, in contrast to classic random mixture AONs, PS stereodefined

oligonucleotides offer a unique opportunity: it is now possible to

study the complex interactions between AONs and, for example,

proteins by computational modeling.

The understanding of how structural diversity determinants

affect functional properties is essential to guide heuristic

approaches in drug discovery. For example, what is the most

efficient path to highly potent gapmers? Is it scanning the target

RNA comprehensively in 1000 regions with one selected stan-

dard design (broad strategy)? Is it scanning the target in only

100 different regions but testing ten different gapmer designs in

each (narrow strategy)? Or is it evaluating only ten different

target regions but with 100 different gapmer designs in each

110 www.drugdiscoverytoday.com

region (deep strategy)? As calculated in Box 1, thousands of

different modified gapmers can be designed against any target

region, so each of these strategies is clearly possible. The answer

depends on whether nucleobase or architectural diversity affects

the therapeutic index the most, and what balance between them

explores chemical space most efficiently. Traditionally, nucleo-

base diversity has been explored the most, with the aim of

searching for the regions on the transcript most accessible to

the AONs. However, as the examples presented in the sections

above show, we are observing considerable impact on potency

from other diversity determinants. Consequently, we are mov-

ing away from pure broad strategies with only a single fixed

design, towards more balanced and deeper strategies where

Drug Discovery Today �Volume 23, Number 1 � January 2018 REVIEWS

BOX 1

The chemical space of RNA therapeutics

Chemical space can be thought of as an analogy to thecosmological universe, with chemical compounds populating thisspace instead of stars [139]. How many molecules populate thisspace? For small organic molecules with up to 17 atoms of C, N, O,S, and halogens, and taking chemical stability and syntheticfeasibility in account, the number has been estimated to be morethan 1011 compounds [140]. Here, we estimate the number ofpossible gapmers that can be designed against an RNA target. Foran RNA that is n nucleotides long, and where the gapmer is lnucleotides long, in total there will then be n � l + 1 possibletarget regions of length l. Considering next the backbonechemistry, for a gapmer of length l, there will be l � 1 linkagesbetween nucleotides, each of which can be in either Rp- or Sp-form. This gives 2l–1 different stereoisomers. Finally, any type of mpossible modified nucleosides can in principle be used in theregions flanking the DNA gap. Therefore, for a gapmer of length l,with a gap of length g, the number of different modified gapmersthat can be synthesized equals ml–g. Thus, considering all gapmersof lengths between lmin and lmax, taken together the number ofdifferent, fully stereodefined, gapmers is equivalent to Eq. (I):

Xlmax

l¼lmin

ðn � l þ 1Þ2l�1ml�g: ðIÞ

For a standard pre-mRNA that is n = 25 000 nt in length, andconsidering gapmer lengths between lmin = 12 nt and lmax = 20 nt,with a minimal gap of g = 6 nt, and where m = 5 types ofnucleosides (DNA, LNA, MOE, 20OMe, and 20F) are considered,there are 8.89 � 1019 � 1020 different possible gapmers.Equation I can also be used when estimating the number ofmixmers designed to target a narrow region of RNA, such as amiRNA or a splice-site. In this case, with the same parameters as forgapmers but setting n = 20 nt and g = 0 nt, there are 6.17 � 1019

� 1020 different possible mixmers, about the same number as forgapmers. The smaller region of RNA that can be targeted isbalanced out by having the entire oligonucleotide available formodifications, where, for gapmers, there is a gap where only DNAcan be used.

Review

s� K

EYNOTE

REV

IEW

multiple designs are tested in highly overlapping regions on the

target RNA.

Another point to consider is the multidimensional nature of the

local optimization in chemical space, where several properties of

gapmers have to be acceptable and drug-like at the same time.

Should we then measure all functional properties for the entire

library of gapmers? Or are there some properties that are not

necessary to consider early on, because diversity can be introduced

at a later stage to ensure that the wanted functional properties can

be achieved? Say, for example, that exploring nucleobase diversity

has identified potent but not well-tolerated gapmer hits. When

then nucleoside, architectural, or backbone diversity is explored,

will the chance of finding leads that are both potent and well

tolerated be higher if we had not begun with hits only optimized

for potency?

If AONs shared the same properties simply by membership of

the same chemical class (‘portability of chemistry’), only sequence

would differentiate properties and broad gene walks would be

required to have enough compounds to select from (broad strate-

gy). However, the pronounced functional effects that small

changes in chemical structure have (see above) offer new oppor-

tunities for AON drug discovery. Diversity has now led us to search

much deeper and in a narrower target sequence space (narrow or

deep strategy). If we on top of this add the diversity of diastereoi-

somers, then a large diversity/property spectrum will unfold even

from just a single classic AON PS compound mixture (ultra-deep

strategy). Deep or ultra-deep strategies offer not only an introduc-

tion of a much larger structural space to search in, but more

importantly, also a possibility to begin with much ‘tougher’ selec-

tion criteria. It is possible to begin with higher sequence restric-

tions and/or requirements. If, for instance, many and strict

requirements are applied, such as species crossreactivity, sequence

specificity to only the intended target [15,18], deselection of

predicted sequence motifs associated with increased toxic poten-

tial [18,19,136], deselection of unwanted sequence motifs from a

chemistry, manufacturing, and control perspective, and so on, only

a very few sequences will finally comply. In the case where a few

16mer compound mixtures have been identified complying with

all selection criteria, it should then be possible to find compounds

with even better properties among the 32 768 structurally different

diastereoisomers of each 16mer. Deep or ultra-deep strategies will

also be advantageous for discovery programs focused on, for exam-

ple, splice-switching or miRNA inhibition, because here the rele-

vant RNA target regions sequence wise are very narrow.

There is no doubt that harnessing the full potential offered by

diversity drives AON drug discovery in the direction of small-

molecule concepts. This is with respect to ‘higher’-throughput

screenings and working with single molecules (fully stereodefined

AONs). However, AON drug discovery exhibits inherent advan-

tages over small-molecule principles. AON drug discovery con-

cepts are more ‘rational’. Watson–Crick base-pairing rules govern

target RNA binding, whereas, for small molecules, complex

computational docking studies must be undertaken. Also, impor-

tantly, oligonucleotide production by solid-phase synthesis fol-

lows shared principles for all compounds. Furthermore, as we

develop our understanding of these new SARs, we expect to apply

an even more ‘rational’ approach to AON drug discovery, such as

by developing in silico algorithms that are more predictive for AON

screening outcomes taking diversity into account. This has already

been done for hepatotoxic potential [19]. In this way, bioinfor-

matics and computational modeling can enable faster discovery

processes and, throughout the process, more informative deci-

sions, thereby creating stronger lead candidates.

Concluding remarksThe fact that structural differences also define functional diversi-

ties is a given for all compound classes. We have shown that not

only sequence, but also the design of an AON is a strong property

determinant (Fig. 2). A clear SAR exists within a defined target

nucleobase sequence simply by varying the sugar and phosphate

backbone within that sequence (Fig. 4). This creates AONs with

different pharmacological properties (Figs 3 and 5). Exploration of

the large property space is the reason why drug discovery libraries

in our laboratories have expanded greatly. Initially, we used

mainly a broad screening strategy (gene walk), but now we are

also exploring compound diversities within selected nucleobase

sequences (deep strategy) (Fig. 6).

www.drugdiscoverytoday.com 111

REVIEWS Drug Discovery Today �Volume 23, Number 1 � January 2018

Reviews�K

EYNOTE

REV

IEW

The diversity we experienced for stereodefined LNAs (with the

same sequence and nucleoside design) represents an extra dimen-

sion by which even more unique leads might be identified. The

basis for their uniqueness might, in part, be created by the positive

heavy atom effects of sulfur and the introduction of chirality on

phosphorous, which we think act as structural diversity parame-

ters in themselves. A chiral phosphorous scaffold might be ideal

for designing drugs interacting with the chiral world of biology.

Given that AONs are based on something as predictable as

Watson–Crick base-pairing to RNA, binding to intended and

unintended RNA targets can be predicted computationally and

measured experimentally [18]. This provides a means for assessing

unintended hybridization-induced biological effects of AONs

[105]. Still, they are, perhaps unsurprisingly, unique molecules

that, in a therapeutic setting, provide unique and yet ‘hard-to-

predict’ pharmacological properties. Clearly, more work is needed

to build understanding of the underlying mechanism of AON

diversity, and to determine how much the TI can be expanded

by these new strategies.

However, what is clear at this point is that antisense has devel-

oped immensely over the past four decades, and the new drug

112 www.drugdiscoverytoday.com

discovery strategies are producing increasingly better RNA thera-

peutics. We have realized that the old concept in antisense of

‘portability of chemistry’ only provides a starting point for RNA

drug discovery and, as shown here, structural diversity at all levels

(nucleobase, nucleoside, backbone, and architectural) can be used

to produce AONs with improved drug properties.

Conflict of interestAll authors except N.P. are full time employees at Roche Innova-

tion Centre Copenhagen A/S.

AcknowledgmentsWe would like to thank Cy A. Stein for helpful comments on the

manuscript, and Mikkel Adriansen for in-depth analysis of the

mass spectra of stereo-defined oligonucleotides. For their excellent

technical assistance, we would like to thank Helle Nymark and

Annette Glidov for qPCR high-throughput screening, Charlotte

Øverup for mouse liver and serum measurements, Rikke Sølberg,

Camilla T. Haugaard, and Lisbeth Bang for handling of mice in the

animal facilities, and Valesi Mukaka for the synthesis and

formulation of oligonucleotides for mouse studies.

References

1 Curwen, V. et al. (2004) The Ensembl automatic gene annotation system. Genome

Res. 14, 942–950

2 Morris, K.V. and Mattick, J.S. (2014) The rise of regulatory RNA. Nat. Rev. Genet. 15,

423–437

3 Lin, M.F. et al. (2011) Locating protein-coding sequences under selection for

additional, overlapping functions in 29 mammalian genomes. Genome Res. 21,

1916–1928

4 Esteller, M. (2011) Epigenetic changes in cancer. F1000 Biol. Rep. 3, 9

5 Alexa Dickson, M. and Jeffrey Wilusz, C.J.W. (2009) mRNA turnover. In

Encyclopedia of Life Sciences. John Wiley & Sons Ltd.

6 Zamecnik, P.C. and Stephenson, M.L. (1978) Inhibition of Rous sarcoma virus

replication and cell transformation by a specific oligonucleotide. Proc. Natl. Acad.

Sci. U. S. A. 75, 280–284

7 Cook, P.D. (1999) Making drugs out of oligonucleotides: a brief review and

perspective. Nucleosides Nucleotides 18, 1141–1162

8 Uhlmann, E. and Peyman, A. (1990) Antisense oligonucleotides: a new therapeutic

principle. Chem. Rev. 90, 544–579

9 Uhlmann, E. (2000) Recent advances in the medicinal chemistry of antisense

oligonucleotides. Curr. Opin. Drug Discov. Dev. 3, 203–213

10 Eckstein, F. (2007) The versatility of oligonucleotides as potential therapeutics.

Expert Opin. Biol. Ther. 7, 1021–1034

11 Khvorova, A. and Watts, J.K. (2017) The chemical evolution of oligonucleotide

therapies of clinical utility. Nat. Biotechnol. 35, 238–248

12 McClorey, G. and Wood, M.J. (2015) An overview of the clinical application of

antisense oligonucleotides for RNA-targeting therapies. Curr. Opin. Pharmacol. 24,

52–58

13 Koch, T. (2013) LNA antisense: a review. Curr. Phys. Chem. 3, 55–68

14 Obika, S. et al. (1998) Stability and structural features of the deplexes containing

nucleoside analogues with a fixed N-type conformation, 20-0,40-C-methyleneribonucleosides. Tetrahedron Lett. 39, 5401–5404

15 Singh, S.K. et al. (1998) LNA (locked nucleic acids): synthesis and high-affinity

nucleic acid recognition. Chem. Commun. 0, 455–456

16 Wahlestedt, C. et al. (2000) Potent and nontoxic antisense oligonucleotides

containing locked nucleic acids. Proc. Natl. Acad. Sci. U. S. A. 97, 5633–5638

17 Swayse, E.E. et al. (2007) Antisense oligonucleotides containing locked nucleic acid

improve potency but cause significant hepatotoxicity in animals. Nucleic Acids Res.

35, 687–700

18 Hagedorn, H.P. et al. (2017) Managing the sequence-specificity of antisense

oligonucleotides in drug discovery. Nucleic Acid Res. 2262–2282

19 Hagedorn, P.H. et al. (2013) Hepatotoxic potential of therapeutic oligonucleotides

can be predicted from their sequence and modification pattern. Nucleic Acid Ther.

23, 302–310

20 Crooke, S.T. et al. (2017) The effects of 20-O-methoxyethyl containing

antisense oligonucleotides on platelets in human clinical trials. Nucleic Acid Ther.

121–129

21 Beaucage, S.L. et al. (1999) Current Protocols in Nucleic Acid Chemistry. John Wiley &

Sons, Inc.

22 Brown, T. and Brown, D.J.S. (1991) Modern machine-aided methods of

oligodeoxiribonucleotide synthesis. In Ligonucleotides and Analogues: A Practical

Approach (Eckstein, F., ed.), pp. 13–14, IRL Press

23 Cohen, J.S. (1993) Phosphorothioate oligonucleotides. In Antisense Research and

Applications (Crooke, S.T. and Lebleu, B., eds), pp. 205–223, CRC Press

24 Wengel, J. (2001) LNA (locked nucleic acid). In Antisense Drug Technology;

Principles, Strategies, and Applications (Crooke, S.T., ed.), pp. 339–357, Marcel

Dekker

25 Burgers, P.M. and Eckstein, F. (1978) Absolute configuration of the diastereomers

of adenosine 50-O-(1-thiotriphosphate): consequences for the stereochemistry of

polymerization by DNA-dependent RNA polymerase from Escherichia coli. Proc.

Natl. Acad. Sci. U. S. A 75, 4798–4800

26 Eckstein, F. et al. (1976) Stereochemistry of polymerization by DNA-dependent

RNA-polymerase from Escherichia coli: an investigation with a diastereomeric ATP-

analogue. Proc. Natl. Acad. Sci. U. S. A. 73, 2987–2990

27 Eckstein, F. and Gindl, H. (1968) Uridin-20,30-O,O-cyclothiophosphate. Chem. Ber.

101, 1670–1673

28 Eckstein, F. and Gindl, H. (1969) Polyribonucleotides containing a thiophosphate

backbone. FEBS Lett. 2, 262–264

29 Saenger, W. and Eckstein, F. (1970) Stereochemistry of a substrate for pancreatic

ribonuclease Crystal and molecular structure of the triethylammonium salt of

uridine 20,30-0,0-cyclophosphorothioate. J. Am. Chem. Soc. 92, 4712–4718

30 Wilk, A. and Stec, W.J. (1995) Analysis of oligo(deoxynucleoside

phosphorothioates)s and their diastereomeric composition. Nucleic Acid Res. 23,

530–534

31 Iyer, R.P. et al. (1998) Solid-phase stereoselective synthesis of oligonucleoside

phosphorothioates: The nucleoside bicyclic oxazaphospholidines as novel

synthons. Tetrahedron Lett. 39, 2491–2494

32 Wang, J.-C. and Just, G. (1997) A stereoselective synthesis of dinucleotide

phosphorothioates, using chiral indol-oxazaphosphorine intermediates.

Tetrahedron Lett. 38, 3797–3800

33 Iwamoto, N. et al. (2017) Control of phosphorothioate stereochemistry

substantially increases the efficacy of antisense oligonucleotides. Nat. Biotech. 845–

851

34 Wan, W.B. et al. (2014) Synthesis, biophysical properties and biological activity of

second generation antisense oligonucleotides containing chiral phosphorothioate

linkages. Nucleic Acids Res. 42, 13456–13468

Drug Discovery Today �Volume 23, Number 1 � January 2018 REVIEWS

Review

s� K

EYNOTE

REV

IEW

35 Oka, N. et al. (2008) Solid-phase synthesis of stereoregular

oligodeoxyribonucleoside phosphorothioates using bicyclic oxazaphospholidine

derivatives as monomer units. J. Am. Chem. Soc. 130 (47), 16031–16037

36 Stec, W.J. et al. (1998) Deoxyribonucleoside 30-O-(2-Thio- and 2-Oxo-’spiro’-4,4-

pentamethylene-1,3,2-oxathiaphospholane)s: monomers for stereocontrolled

synthesis of oligo(deoxyribonucleoside phosphorothioate)s and chimeric PS/PO

oligonucleotides. J. Am. Chem. Soc. 120, 7156–7167

37 Oka, N. et al. (2002) Diastereocontrolled synthesis of dinucleoside

phosphorothioates using a novel class of activators, dialkyl(cyanomethyl)

ammonium tetrafluoroborates. J. Am. Chem. Soc. 124, 4962–4963

38 Koziolkiewicz, M. et al. (1995) Stereodifferentiation-the effect of P chirality of oligo

(nucleoside phosphorothioates) on the activity of bacterial RNase H. Nucleic Acids

Res. 23, 5000–5005

39 Koziolkiewicz, M. et al. (1997) Stability of stereoregular oligo(nucleoside

phosphorothioate)s in human plasma: diastereoselectivity of plasma 30-exonuclease. Antisense Nucleic Acid Drug Dev. 7, 43–48

40 Krieg, A.M. et al. (2003) P-chirality-dependent immune activation by

phosphorothioate CpG oligodeoxynucleotides. Oligonucleotides 13, 491–499

41 Monteleone, G. et al. (2015) Mongersen, an oral SMAD7 antisense oligonucleotide,

and Crohn’s disease. N. Engl. J. Med. 372, 1104–1113

42 Gura, T. (1995) Antisense has growing pains. Science 270, 575–577

43 Eckstein, F. (2014) Phosphorothioates, essential components of therapeutic

oligonucleotides. Nucleic Acid Ther. 24, 374–387

44 Freier, S.M. and Altmann, K.-H. (1997) The ups and downs of nucleic acid duplex

stability: structure-stability studies on chemically-modified DNA:RNA duplexes.

Nucleic Acid Res. 25, 4429–4443

45 Galderisi, U. et al. (2001) Clinical trials of a new class of therapeutic agents:

antisense oligonucleotides. Emerg. Drugs 6, 69–79

46 Martin, P. (1995) Ein neuer Zugang zu 20-O-Alkylribonucleosiden und

Eigenschaften deren Oligonucleotiden. Helv. Chim. Acta 78, 486–504

47 Thuong, N.T. and Helene, C. (1993) Sequenzspezifische Erkennung und

Modifikation von Doppelhelix-DNA durch Oligonucleotide. Angw. Chem. 105,

697–723

48 Raal, F.J. et al. (2010) Mipomersen, an apolipoprotein B synthesis inhibitor, for

lowering of LDL cholesterol concentrations in patients with homozygous familial

hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial.

Lancet 375, 998–1006

49 Chiriboga, C.A. et al. (2016) Results from a phase 1 study of nusinersen (ISIS-SMN

(Rx)) in children with spinal muscular atrophy. Neurology 86, 890–897

50 Obika, S. et al. (1997) Synthesis and conformation of 30-0,40-C-methyleneribonucleosides, novel bicyclic nucleoside analogues for 20,50-linkedoligonucleotide modification. Chem. Commun. 1643–1644

51 Wengel, J. et al. (1999) LNA (locked nucleic acid). Nucleosides Nucleotides 18, 1365–

1370

52 Singh, S.K. and Wengel, J. (1998) Universality of LNA-mediated high-affinity

nucleic acid recognition. Chem. Commun. 0, 1247–1248

53 Koshkin, A. et al. (1998) LNA (locked nucleic acids): synthesis of the adenine,

cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside

monomers, oligomerisation, and unprecedented nucleic acid recognition.

Tetrahedron 54, 3607–3630

54 Wengel, J. et al. (2001) LNA (locked nucleic acid) and the diastereoisomeric alpha-

L-LNA: conformational tuning and high-affinity recognition of DNA/RNA targets.

Nucleosides Nucleotides Nucleic Acids 20, 389–396

55 Fluiter, K. et al. (2005) On the in vitro and in vivo properties of four locked nucleic

acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide.

Chembiochem 6, 1104–1109

56 Koch, T. and Ørum, H. (2007) Locked nucleic acid. In Antisense Drug Technology

(Crooke, S.T., ed.), pp. 519–565, CRC Press

57 Koch, T. et al. (2008) Locked nucleic acid: properties and therapeutic aspects. In

Therapeutic Oligonucleotides (Kurreck, J., ed.), pp. 103–141, RSC Publishing

58 Yamamoto, T. et al. (2011) Antisense drug discovery and development. Future Med.

Chem. 3, 339–365

59 Oerum, H. and Wengel, J. (2001) Locked nucleic acids: a promising molecular

family for gene-function analysis and antisense drug development. Curr. Opin.

Mol. Ther. 3, 239–243

60 Oerum, H. et al. (2003) Locked nucleic acids (LNA) and medical applications. Lett.

Pep. Sci. 10, 325–334

61 Kloosterman, W.P. et al. (2006) In situ detection of miRNAs in animal embryos

using LNA-modified oligonucleotide probes. Nat. Methods 3, 27–29

62 Obika, S. (2004) Development of bridged nucleic acid analogues for antigene

technology. Chem. Pharm. Bull. (Tokyo) 52, 1399–1404

63 Orom, U.A. et al. (2006) LNA-modified oligonucleotides mediate specific

inhibition of microRNA function. Gene 372, 137–141

64 Rajwanshi, V.K. et al. (2000) The eight stereoisomers of LNA (locked nucleic acid): a

remarkable family of strong RNA binding molecules. Angew. Chem. Int. Ed. 39,

1656–1659

65 Rosenbohm, C. et al. (2003) Synthesis of 20-amino-LNA: a new strategy. Org.

Biomol. Chem. 1, 655–663

66 Singh, S.K. et al. (1998) Synthesis of novel bicyclo [2.2.1] ribonucleosides:

20-amino-and 20-thio-LNA monomeric nucleosides. J. Org. Chem. 63, 6078–6079

67 Singh, S.K. et al. (1998) Synthesis of 20-amino-LNA: a novel conformationally

restricted high-affinity oligonucleotide analogue with a handle. J. Org. Chem. 63,

10035–10039

68 Soerensen, M.D. et al. (2002) Alpha-L-ribo-configures locked nucleic acid (alpha-L-

LNA): synthesis and properties. J. Am. Chem. Soc. 124, 2164–2176

69 Egli, M. et al. (2001) X-ray crystal structure of a locked nucleic acid (LNA) duplex

composed of a palindromic 10-mer DNA strand containing one LNA thymine

monomer. Chem. Commun. 0, 651–652

70 Nielsen, K.E. et al. (2000) Solution structure of an LNA hybridized to DNA: NMR

study of the d(CT(L)GCT(L)T(L)CT(L)GC):d(GCAGAAGCAG) duplex containing

four locked nucleotides. Bioconjug. Chem. 11, 228–238

71 Nielsen, K.E. et al. (2004) NMR studies of fully modified locked nucleic acid (LNA)

hybrids: solution structure of an LNA:RNA hybrid and characterization of an LNA:

DNA hybrid. Bioconjug. Chem. 15, 449–457

72 Petersen, M. et al. (2000) The conformations of locked nucleic acids (LNA). J. Mol.

Recognit. 13, 44–53

73 Petersen, M. et al. (2002) Locked nucleic acid (LNA) recognition of RNA: NMR

solution structures of LNA:RNA hybrids. J. Am. Chem. Soc. 124, 5974–5982

74 Petersen, M. et al. (2003) Structural characterization of LNA and alpha-L-LNA

hybridized to RNA. Nucleosides Nucleotides Nucleic Acids 22, 1691–1693

75 Xu, Y. et al. (2017) The free energy of locking a ring: changing a

deoxyribonucleoside to a locked nucleic acid. J. Comput. Chem. 38, 1147–1157

76 Yildirim, I. et al. (2014) Interplay of LNA and 20-O-methyl RNA in the structure and

thermodynamics of RNA hybrid systems: a molecular dynamics study using the

revised AMBER force field and comparison with experimental results. J. Phys.

Chem. B 118, 14177–14187

77 Eichert, A. et al. (2010) The crystal structure of an ‘All Locked’ nucleic acid duplex.

Nucleic Acids Res. 38, 6729–6736

78 Hughesman, C.B. et al. (2011) Role of the heat capacity change in understanding

and modeling melting thermodynamics of complementary duplexes containing

standard and nucleobase-modified LNA. Biochemistry 50, 5354–5368

79 Frieden, M. et al. (2003) Expanding the design horizon of antisense

oligonucleotides with alpha-L-LNA. Nucleic Acids Res. 31, 6365–6372

80 Cerritelli, S.M. and Crouch, R.J. (2009) Ribonuclease H: the enzymes in

Eukaryotes. FEBS J. 276, 1494–1505

81 Crooke, S.T. (1998) Molecular mechanisms of antisense drugs: RNase H. Antisense

Nucleic Acid Drug Dev. 8, 133–134

82 Wu, H. et al. (2004) Determination of the role of the human RNase H1

in the pharmacology of DNA-like antisense drugs. J. Biol. Chem. 279,

17181–17189

83 Braasch, D.A. and Corey, R. (2000) Locked nucleic acid (LNA): fine-tuning the

recognition of DNA and RNA. Chem. Biol. 55, 1–7

84 Braasch, D.A. et al. (2002) Antisense inhibition of gene expression in cells by

oligonucleotides incorporating locked nucleic acids: effect of mRNA target

sequence and chimera design. Nucleic Acids Res. 30, 5160–5167

85 Castanotto, D. et al. (2015) A cytoplasmic pathway for gapmer antisense

oligonucleotide-mediated gene silencing in mammalian cells. Nucleic Acids Res. 43,

9350–9361

86 Liang, X.H. et al. (2017) RNase H1-dependent antisense oligonucleotides are

robustly active in directing RNA cleavage in both the cytoplasm and the nucleus.

Mol Ther. 2075–2092

87 Dominski, Z. and Kole, R. (1993) Restoration of correct splicing in thalassemic pre-

mRNA by antisense oligonucleotides. Proc. Natl. Acad. Sci. U. S. A. 90, 8673–8677

88 Rigo, F. et al. (2014) Antisense oligonucleotide-based therapies for diseases caused

by pre-mRNA processing defects. Adv. Exp. Med. Biol. 825, 303–352

89 Sierakowska, H. et al. (1996) Repair of thalassemic human beta-globin mRNA in

mammalian cells by antisense oligonucleotides. Proc. Natl. Acad. Sci. U. S. A. 93,

12840–12844

90 Passini, M.A. et al. (2011) Antisense oligonucleotides delivered to the mouse CNS

ameliorate symptoms of severe spinal muscular atrophy. Sci. Transl. Med. 3, 72ra18

91 Kole, R. and Krieg, A.M. (2015) Exon skipping therapy for Duchenne muscular

dystrophy. Adv. Drug Deliv. Rev. 87, 104–107

92 Li, L. et al. (2016) Activating frataxin expression by repeat-targeted nucleic acids.

Nat. Commun. 7, 10606

93 Wahlestedt, C. (2013) Targeting long non-coding RNA to therapeutically

upregulate gene expression. Nat. Rev. Drug Discov. 12, 433–446

www.drugdiscoverytoday.com 113

REVIEWS Drug Discovery Today �Volume 23, Number 1 � January 2018

Reviews�K

EYNOTE

REV

IEW

94 Jonas, S. and Izaurralde, E. (2015) Towards a molecular understanding of

microRNA-mediated gene silencing. Nat. Rev. Genet. 16, 421–433

95 Li, Z. and Rana, T.M. (2014) Therapeutic targeting of microRNAs: current status

and future challenges. Nat. Rev. Drug Discov. 13, 622–638

96 Lanford, R. et al. (2010) Therapeutic silencing of microRNA-122 in primates with

chronic hepatitis C virus infection. Science 327, 198–201

97 Janssen, H.L. et al. (2013) Treatment of HCV infection by targeting microRNA. N.

Engl. J. Med. 368, 1685–1694

98 van Rooij, E. and Kauppinen, S. (2014) Development of microRNA therapeutics is

coming of age. EMBO Mol. Med. 6, 851–864

99 Straarup, E.M. et al. (2010) Short locked nucleic acid antisense oligonucleotides

potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-

human primates. Nucleic Acids Res. 38, 7100–7111

100 Pedersen, L. et al. (2014) A kinetic model explains why shorter and less affine

enzyme-recruiting oligonucleotides can be more potent. Mol. Ther. Nucleic Acids 3,

e149

101 Obad, S. et al. (2011) Silencing of microRNA families by seed-targeting tiny LNAs.

Nat. Genet. 43, 371–378

102 Kakiuchi-Kiyota, S. et al. (2014) Comparison of hepatic transcription profiles of

locked ribonucleic acid antisense oligonucleotides: evidence of distinct pathways

contributing to non-target mediated toxicity in mice. Toxicol. Sci. 138, 234–248

103 Burel, S.A. et al. (2016) Hepatotoxicity of high affinity gapmer antisense

oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of

very long pre-mRNA transcripts. Nucleic Acids Res. 44, 2093–2109

104 Kasuya, T. et al. (2016) Ribonuclease H1-dependent hepatotoxicity caused by

locked nucleic acid-modified gapmer antisense oligonucleotides. Sci. Rep. 6, 30377

105 Lindow, M. et al. (2012) Assessing unintended hybridization-induced biological

effects of oligonucleotides. Nat. Biotechnol. 30, 920–923

106 Lebedeva, I. et al. (2000) Cellular delivery of antisense oligonucleotides. Eur. J.

Pharm. Biopharm. 50, 101–119

107 Akhtar, S. et al. (1991) Interactions of antisense DNA oligonucleotide analogs with

phospholipid membranes (liposomes). Nucleic Acids Res. 19, 5551–5559

108 Juliano, R.L. (2016) The delivery of therapeutic oligonucleotides. Nucleic Acids Res.

44, 6518–6548

109 Crooke, S.T. et al. (2017) Cellular uptake and trafficking of antisense

oligonucleotides. Nat. Biotechnol. 35, 230–237

110 Manoharan, M. (2002) Oligonucleotide conjugates as potential antisense drugs

with improved uptake, biodistribution, targeted delivery, and mechanism of

action. Antisense Nucleic Acid Drug Dev. 12, 103–128

111 Debart, F. et al. (2007) Chemical modifications to improve the cellular uptake of

oligonucleotides. Curr. Top. Med. Chem. 7, 727–737

112 Eckstein, F. (2007) The versatility of oligonucleotides as potential therapeutics.

Expert Opin. Biol. Ther. 7, 1021–1034

113 Stein, C.A. et al. (2010) Efficient gene silencing by delivery of locked nucleic acid

antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res.

38, e3

114 Moghimi, S.M. et al. (2005) A two-stage poly(ethylenimine)-mediated

cytotoxicity: implications for gene transfer/therapy. Mol. Ther. 11, 990–995

115 Symonds, P. et al. (2005) Low and high molecular weight poly(L-lysine)s/poly(L-

lysine)-DNA complexes initiate mitochondrial-mediated apoptosis differently.

FEBS Lett. 579, 6191–6198

116 Mettlen, M. et al. (2009) Dissecting dynamin’s role in clathrin-mediated

endocytosis. Biochem. Soc. Trans. 37, 1022–1026

117 Lajoie, P. and Nabi, I.R. (2010) Lipid rafts, caveolae, and their endocytosis. Int. Rev.

Cell. Mol. Biol. 282, 135–163

118 Howes, M.T. et al. (2010) Molecules, mechanisms, and cellular roles of clathrin-

independent endocytosis. Curr. Opin. Cell Biol. 22, 519–527

114 www.drugdiscoverytoday.com

119 Koller, E. et al. (2011) Mechanisms of single-stranded phosphorothioate modified

antisense oligonucleotide accumulation in hepatocytes. Nucleic Acids Res. 39,

4795–4807

120 Fazil, M.H. et al. (2016) GapmeR cellular internalization by macropinocytosis

induces sequence-specific gene silencing in human primary T-cells. Sci. Rep. 6,

37721

121 Castanotto, D. et al. (2016) Protein kinase C-alpha is a critical protein for antisense

oligonucleotide-mediated silencing in mammalian cells. Mol. Ther. 24, 1117–1125

122 Mechti, N. et al. (1991) Nuclear location of synthetic oligonucleotides

microinjected somatic cells: its implication in an antisense strategy. Nucleic Acids

Symp. Ser. 24, 147–150

123 Leonetti, J.P. et al. (1991) Intracellular distribution of microinjected antisense

oligonucleotides. Proc. Natl. Acad. Sci. U. S. A. 88, 2702–2706

124 D’Souza, A.A. and Devarajan, P.V. (2015) Asialoglycoprotein receptor mediated

hepatocyte targeting � strategies and applications. J. Control. Release 203, 126–139

125 Shi, F. et al. (2007) In situ entry of oligonucleotides into brain cells can occur

through a nucleic acid channel. Oligonucleotides 17, 122–133

126 Galloway, W.R. et al. (2010) Diversity-oriented synthesis as a tool for the discovery

of novel biologically active small molecules. Nat. Commun. 1, 80

127 Koch, T. et al. (2014) Quantum mechanical studies of DNA and LNA. Nucleic Acid

Ther. 24, 139–148

128 Bohr, H.G. et al. (2017) Electronic structures of LNA phosphorothioate

oligonucleotides. Mol. Ther. Nucleic Acids 428–441

129 Vickers, T.A. et al. (2000) Effects of RNA secondary structure on cellular antisense

activity. Nucleic Acids Res. 28, 1340–1347

130 Watt, A. and Freier, S. (2007) Basic principles of antisense drug discovery. In

Antisense Drug Technology (Crooke, S.T., ed.), pp. 117–141, CRC Press

131 Laxton, C. et al. (2011) Selection, optimization, and pharmacokinetic properties of

a novel, potent antiviral locked nucleic acid-based antisense oligomer targeting

hepatitis C virus internal ribosome entry site. Antimicrob. Agents Chemother. 55,

3105–3114

132 Stanton, R. et al. (2012) Chemical modification study of antisense gapmers. Nucleic

Acid Ther. 22, 344–359

133 Yu, R.Z. et al. (2015) Predictive dose-based estimation of systemic exposure

multiples in mouse and monkey relative to human for antisense oligonucleotides

with 20-o-(2–methoxyethyl) modifications. Mol. Ther. Nucleic Acids 4, e218

134 Watanabe, T.A. et al. (2006) Plasma protein binding of an antisense

oligonucleotide targeting human ICAM-1 (ISIS 2302). Oligonucleotides 16,

169–180

135 Seth, P.P. et al. (2009) Short antisense oligonucleotides with novel 20-40

conformationaly restricted nucleoside analogues show improved potency without

increased toxicity in animals. J. Med. Chem. 52, 10–13

136 Burdick, A.D. et al. (2014) Sequence motifs associated with hepatotoxicity of

locked nucleic acid-modified antisense oligonucleotides. Nucleic Acids Res. 42,

4882–4891

137 Kamola, P.J. et al. (2015) In silico and in vitro evaluation of exonic and intronic off-

target effects form a critical element of therapeutic ASO gapmer optimization.

Nucleic Acids Res. 43, 8638–8650

138 Li, M. et al. (2017) Synthesis and cellular activity of stereochemically-pure 2[prime

or minute]-O-(2-methoxyethyl)-phosphorothioate oligonucleotides. Chem.

Commun. 53, 541–544

139 Lipinski, C. and Hopkins, A. (2004) Navigating chemical space for biology and

medicine. Nature 432, 855–861

140 Reymond, J.L. (2015) The chemical space project. ACC Chem. Res. 48, 722–730

141 Chan, J.H. et al. (2006) Antisense oligonucleotides: from design to therapeutic

application. Clin. Exp. Pharmacol. Physiol. 33, 533–540