locked nucleic acid: modality, diversity, and drug …...stability, the driver behind the many...
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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 VersionPeer reviewed version
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
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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.
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, and drug discovery, Drug Discov Today (2017), https://doi.org/10.1016/j.drudis.2017.09.018
Corresponding author: Koch, T. ([email protected])
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 1
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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.
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, a
2 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
nd drug discovery, Drug Discov Today (2017), https://doi.org/10.1016/j.drudis.2017.09.018
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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].
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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].
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, a
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
nd drug discovery, Drug Discov Today (2017), https://doi.org/10.1016/j.drudis.2017.09.018
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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
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, a
4 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
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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
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, a
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
nd drug discovery, Drug Discov Today (2017), https://doi.org/10.1016/j.drudis.2017.09.018
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DRUDIS-2093; No of Pages 14
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.
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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
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, a
6 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.
nd drug discovery, Drug Discov Today (2017), https://doi.org/10.1016/j.drudis.2017.09.018
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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.).
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, a
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
nd drug discovery, Drug Discov Today (2017), https://doi.org/10.1016/j.drudis.2017.09.018
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DRUDIS-2093; No of Pages 14
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.
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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.
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, a
8 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
nd drug discovery, Drug Discov Today (2017), https://doi.org/10.1016/j.drudis.2017.09.018
Drug Discovery Today �Volume 00, Number 00 �October 2017 REVIEWS
DRUDIS-2093; No of Pages 14
Review
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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.
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, a
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.
nd drug discovery, Drug Discov Today (2017), https://doi.org/10.1016/j.drudis.2017.09.018
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DRUDIS-2093; No of Pages 14
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.
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Also, in contrast to classic random mixture AONs, PS stereodefinedoligonucleotides 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
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, a
10 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
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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
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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-
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, a
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).
nd drug discovery, Drug Discov Today (2017), https://doi.org/10.1016/j.drudis.2017.09.018
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REVIEWS Drug Discovery Today �Volume 00, Number 00 �October 2017
DRUDIS-2093; No of Pages 14
Reviews�K
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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
Please cite this article in press as: Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, a
12 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.
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