Impact of Phosphorothioate Chirality on Double-Stranded siRNAs: A Systematic Evaluation of Stereopure siRNA Designs

Article abstract of DOI:10.1002/cbic.201900630

Oligonucleotides are important therapeutic approaches, as evidenced by recent clinical successes with antisense oligonucleotides (ASOs) and double-stranded short interfering RNAs (siRNAs). Phosphorothioate (PS) modifications are a standard feature in the

Full text of DOI:10.1002/cbic.201900630

Accepted Article
Title: Impact of Phosphorothioate Chirality on Double-stranded siRNAs:
A Systematic Evaluation of Stereopure siRNA Designs
Authors: Sukumar Sakamuri, Laxman Eltepu, Dingguo Liu, Son Lam,
Bryan R Meade, Bin Liu, Giuseppe Dello Iacono, Ayman
Kabakibi, Lena Luukkonen, Tom Leedom, Mark Foster, and
Curt W Bradshaw
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content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201900630
Link to VoR: http://dx.doi.org/10.1002/cbic.201900630
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10.1002/cbic.201900630
ChemBioChem
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Impact of Phosphorothioate Chirality on Double-stranded
siRNAs: A Systematic Evaluation of Stereopure siRNA Designs
Sukumar Sakamuri,* Laxman Eltepu, Dingguo Liu, Son Lam, Bryan R Meade, Bin Liu, Giuseppe Dello
Iacono, Ayman Kabakibi, Lena Luukkonen, Tom Leedom, Mark Foster, and Curt W Bradshaw
Abstract: Oligonucleotides are important therapeutic approaches as
variations were explored in early optimization including significant
modifications in size (19- to 27-mers) and shape (DsiRNA,
asymmetric and blunt ended designs).[4] In addition, the siRNA
components such as sugars, nucleobases and phosphate
backbone were subjected to modifications in an effort to impart
nuclease stability, proper strand loading etc.[5] Ever since
Kornberg and De-Clercq’s early work on phosphorothioate
nuclease stability, thiol modification of phosphodiester (PO)
evidenced by recent clinical successes with anti-sense
oligonucleotides
(ASOs)
and
double-stranded
siRNAs.
Phosphorothioate (PS) modifications are a standard feature in the
current generation of oligo therapeutics but generates isomeric
mixtures leading to 2n isomers. All currently marketed therapeutic
oligonucleotides (ASOs and siRNAs) are complex isomeric mixtures.
Recent chemical methodologies for stereopure PS insertions have
resulted in preliminary rules for ASOs with multiple stereopure ASOs
moving into clinical development. Although siRNAs have
comparatively fewer PS, the field has yet to embrace the idea of
stereopure siRNAs. We investigated whether the individual isomers
contribute equally to in vivo activity of a representative siRNA. Here,
we report the results of a systematic evaluation of stereopure PS
incorporation into antithrombin-3 (AT3) siRNA demonstrating that
individual PS isomers dramatically affect in vivo activity. We evaluated
a standard siRNA design having six Phosphorothioate insertions and
found only ~10% of the 64 possible isomers are as efficacious as the
stereorandom control. Based on our data we conclude that G1R
stereochemistry is critical, G2R is important, G21S is preferable and
G22 and P1/P2 tolerates both isomers. To our surprise, the
disproportionate loss of efficacy for most isomers does not translate
to significant gain for the productive isomers, warrants further
mechanistic studies.
became
a
critical component of siRNA designs.[6]
Phosphorothioates are the most atom economical modification to
increase nuclease stability and, as recently reported, contribute
binding to various proteins and transporters which influences
biodistribution and efficacy.[7] One underappreciated outcome of
PS insertions is the introduction of stereoisomers compared to the
non-stereomeric PO backbone (Figure 1). Standard oligo
synthesis lacks stereocontrol[8a] but the issue was ignored
primarily due to the success of PS containing oligonucleotides in
clinic and the lack of practical stereopure PS synthetic
methodology. Stereopure PS containing oligonucleotides became
a reality with the development of oxazaphospholidine methods.[8]
The anti-sense oligonucleotide field was first to build on this
opportunity by exploring structure activity relationships and began
defining rules for stereopure PS insertions.[9]
Since the discovery that double-stranded siRNA can cleave
mRNA and inhibit protein translation by Fire and Mello in 1998,
this new class of therapeutics has moved towards clinical utility.[1]
The advent of double-stranded siRNAs as a modality to harness
a natural catalytic pathway called RNAi excited scientific and
business communities alike because of its implications in
therapeutics, particularly for targets difficult to drug using small
molecules and proteins.[2] Interest in siRNAs has had periodic
boom and bust cycles following recognition of and solutions for
technical challenges. One of the significant challenges is delivery
of these large, charged siRNA molecules across the cell
membrane. Both lipid nanoparticle and ligand-based approaches
are clinically validated with a recent approval of first RNAi
≠
Figure 1. Generic Examples of Phosphodiester and Phosphorothioate
Oligonucleotides. Phosphorothioate Isomers Shown in Highlighted Circles.
therapeutic, Patisiran,
a lipid complex from Alnylam and
numerous GalNAc-targeted siRNAs in clinical trials.[3]
siRNAs require fewer PS insertions than ASO with 15-20%
of the phosphate backbone at the 5’ and/or 3’ terminus of the
passenger (P-strand, sense) and guide (G-strand, antisense)
strands. Alnylam’s antithrombin-3 (AT3) siRNA design is shown
in Figure 2 with four guide strand PS insertions at G1, G2, G21
and G22, and two passenger strand PS insertions at P1 and P2.[3a,
Naturally occurring siRNA molecules have two
complementary strands (19 base pairs) with an additional two
unpaired bases at the 3’ ends, so called 21/21. A wide variety of
10]
For ease of comparison, we used this design for our stereopure
S. Sakamuri, L. Eltepu, D. Liu, S. Lam, B. R. Meade, B. Liu, G. D.
Iacono, A. Kabakibi, L. Luukkonen, T. Leedom, M. Foster, C. W.
Bradshaw. Solstice Biologics, 9535 Waples St, San Diego, CA
92121, USA
PS evaluations. To our surprise there was no systematic
evaluation of stereopure PS insertions into siRNA designs.
Preliminary work by Wave Life Sciences where a single
stereopure PS was introduced into the strands with limited in vitro
data suggested a potential benefit.[11] Earlier work primarily
[*] Corresponding Author: E-mail: ssakamuri01@gmail.com
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cbic.201900630
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focused on DNA, methods to generate stereopure PS insertions,
with minimal/no systematic work carried out on double-stranded
siRNAs.[8c, 9b, 12] This prompted us to systematically evaluate the
effect of PS chirality on the function of double-stranded siRNA and
identify
a practical method to synthesize stereopure PS
containing oligonucleotides. This article summarizes our efforts
on the former topic and the latter is forthcoming.[16b]
Scheme 2. Synthesis of Monomeric GalNAc-Azide (10).
Our systematic investigation started with guide (G)-strand
PS insertions followed by passenger (P)-strand insertions and
then combining G-strand and P-strand variations to generate
stereopure siRNAs. Synthesis of all sixty-four (26) stereopure
isomers generated from 6 PS insertions is required to establish
the role of chirality at individual locations as well as
interdependency from different locations. The X2-linker insertion
also created a new phosphotriester stereocenter. To rule out any
potential role of phosphotriester chirality on P-strand 3’-GalNAc
conjugates we determined that chiral phosphotriester-GalNAc
conjugates had no measurable effect on biological outcome
(unpublished data). It’s important to highlight that our X2-linker
and GalNAc monomer were synthesized in 4 and 5 simple steps
respectively. Trivalent GalNAc conjugate is made by click reaction
of monomer GalNAc-azide (10) to X2-linked oligonucleotide.
Figure 2. Structures of siRNA and X2 linker-GalNAc. P1, P2, G1, G2, G21 and
G22 Identify PS Locations. Structure of X2-GalNAc Used in this Study is
Attached to Passenger Strand 3’-End.
For our current investigation, we evaluated an AT3 siRNA
as shown in Figure 2 which features fully 2’-modified sequences
having six phosphorothioates, two at 5’- end of the passenger
strand, the 5’-, and 3’- ends of the guide strand.[10] Monomeric
GalNAc ligands were linked to novel X2 linker via click
conjugation.[13] Synthesis of X2-linker phosphoramidite 5 and
monomeric GalNAc-azide 10 is illustrated in Scheme 1 and 2
respectively. Experimental protocols along with assembly of
oligonucleotides, click conjugation and biological testing can be
All isomers of AT3 siRNA-GalNAc conjugates and controls
were tested in vitro in primary mouse hepatocytes and in vivo
(protocols described in supplementary information). Our
preliminary observations of in vitro and in vivo data suggested
similar trends (Figure 5a and Table 2, supplementary information),
but the narrow range of in vitro data precluded meaningful
conclusions. We primarily limited the in vitro data to general
comparisons.
found in the supporting information.
We used Wada’s
oxazaphospholidine P(III) chemistry, well known for excellent
diastereoselectivity (≥99%) to insert stereopure PS into 2’-
substituted (2’-OTBDMS, 2’-OMOE, 2’-OCEM and 2’-Deoxy)
oligonucleotides.[8b-f]
We opted to focus on in vivo studies which demonstrate a
combination of stability and duration of action to understand
potential therapeutic benefits. In early experiments, we compared
the impact of compounds on mouse hepatocyte AT3 gene
expression as well as on secreted AT3 protein in the circulation.
We found nearly perfect correlation between the two parameters
and thus limited all subsequent experiments to measuring plasma
AT3 activity. We tested all compounds in vitro at three
concentrations (1, 3.3, and 10.0 nM) and dosed in vivo at 0.25
mg/kg which represented the EC50 value for AT3 mRNA
knockdown of the racemic control SB1391. Statistical analysis on
some of the compounds efficacy is included in the supplementary
information.
We first explored guide-strand PS insertions at G1 and/or
G2 (Table 1). Unexpectedly, the in vivo data revealed nearly half
of the isomers were not efficacious or poorly efficacious (Figure 3
and Table 2) indicating a critical role for guide-strand 5’- PS
stereochemistry for the sequence evaluated. In particular, G1S
Scheme 1. Synthesis of X2-Phosphoramidite (5).
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10.1002/cbic.201900630
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PS stereochemistry is detrimental to the efficacy and was
common to all less efficacious/inactive molecules. We can infer
that half of all isomers (32/64) in a typical SB1391 design
contribute very little to in vivo pharmacology. It is important to note
some of these isomers with G1S PS stereochemistry show
moderate in vitro activity (supplementary information, Table 2). It
is known that R- and S- PS isomers of DNA have different
nuclease stability and binding characteristics.[6c] Our data highlight
the efficacy of stereopure PS insertion at guide-strand G1 position
does not depend entirely on nuclease stability. It is possible 5’
guide-strand PS stereochemistry could influence the G1
phosphorylation required for RISC loading or Ago2 binding.
As the siRNA field continues to modify the pattern of 2’-F
and 2’-OMe ribose substitutions, generally increasing the 2’-
OMe/2’-F ratio[14], we optimized 2’-OMe substitutions on the same
AT3 siRNA sequence. We investigated whether the superior
chiral PS performance would hold for SB2333 with only seven 2’-
F substitutions versus nineteen in SB1391 (Table 1 and Figure 6).
Molecule SB2477 incorporated the improved 2’OMe/2’-F ratio and
the chiral PS pattern from SB2222 (G1R, G2R, G21S, G22R;
Table 1). SB2477 recapitulated the strong in vivo efficacy of
SB2222 suggesting the translatability of our findings to the current
2’-mod pattern (Figure 7 and Figure 7SI in Supplementary info).
0.25 mg/kg S.C. Single Dose
Saline
0.25 mg/kg S.C. Single Dose
Saline
SB1391 (mixture)
SB1391 (mixture)
100
100
SB2210 (G21S)
SB2078 (G1R)
SB2115 (G1S)
SB2211 (G21R)
75
75
SB2116 (G2S)
SB2212 (G22S)
50
SB2117 (G2R)
50
SB2213 (G22R)
SB2079 (G1R, G2R)
SB2214 (G21S, G22S)
SB2215 (G21S, G22R)
25
0
25
0
SB2118 (G1S, G2S)
SB2119 (G1S, G2R)
SB2120 (G1R, G2S)
SB2216 (G21R, G22S)
SB2217 (G21R, G22R)
0
5
10 15 20 25 30 35 40 45 50 55 60 65
Time (d)
0
5
10 15 20 25 30 35 40 45 50 55 60 65
Time (d)
Figure 4. Mouse In Vivo Efficacy Data of G-Strand 3‘-Stereopure PS
Insertions Show G21S,G22S Preferred Orver R-Isomers.
Figure 3. Mouse In Vivo Efficacy Data of G-Strand 5‘-Stereopure PS
Insertions Show G1S is Detrimental to Efficacy.
0.25 mg/kg S.C. Single Dose
Saline
SB1391 (mixture)
100
Next, we examined stereopure PS insertions at guide-
strand 3’-locations, G21 and G22 in isolation with all other PS
racemic. The impact of PS stereochemistry in this data set (Figure
4 and Table 2) was less pronounced than guide-strand 5’-end
modifications but mirrors likely higher nuclease stability of S-
isomers as the S- isomer combinations were found to be more
efficacious with longer duration of action. For example, SB2214
(SS isomer) and SB2217 (RR isomer) have similar in vitro potency
but significantly different in vivo profiles (Table 2 and Figure 4). In
addition, as an independent location or in combination with other
G-strand 3’- stereopure PS positions, G21S was preferred over
G21R (Figure 4SI, Figure 5SI, Supplementary info).
SB2220 (G1R, G2R, G21R, G22R)
SB2221 (G1R, G2R, G21R, G22S)
75
SB2222 (G1R, G2R, G21S, G22R)
50
SB2223 (G1R, G2R, G21S, G22S)
SB2224 (G1S, G2S, G21R, G22R)
25
0
SB2225 (G1S, G2S, G21R, G22S)
SB2226 (G1S, G2S, G21S, G22R)
SB2227 (G1S, G2S, G21S, G22S)
SB2228 (G1R, G2S, G21S, G22S)
0
10
20
30
40
50
60
70
Time (d)
SB2229 (G1S, G2R, G21S, G22S)
SB2230 (G1R, G2S, G21R, G22R)
SB2231 (G1S, G2R, G21R, G22R)
Figure 5. Mouse In Vivo Efficacy Data of Steropure G-Strand PS Insertions
Demonstrate only SB2221, SB2222 and SB2223 Show Comparable or
Superior Efficacy Than Sterorandom control (SB1391).
Since both 5’- and 3’- G-strand isomers contributed to in vivo
profile, we next combined stereopure insertions at all four
locations while maintaining a racemic passenger strand. Strikingly,
twelve of the sixteen siRNA isomers made, only three stereopure
G-strands SB2221 (G1R, G2R, G21R, G22S), SB2222 (G1R,
G2R, G21S, G22R) and SB2223 (G1R, G2R, G21S, G22S)
showed comparable or superior efficacy versus racemic control
SB1391 (Figure 5 and Table 2). The remaining isomers were not
efficacious or less efficacious implying the in vivo activity of the
racemic mixture significantly relies upon a fraction of the total drug
product. All molecules with poor efficacy contain G1S
stereochemistry, further confirming our learning from Figure 3.
Based on this data we concluded that G1R stereochemistry is
critical, G2R is important, G21S is preferable and G22 tolerates
R- and S- isomers.
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Figure 5a. In Vitro and In Vivo Data (Mouse) Comparison of Steropure G-
Strand PS Insertions Show Similar Trends.
and stereopure guide-strands (Figure 8a, Figure 8b and Table 2).
Based on our findings that G1S is detrimental to efficacy, we
limited this set to G1R containing isomers reducing the total
compounds to 32, of which 25 were synthesized. Consistent with
earlier data, a significant number of stereopure siRNAs have poor
efficacy when compared to the stereorandom control SB1391.
Only a subset of stereopure siRNAs (SB2277, SB2284, SB2301,
SB2308, SB2313 and SB2320) were comparable in efficacy to the
control. To our surprise, the disproportionate loss of efficacy for
most isomers did not translate to significant gain for the active
isomers. It is possible that extracellular stability, unknown uptake
mechanisms, limited Ago2 binding/loading, selective transport of
preferred isomers out of the endosome and other factors may be
influencing the stereopure siRNA efficacy and warrants further
mechanistic investigation. We also confirmed the impact of
increased 2’-OMe/2’F ratio and chiral PS insertions of key AT3
sequence and an additional gene target (PCSK9) in cynomolgus
monkey primary hepatocytes (Table 1 and Table 3).
Figure 6. AT3 siRNA Sequences With Varying 2‘-OMe Modifications.
0 .5 m g /k g S .C . S in g le D o s e
1 0 0
S aline
S B 1 3 91 (M ixtu re)
7 5
S B 2 3 33 (M ixtu re)
5 0
S B 2222 (G 1R ,G 2R ,G 21S ,G 22R )
S B 2477 (G 1R ,G 2R ,G 21S ,G 22R )
2 5
0
Since it appears that only ~10% of the 64 possible isomers
disproportionately contribute to efficacy, future studies would be
important to understand underlining reasons to help design better
molecules. As advances in siRNA dosing and delivery beyond
liver into more technically demanding tissues emerge, the
contribution of isomers to clinical profile may increase in
importance.[15]
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
8 0
9 0
T im e (d )
Figure 7. Mouse In Vivo Efficacy Data Comparison of Steropure G-Strand PS
Insertions With Two Different 2‘-Mod Patterns Confrim Translatability of Our
Learnings .
In summary, our systematic evaluation of stereopure PS
insertions into double-stranded siRNA demonstrated only certain
isomers have comparable efficacy to the stereorandom control.
Based on our data we conclude that G1R stereochemistry is
critical, G2R is important, G21S is preferable, G22 and P1/P2
tolerates both isomers. Importantly, the approaches translate
across two different sequences and ribose 2’-modification
patterns using 2’-Fluoro and 2’-OMe substitutions. In addition,
recent progress towards simplified synthetic methods[16] could
complement our studies and are a step in the right direction
towards stereopure siRNA therapeutics.
Saline
0.25 mg/kg S.C. Single Dose
SB1391
SB2274 (G1R,G2R,G21R,G22R,P1R,P2R)
SB2276 (G1R,G2S,G21R,G22R,P1R,P2R)
100
SB2277 (G1R,G2R,G21S,G22R,P1R,P2R)
75
SB2278 (G1R,G2R,G21R,G22S,P1R,P2R)
SB2280 (G1R,G2S,G21S,G22S,P1R,P2R)
50
SB2284 (G1R,G2R,G21S,G22S,P1R,P2R)
25
SB2289 (G1R,G2R,G21S,G22R,P1S,P2R)
SB2296 (G1R,G2R,G21S,G22S,P1S,P2R)
0
SB2298 (G1R,G2R,G21R,G22R,P1R,P2S)
SB2300 (G1R,G2S,G21R,G22R,P1R,P2S)
0
10 20 30 40 50 60 70 80
Time (d)
Figure 8a. Mouse In Vivo Efficacy Data of Steropure siRNAs Show Only a
Small Number of Isomers Contribute to Efficacy.
Keywords: Oligonucleotides • Stereopure Phosphorothioates •
Stereopure siRNA Design •
0.25 mg/kg S.C. Single Dose
Saline
SB1391
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75
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COMMUNICATION
Sukumar Sakamuri,* Laxman Eltepu,
Dingguo Liu, Son Lam, Bryan R Meade,
Bin Liu, Giuseppe Dello Iacono, Ayman
Kabakibi, Lena Luukkonen, Tom
Leedom, Mark Foster, and Curt W
Bradshaw
P1 P2
Ligand
G21
G22
G1
G2
(2)6 = 64 Isomerically pure compounds
Potential for future stereopure siRNA therapeutics!
Phosphorothioate (PS) isomers
Page No. – Page No.
Impact of Phosphorothioate Chirality
on Double-stranded siRNAs: A
Systematic Evaluation of Stereopure
siRNA Designs
Current siRNA designs contain phosphorothioate modifications for nuclease stability generating isomeric mixtures. We carried out
first systematic evaluation to understand the impact of stereopure phosphorothioate incorporations into siRNA and found only ~10%
of the 64 possible isomers disproportionately contribute to efficacy. We defined preliminary design rules for stereopure siRNAs.
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