Conjugation of mono and di-GalNAc sugars enhances the potency of antisense oligonucleotides via ASGR mediated delivery to hepatocytes

Article abstract of DOI:10.1016/j.bmcl.2016.05.084

Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a high affinity ligand for the asialoglycoprotein receptor (ASGR)—a C-type lectin expressed almost exclusively on hepatocytes in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single stranded chemically modified ASOs can enhance potency 5–10 fold in mice. Evaluation of the mono- and di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance binding to the receptor and provide a rationale for the enhanced potency.

Full text of DOI:10.1016/j.bmcl.2016.05.084

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Conjugation of mono and di-GalNAc sugars enhances the potency of
antisense oligonucleotides via ASGR mediated delivery to
hepatocytes
Garth A. Kinberger, Thazha P. Prakash, Jinghua Yu, Guillermo Vasquez, Audrey Low, Alfred Chappell,
⇑
Karsten Schmidt, Heather M. Murray, Hans Gaus, Eric E. Swayze, Punit P. Seth
Ionis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced
potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a high affinity ligand
for the asialoglycoprotein receptor (ASGR)—a C-type lectin expressed almost exclusively on hepatocytes
in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single
stranded chemically modified ASOs can enhance potency 5–10 fold in mice. Evaluation of the mono- and
di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance
binding to the receptor and provide a rationale for the enhanced potency.
Received 5 May 2016
Revised 24 May 2016
Accepted 27 May 2016
Available online xxxx
Keywords:
ASGR
GalNAc
ASO
Ó 2016 Elsevier Ltd. All rights reserved.
Delivery
Hepatocytes
Antisense oligonucleotides bind their cognate mRNA in cells by
Watson–Crick base pairing. Upon binding, they modulate function
of the mRNA via one or more antisense mechanisms to produce a
somal compartments.11 Acidification of endosomal compartments
promotes dissociation of the ligand–receptor complex. The recep-
tor is recycled back to the plasma membrane while the cargo is
sorted to lysosomes for degradation.
1
pharmacological effect. Second generation ASOs are phosphoroth-
ioate (PS) backbone modified with a central gap region of 7–14
Mice lacking the ASGR2 subunit are viable and fertile but
express the ASGR1 subunit at reduced levels. Cells expressing
0
12
DNA nucleotides flanked on either end with 2 -modified
2
nucleotides. The DNA gap region promotes cleavage of the tar-
ASGR1 alone are capable of binding ligand but substantial binding
3
0
13
geted mRNA by RNase H1 mediated hydrolysis, while the 2 -mod-
is dependent on the level of expression of the protein. In contrast,
4
ified nucleotides enhance ASO affinity for cognate RNA. We
mice lacking the ASGR1 subunit do not bind ligand and do not
1
4
recently showed that the potency of second generation ASOs for
suppressing gene targets expressed in hepatocytes can be
enhanced 10-fold by targeted delivery via the Asialoglycoprotein
express ASGR2 on the plasma membrane. The ASGR1 possesses
the carbohydrate recognition domain (CRD) for calcium mediated
1
5
sugar binding and the cytoplasmic signal for binding clathrin
5
,6
16
receptor (ASGR).
adaptor proteins within coated pits.
The ASGR is a C-type lectin which is abundantly expressed on
hepatocytes of all mammals and regulates levels of plasma glyco-
Elegant work by Lee showed that synthetic glycosides with
branched tethers bind the ASGR with high affinity.1
7,18
Binding
proteins terminating with sialic acid
a
2,6 galactose and N-acetyl
affinity was dependent on the nature of the sugar (GalNAc > galac-
tose), number of sugars (4 = 3 > 2 > 1) and the geometrical spacing
galactosamine (GalNAc) sugars.7 The functional receptor in mam-
,8
1
9
mals is comprised of two subunits (ASGR1 and ASGR2) which form
a hetero-oligomeric complex with varying ratios (2–5:1). The
ASGR is internalized via clathrin mediated endocytosis in coated
pits on the basolateral membrane of hepatocytes.10 Upon internal-
ization, the ligand–receptor complex is transported to endo-lyso-
between the sugar moieties. Given the complexity of ASGR bind-
ing and trafficking pathways, we wished to determine optimal
valency of GalNAc sugars required for efficient ASGR-mediated
delivery of ASOs to hepatocytes. In this manuscript, we show that
second generation ASOs of different configurations can be effec-
tively delivered to hepatocytes in mice using two and even a single
GalNAc sugar. Our results suggest that the chemical features of
9
⇑
Corresponding author.
E-mail address: pseth@ionisph.com (P.P. Seth).
http://dx.doi.org/10.1016/j.bmcl.2016.05.084
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Kinberger, G. A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.084

2
G. A. Kinberger et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
R2
HN
second generation ASOs enhance interactions of the sugar ligand
with the ASGR which facilitates delivery to hepatocytes.
R2
HN
(
CH
2
)
4
2 4
(CH )
i
O
For the current study, we prepared GalNAc-ASO conjugates
with three (GN3), two (GN2) and one (GN1) sugars using a
lysine-based scaffold similar to that described by Valentijn for
preparation or tri-antennary GalNAc clusters (Fig. 1).20 Briefly,
the syntheses commenced with the mono-GalNAc structure
H
N
v
R1
R1
OH
2
3
N
H
N
H
R
m
O
O
(
2 4
CH )
HN
1
2
1
1
: R =Ac; R =Boc
R
1
2
1
2
3
0: R ,R =Boc
3: R =Ac; R =Boc; m=0; R =2, 93%
a
e
derived from N -acetyl-N -Boc-lysine (1), the di-GalNAc com-
1
2
3
1
1: R ,R =Boc; m=0; R =2, 85%
a
e
pound derived from N -Boc-N -Boc-
L-lysine (10) and the tri-
ii
1
2
3
1
7: R ,R =Boc; m=1; R =OMe, 78%
GalNAc cluster derived from the HBTU facilitated condensation of
iii
i
1
2
3
e
18: R ,R =Boc; m=1; R =OH, quant
compound 10 with N -Boc-
The methyl ester of 17 was saponified to prepare the free acid
8. At which point, compounds 1, 10 and 18, were subjected to
L-lysine methyl ester (17) (Scheme 1).
1
2
3
R2
19: R ,R =Boc; m=1; R =2, 94%
HN
1
2 4
(CH )
O
OAc
OAc
H
N
analogous synthetic routes. Uronium mediated coupling of the
lysine derivatives to benzyl 6-aminohexanoate afforded com-
pounds 3, 11, and 19. Subsequent acid treatment provided the
mono, di and tri-amine structures 6, 12 and 20. GalNAc hexanoic
R1
O
3
O
N
H
R
AcO
O
m
(
CH
)
R4
O
AcHN
2
5
(CH
2
)
4
HN
(GalNAc-C6)
2
R
2
1
1
2
3
4
4
acid (4) as well as the corresponding pentafluorophenyl (Pfp)
ester (5) were utilized to prepare 7, 13 and 21 according to
Scheme 1. The benzyl esters were removed via catalytic hydro-
genation and the Pfp esters were prepared utilizing PFPTFA to
6
1
2
. R =Ac; R =H; m=0; R =2, quant
iv
: R =OH
1
2
3
5: R =OPfp, 87%
4
2. R ,R =H; m=0; R =2, quant
1
2
3
0. R ,R =H; m=1; R =2, quant
R2
HN
vi
2
2
6
1
2
synthesize the mono, di and tri-GalNAc clusters 9, 15 and 23.
2 4
(CH )
O
viii, iv
vi
H
N
2
0
R
1
3
N
H
R
vii
m
O
(
CH
2
)
4
HN
R
O
2
A
1
2
3
O
H C
NH
7. R =Ac; R =GalNAc-C6; m=0; R =2, 79%
3
R2
1
2
3
13. R ,R =GalNAc-C6; m=0; R =2, 59%
OH
O
O
O
N
H
1
2
3
O
O
O
HN
21. R ,R =GalNAc-C6; m=1; R =2, 38%
HO
HO
HN
2 4
(CH )
O
GN1-ASO
H
N
NHAc
ASO
ASO
R1
OPfp
2
2 5
(CH )
OH
OH
N
H
N
mH
O
O
O
(
2 4
CH )
HO
AcHN
O
O
1
HN
R
9. R =Ac; R =GalNAc-C6; m=0, 82% (GN1-Pfp)
2
1
2
O
HN
15
. R ,R =GalNAc-C6; m=0, 97% (GN2-Pfp)
1
2
OH
O
23. R ,R =GalNAc-C6; m=1, 93% (GN3-Pfp)
1. 9, 15 or 23,
N
H
O
HO
HO
HN
O
O
GN2-ASO
DMSO, borate buffer,
pH 8.5
2
NHAc
H
2
N
P
O
O
4 2
. 30% NH OH/H O
OH
OH
PS, MBB or CET
O
HO
AcHN
O
O
O
O
H
N
OH
HO
OH
O
Conjugate
P
O
O
O
NH
PS, MBB or CET
O
Conjugate = GN1, GN2 or GN3
N
O
AcHN
H
Scheme 1. Reagents and conditions: (i) Benzyl 6-aminohexanoate (2), HBTU,
iPr EtN, DMF; (ii) H-Lys(Boc)-OMe, HBTU, Et N, DMF; (iii) LiOH, MeOH, THF, H O,
quant; (iv) PFPTFA, iPr EtN, CH Cl ; (v) 50%TFA/CH Cl ; (vi) 5, iPr EtN, DMF; (vii) 4,
HATU, iPr EtN, DMF; (viii) H , Pd/C, MeOH.
O
HN
2
3
2
O
OH
O
N
H
2
2
2
2
2
2
O
O
HO
HO
HN
2
2
GN3-ASO
ASO
NHAc
The pentafluorophenyl activated peracetylated GalNAc clusters
, 15 and 23 were conjugated to 5 -aminohexyl-modified ASOs fol-
B
0
9
H
N
(
)5oAoGsCsTsTsCsAsGsTsCsAsTsGsAsCsTsTsCsCsTsT (PS)
lowed by hydrolysis of the acetate protecting groups to provide the
conjugates represented by GN3-ASO, GN2-ASO and GN1-ASO,
respectively (Scheme 1). Three distinct ASO designs targeting the
scavenger receptor B-1 mRNA (SRB-1) were employed for the ini-
H
N
(
)5
oAoGsCoToToCoAsGsTsCsAsTsGsAsCsTsToCoCsTsT (MBB)
oAoTsCsAsGsTsCsAsTsGsAsCsTsTsCs (CET)
H
N
23
tial evaluation (Fig. 1). These included a fully phosphorothioate
(
)5
(
(
PS) modified 5-10-5 MOE gapmer (PS0) and its mixed-backbone
MBB) variant where six PS linkages in the flanks were replaced
C
O
O
O
with natural phosphodiester (PO) linkages. MBB ASOs show
reduced non-specific binding to intra and extra-cellular proteins
and GalNAc conjugation enhances potency of these designs relative
O
O
O
O
Bx
Bx
Bx
Me
O
P
O
P
O
O
P
6
O
O
O
to their full PS parents. We also investigated a truncated version of
O
O
MeO
O
X
X
X
ASO PS0 where the MOE nucleotides in the wings were replaced
with higher affinity constrained Ethyl (cEt) nucleotides (CET0).24
Shorter cEt ASOs show enhanced potency in animal models relative
X = O, PO DNA
X = S, PS DNA
X = O, PO MOE
X = S, PS MOE
X = O, PO cEt
X = S, PS cEt
5
,23
to their longer 5-10-5 MOE counterparts.
Presumably, the
Figure 1. (A) Structures of mono-, di- and tri-GalNAc conjugated ASOs (B) sequence
and backbone composition of ASOs and (C) structures of nucleoside modifications
used in ASOs.
reduced charge and molecular weight facilitates escape of shorter
ASOs from endosomal compartments.
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G. A. Kinberger et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Mice (C57Bl-6, n = 4/group) were injected subcutaneously with
A
Primary hepatocytes
ASO PS0 at 3, 10 and 30 mg/kg and its mono, di and tri GalNAc con-
jugates PS1, PS2 and PS3, respectively, at 0.5, 1.5, 5 and 15 mg/kg.
Additional mice were injected with the MBB mono, di and tri
GalNAc conjugates MBB1, MBB2 and MBB3, respectively, at 0.5,
1
20
PS0
100
MBB0
MBB3
MBB2
MBB1
8
0
1
.5, 5 and 15 mg/kg. Mice were sacrificed after 72 h and livers were
homogenized and analyzed for reduction in SRB-1 mRNA by quan-
titative RT-PCR. As observed previously, the full PS GN3-conjugate
PS3 exhibited 7-fold enhanced potency relative to the parent ASO
PS0 (Table 1, Supporting information). We also observed a 7-fold
enhancement in potency with the di-GalNAc conjugate PS2 and a
60
5
40
20
0
4
-fold enhancement with the mono-GalNAc conjugate PS1.
-9
-8
-7
-6
-5
Additionally, we also observed a 15-fold enhancement in potency
for MBB2 and MBB3 and a 5-fold enhancement for MBB1 relative
to the parent ASO PS0. The parent MBB0 showed very poor activity
in mice (<10% mRNA reduction at 30 mg/kg, Supporting
information).
Log Conc. (M)
B
ASGR Binding (Ki)
1
.2
MBB3
MBB2
MBB1
1.0
In a parallel experiment, mice (C57Bl-6, n = 4/group) were
injected subcutaneously with the cEt ASO CET0 at 0.6, 2.0, 6 and
0.8
2
0 mg/kg and its mono, di and tri GalNAc conjugates CET1, CET2
0
.6
.4
and CET3, respectively, at 0.2, 0.6, 2.0, 6 mg/kg. Once again, we
observed a 10-fold enhancement in potency with the di and tri-
GalNAc conjugates CET2 and CET3 while the mono-GalNAc
conjugate CET1 was 7-fold more potent as compared to the uncon-
jugated parent (Table 1).
0
0.2
.0
0
-11 -10 -9
-8
-7
-6
-5
-4
-3
Log Conc. (M)
To determine if the enhanced potency of the MBB ASOs with
mono, di and tri-GalNAc conjugation is also observed in cells, we
evaluated these conjugates in mouse primary hepatocytes which
retain ASGR expression.25 Freshly plated mouse hepatocytes were
treated with increasing concentrations of PS0, MBB0 and the con-
jugates MBB1, MBB2 and MBB3 in the absence of any transfection
agent to help deliver the ASO across the plasma membrane. After
incubation for 24 hours, the cells were lysed and the reduction in
SRB-1 mRNA was analyzed by quantitative RT-PCR. The di- and
tri-GalNAc conjugates showed a 30-fold increase in potency while
the mono-GalNAc conjugate showed a 6-fold increase relative to
the full PS parent ASO PS0 (Fig. 2 and Table 2). Interestingly, the
di- and tri-GalNAc conjugated MBB ASOs showed almost 180-fold
increase in potency relative to the parent MBB0, while MBB1 was
Figure 2. Dose–response curves for (A) reducing SRB-1 mRNA in mouse hepato-
cytes in culture (note overlapping curves for MBB2 and MBB3) (B) competition
125
binding assay for displacing
I-ASOR using ASO-conjugates.
Table 2
Activity of ASO and GalNAc ASO conjugates for reducing SRB1 mRNA in the liver in
mice
ASO
ASO backbone
GalNAc conjugate
Ki (nM)a
IC50 (nM)b
PS0
Full PS
PO/PS
PO/PS
PO/PS
PO/PS
None
None
None
GN1
GN2
GN3
Mono GalNAc
>10,000
>10,000
610
120
720
23
3.9
4.1
NA
MBB0
MBB1
MBB2
MBB3
None
40
15
3
0-fold more potent. The greater fold-enhancement in potency in
M29
870 l
the cells relative to the animal experiment, could be a result of
enhanced exposure (24 h) in culture which is not seen in animals
as ASOs and conjugates are rapidly cleared from the blood com-
partment into tissues after systemic injection.26
a
Inhibition constant for displacing 125I-ASOR in a competition binding assay.
ASO concentration for reducing SRB1-mRNA in fresh mouse hepatocytes in
b
culture without any transfection agent.
To determine if the enhanced potency of the GalNAc conjugates
was a result of enhanced interaction with the ASGR, ASO conju-
gates MBB1, MBB2 and MBB3 were evaluated for their ability to
displace 125I-labeled asialoorosomucoid (ASOR, K
10 nM)27 from
D
5
mouse hepatocytes as described previously. The MBB designs
were evaluated because they were expected to show lower non-
specific binding to membrane proteins as compared to ASOs with
Table 1
28
full PS designs. Both, the MBB2 and MBB3 ASO-conjugates were
Activity of ASO and GalNAc ASO conjugates for reducing SRB1 mRNA in the liver in
mice
125
able to displace
I-ASOR efficiently with inhibitory constants of
4
0 and 15 nM, respectively (Fig. 2 and Table 2). In contrast,
ASO
ASO backbonea
ASO length
GalNAc conjugate
ED50 (mg/kg)b
MBB1 showed a slightly lower inhibitory constant of 610 nM.
The above data were surprising because previous reports have
shown that di- and mono-galactose and GalNAc motifs have
PS0
PS1
PS2
PS3
MBB0
MBB1
MBB2
MBB3
CET0
CET1
CET2
CET3
PS
PS
PS
PS
PO/PS
PO/PS
PO/PS
PO/PS
PS
20-mer
20-mer
20-mer
20-mer
20-mer
20-mer
20-mer
20-mer
14-mer
14-mer
14-mer
14-mer
None
GN1
GN2
GN3
None
GN1
GN2
GN3
None
GN1
GN2
GN3
30
8.3
4.3
4.9
ꢀ30
6.1
1.8
2.2
4.3
0.6
1
0- to >1000-fold lower binding affinity for the ASGR compared
1
8,29
to the tri-valent GalNAc clusters.
An X-ray crystal structure
of the ASGR1 ectodomain shows that the sugar binding pocket is
1
5
relatively shallow and solvent exposed. As a result, monovalent
sugar ligands do not display high binding affinity for the ASGR
and multiple interactions with the hetero-oligomeric receptor are
PS
PS
PS
3
0
0.44
0.39
required to enhance avidity.
Our data suggest that the chemical features of the ASO perhaps
enhance interactions of the single GalNAc sugar with the hetero-
oligomeric receptor. However, at this time it is not clear if some
part of the ASO binds within the sugar binding pocket of another
a
PS indicates phosphorothioate, PO indicates phosphodiester.
Dose required for reducing SRB-1 mRNA in mouse liver by 50% relative to
b
untreated control mice.
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4
G. A. Kinberger et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
ASGR-1 sub-unit or if the negatively charged backbone interacts
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5
with basic amino acids on the exposed surface of the receptor.
Furthermore, our results are different from a recent report employ-
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Stockert, R. J. Physiol. Rev. 1995, 75, 591.
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3
1
ing a 13-mer gapmer ASO of a slightly altered chemical design. In
this report, the mono- and di-GalNAc conjugates showed reduced
activity relative to the tri-valent GalNAc ASO conjugate suggesting
that linker and ASO design could be additional contributors to
ASGR binding and uptake. Additionally, recent studies with double
stranded siRNA GalNAc conjugates have also demonstrated the
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3
1
2
1
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In conclusion, single stranded chemically modified ASOs can be
effectively delivered to hepatocytes using two and even a single
GalNAc sugar. This can significantly reduce the structural complex-
ity associated with manufacturing such conjugates for clinical
applications. While single GalNAc ASO conjugates are 5-fold more
potent than the parent ASO, they are still 2-fold less potent as com-
pared to the trivalent GalNAc designs. However, modified mono-
meric GalNAc sugars which enhance binding affinity for the
ASGR have been reported.34 Conjugates prepared using such mod-
ified GalNAc sugars could enhance the ASGR binding affinity and
activity of mono GalNAc ASO conjugates to match the activity pro-
file of the tri-valent designs. These efforts are currently underway
and the results will be reported in due course.
2
1
1
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Supplementary data
Supplementary data (experimental protocols for synthesis of
conjugates and for cell and animal experiments is provided) asso-
ciated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2016.05.084.
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Please cite this article in press as: Kinberger, G. A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.084