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T. Yamamoto et al. / Bioorg. Med. Chem. 24 (2016) 26–32
Table 1
72 h post-injection. As shown in Figure 2a, the knockdown
activities of GN1 and GN2 were similar to or lower than that of
the parent ApoB-ASO, whereas GN3 exhibited significantly
improved in vivo potency. In addition, serum total cholesterol
levels were consistent with the hepatic mRNA levels of GN1, GN2
and GN3, supporting their systemic activity (Fig. 2b). These results
indicate that the simplified monovalent GalNAc 1 can strongly
activate ASO in liver provided that three 1s are tethered to ASO
in a serial manner with bio-labile linkers.
Sequences and Tm values of GalNAc-conjugated or -unconjugated ASOs used in this
study
ID
Sequence (50–30)
Tm (°C)
ApoB
GN1
GN2
GN3
GN4
GN5
GN6
GN7
GN8
G^C^a^t^t^g^g^t^a^t^T^C^A
61
59
59
60
57
57
57
60
58
G^C^a^t^t^g^g^t^a^t^T^C^A^R^R^R
G^C^a^t^t^g^g^t^a^t^T^C^AR^R^R
G^C^a^t^t^g^g^t^a^t^T^C^ARRR
R^R^R^G^C^a^t^t^g^g^t^a^t^T^C^A
R^R^RG^C^a^t^t^g^g^t^a^t^T^C^A
RRRG^C^a^t^t^g^g^t^a^t^T^C^A
RG^C^a^t^t^g^g^t^a^t^T^C^A
2.4. Enhanced effect of 50-conjugation over 30-conjugation
RRRRRG^C^a^t^t^g^g^t^a^t^T^C^A
Studies using a conventional triantennary GalNAc with a single
branching point11 demonstrated that a different attachment direc-
tion (30-end or 50-end) of the monovalent GalNAc 1 can affect activ-
ity. The in vivo efficacies of GN4–6 were compared to determine in
which direction 1 should be elongated and how the three 1s should
be linked to each other. ApoB-ASO, GN4, GN5 and GN6 were singly
administered to male mice at doses of 35–140 nmol/kg. On day 3,
the livers were harvested and apoB expression was measured, and
blood was collected and total cholesterol was analyzed. GalNAc-
conjugated ASOs with bio-labile phosphodiester linkers (GN5 and
GN6) surprisingly enhanced apoB mRNA degradation (Fig. 3a).
GN6 was the most potent of the six ASOs (GN1-GN6) and provided
93% knockdown even at a dose of 35 nmol/kg (ꢀ0.2 mg/kg). In con-
trast, the 50-trimer linked using PS chemistry, represented by GN4,
did not significantly enhance the potency of the parent ASO. Serum
total cholesterol levels were consistent with the mRNA levels
(Fig. 3b). The advantage of 50-GalNAc over 30-congeners has been
reported for a conventional 3-in-1 type GalNAc, and a possible
mechanism underlying the superiority of 50-conjugation with
phosphodiester bonds suggests that their potential metabolic
susceptibility affects their fast liberation kinetics.11
Conditions: for a duplex formation with targeted RNA, 10 mM sodium phosphate
buffer (pH 7.2) containing 100 mM NaCl. The sequence of target; 50-CACU-
GAAUACCAAUGCUGAA-30. Upper and lower case indicate 20,40-BNA/LNA and nature
DNA, respectively.
R represents monovalent GalNAc unit and ^ indicates a
phosphorothioate (PS) linkage. Tm values were determined by averaging three
measurements, which were accurate to within 1 °C.
2.3. Enhanced in vivo efficacy of phosphodiester-linked
simplified GalNAc-conjugated ASO
A superior tethering strategy for the simplified GalNAc conjuga-
tion was identified by first comparing the in vivo knockdown
activities of GN1, GN2 and GN3. Normal chow-fed C57Bl/6J mice
(8-weeks old, male, N = 4 per group) were subcutaneously injected
with ApoB, GN1, GN2 or GN3 at a dose of 35, 70 or 140 nmol/kg
and the expression level of apoB mRNA in the liver was analyzed
a
140 nmol/kg
70 nmol/kg
35 nmol/kg
120
100
80
60
40
20
0
2.5. Comparison of hepatic distribution of GalNAc-conjugated
ASOs
The activity differences between GN1, GN2 and GN3 and GN4,
GN5 and GN6 could be due to the fact that a specific and/or non-
specific interaction between ASOs and endogenous substances
increases as the number of PS linkages increases, which may inter-
fere with a specific interaction of GalNAc with ASGPR.16 The inhi-
bitory effect of PS linkages on the receptor binding of GalNAc
was estimated by fixing the amount of intact ASO accumulated
in the liver using a previously described ELISA method.5 As shown
in Figure 3c, GalNAc conjugation of GN5 and GN6 provides an
ꢀ4-fold activation of hepatic uptake of ASO, but only a 2.5-fold
increase using fully phosphorothioated GN4, indicating that PS
can interfere with receptor binding. Calculations showed that 3%
of the ApoB and 13% of the GN6 dose was distributed in the liver.
GN5 and GN6 were similarly distributed in liver, but GN5 was less
potent than GN6, indicating that efficient GalNAc must be liberated
after the GalNAc-terminated ASO is taken up through the ASGPR
pathway. Further information regarding the precise subcellular
dynamics of GalNAc-terminated ASOs is required to optimize the
linker chemistry.
Saline
ApoB
GN1
GN2
GN3
b
140 nmol/kg
70 nmol/kg
35 nmol/kg
100
90
80
70
60
50
40
30
20
10
0
2.6. Onset of hepatotoxicity
The hepatotoxicity of chemically-modified ASOs is a major
concern regarding ASOs in clinical development17–20 but its
underlying mechanisms are unknown. We hypothesized that the
above-mentioned large influx of GalNAc-conjugated ASOs in the
liver may affect the toxicology of ASO; consequently, serum liver
transaminases (alanine-aminotransferase, ALT and aspartate-
aminotransferase, AST) were measured as biomarkers for acute
Saline
ApoB
GN1
GN2
GN3
Figure 2. Efficacy of GalNAc-conjugated or unconjugated ASOs. (a) Quantitative RT-
PCR analysis of apolipoprotein B (apoB) mRNA levels normalized to Gapdh mRNA
levels following subcutaneous injection of ApoB, GN1, GN2 or GN3 (35, 70 or
140 nmol/kg). (b) Decreased levels of serum total cholesterol after subcutaneous
injection of ASOs. Sera were harvested 3 days after the injection. Data shown are
presented as mean values + SD, N = 4.