Superior Silencing by 2[variant prime],4[variant prime]-BNANC-Based Short Antisense Oligonucleotides Compared to 2[variant prime],4[variant prime]-BNA/LNA-Based Apolipoprotein B Antisense Inhibitors

Abstract

The duplex stability with target mRNA and the gene silencing potential of a novel bridged nucleic acid analogue are described. The analogue, [superscript] 2 [variant prime][/superscript] , [superscript] 4 [variant prime][/superscript] - [superscript] BNA NC[/superscript] antisense oligonucleotides (AONs) ranging from 10- to 20-nt-long, targeted apolipoprotein B. [superscript] 2 [variant prime][/superscript] , [superscript] 4 [variant prime][/superscript] - [superscript] BNA NC[/superscript] was directly compared to its conventional bridged (or locked) nucleic acid ( [superscript] 2 [variant prime][/superscript] , [superscript] 4 [variant prime][/superscript] -BNA/LNA)-based counterparts. Melting temperatures of duplexes formed between [superscript] 2 [variant prime][/superscript] , [superscript] 4 [variant prime][/superscript] - [superscript] BNA NC[/superscript] -based antisense oligonucleotides and the target mRNA surpassed those of 2[variant prime],4[variant prime]-BNA/LNA-based counterparts at all lengths. An in vitrotransfection study revealed that when compared to the identical length [superscript] 2 [variant prime][/superscript] , [superscript] 4 [variant prime][/superscript] -BNA/LNA-based counterpart, the corresponding [superscript] 2 [variant prime][/superscript] , [superscript] 4 [variant prime][/superscript] - [superscript] BNA NC[/superscript] -based antisense oligonucleotide showed significantly stronger inhibitory activity. This inhibitory activity was more pronounced in shorter (13-, 14-, and 16-mer) oligonucleotides. On the other hand, the 2[variant prime],4[variant prime]-BNANC-based 20-mer AON exhibited the highest affinity but the worst [subscript] IC 50[/subscript] value, indicating that very high affinity may undermine antisense potency. These results suggest that the potency of AONs requires a balance between reward term and penalty term. Balance of these two parameters would depend on affinity, length, and the specific chemistry of the AON, and fine-tuning of this balance could lead to improved potency. We demonstrate that [superscript] 2 [variant prime][/superscript] , [superscript] 4 [variant prime][/superscript] - [superscript] BNA NC[/superscript] may be a better alternative to conventional [superscript] 2 [variant prime][/superscript] , [superscript] 4 [variant prime][/superscript] -BNA/LNA, even for "short" antisense oligonucleotides, which are attractive in terms of drug-likeness and cost-effective bulk production.

Tsuyoshi Yamamoto 1, 2 and Hidenori Yasuhara 1, 2 and Fumito Wada 1, 2 and Mariko Harada-Shiba 2 and Takeshi Imanishi 3 and Satoshi Obika 1

Recommended by Masayasu Kuwahara

1, Applied Biopharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
2, Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan
3, BNA Inc, 7-7-20 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan

Received 13 June 2012; Revised 17 August 2012; Accepted 18 August 2012

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Recently designed and synthesized high-performance modified-nucleic-acids (HiPerNAs) such as 2[variant prime]- O -methyl RNA (2[variant prime]-OMe), 2[variant prime]- O -methoxyethyl RNA (MOE), and 2[variant prime],4[variant prime]-bridged nucleic acid/locked nucleic acid (2[variant prime],4[variant prime]-BNA/LNA) have improved performance compared to phosphorothioate antisense oligonucleotides (AONs). HiPerNAs overcome the systemic antisense effects of these earlier antisense oligonucleotides and show promise as antisense therapeutics for the treatment of a variety of diseases [ 1- 5]. However, more potent and less toxic AONs are required, since several clinical trials of AON drugs carrying HiPerNAs have been recently terminated due to the lack of efficacy or because of safety concerns. In addition, toxicity and delivery problems remain [ 6- 8].

We previously described a unique modified nucleic acid, 2[variant prime],4[variant prime]-bridged nucleic acid (2[variant prime],4[variant prime]-BNA; also known as LNA) [ 9, 10]. Its high therapeutic efficacy is based on the extraordinarily high target binding of the original 2[variant prime],4[variant prime]-BNA/LNA-based AON. 2[variant prime],4[variant prime]-BNA/LNA-based AON is widely accepted as one of the most promising antisense drugs, so fine-tuning the structure of BNA is the key for further improving the therapeutic potency and toxicological properties of AON. In this context, Seth and coworkers developed BNA analogues and elucidated their potency and safety in vivo . They reported BNA or LNA analogues with 2[variant prime],4[variant prime]-BNA/LNA-like binding affinities and biological activities with increased nuclease resistance and reduced toxicity [ 11- 14]. We have also reported the synthesis and physicochemical properties of several novel BNAs, including 2[variant prime],4[variant prime]- ^{BNA COC} and 2,[variant prime]4[variant prime]- ^{BNA NC} [ 15- 17], and recently demonstrated the biological activity of a 2[variant prime],4[variant prime]- ^{BNA NC} -based AON. Our results indicate that 2[variant prime],4[variant prime]- ^{BNA NC} may be a better candidate as an antisense therapeutic [ 7]. 2[variant prime],4[variant prime]- ^{BNA NC} is a six-membered bridged structure containing a hydrophilic aminooxy moiety. A wide range of functional groups can be easily introduced, and the introduction of nitrogen atoms would improve the stability of the duplex by reducing repulsions between phosphates in the backbone [ 18, 19] (Figure 1). 2[variant prime],4[variant prime]- ^{BNA NC} -modified oligonucleotides have very high RNA affinity, similar to or even higher than their 2[variant prime],4[variant prime]-BNA/LNA counterparts. Moreover, 2[variant prime],4[variant prime]- ^{BNA NC} -modified oligonucleotides are more resistant to endonucleolytic cleavage by nucleases than their 2[variant prime],4[variant prime]-BNA/LNA counterparts [ 17]. Although there is limited information regarding the biological activity or therapeutic potency of 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs, we have demonstrated the high systemic effects and safety of 2[variant prime],4[variant prime]-BNA/LNA- and 2[variant prime],4[variant prime]- ^{BNA NC} -based 20-nucleotide-long (20-nt-long) AONs that target PCSK9 mRNA [ 7]. Additionally, Prakashet al. independently showed the high potency and the nontoxicity of 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs [ 11].

Figure 1: Structures of the BNAs. The chemical structure on the left is the original BNA, 2[variant prime],4[variant prime]-BNA/LNA, and the structure on the right is 2[variant prime],4[variant prime]- ^{BNA NC} .

[figure omitted; refer to PDF]

Straarup et al. recently shortened the length of 2[variant prime],4[variant prime]-BNA/LNA-based phosphorothioate AONs to eliminate the latent potency of 2[variant prime],4[variant prime]-BNA/LNA drugs [ 20]. These short phosphorothioate AONs contain central 6- to 10-nt-long DNA regions flanked by terminal 2- to 4-nt-long 2[variant prime],4[variant prime]-BNA/LNA segments. These short (12- to 14-nt) AONs would be beneficial in terms of target specificity. The introduction of only small numbers of modifications into short 2[variant prime],4[variant prime]-BNA/LNA-based AONs can greatly increase target affinity. Thus, short 2[variant prime],4[variant prime]-BNA/LNA phosphorothioate AONs can minimize length-dependent disadvantages such as phosphorothioate-related protein binding and RNase H inactivation [ 21- 23] while maintaining satisfactory affinity and specificity. Additionally, short AONs are easier to produce on a bulk scale and could exhibit more drug-like characteristics. Based on the assumption that the strand-shortening strategy is also applicable to 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs, we shortened 2[variant prime],4[variant prime]- ^{BNA NC} -based phosphorothioate AONs and directly compared their silencing activities against the corresponding 2[variant prime],4[variant prime]-BNA/LNA-based apolipoprotein B (apoB)-targeting AONs.

2. Materials and Methods

2.1. Oligonucleotides

A series of 2[variant prime],4[variant prime]-BNA/LNA-based antisense 10- to 20-nucleotide-long phosphorothioate gapmers reported previously by Straarup et al. [ 20] were prepared and used in this study . These AONs were designed with complementary target sites for both cynomolgus monkey and human apoB mRNA sequences. The 10- to 16-nt-long AONs can also target murine apoB mRNA (GenBank accession number NM_000384 and NM_009693 for human and mouse apoB mRNA, resp.). Additionally, we prepared 2[variant prime],4[variant prime]- ^{BNA NC} -based counterparts in which all the 2[variant prime],4[variant prime]-BNA/LNAs were substituted by 2[variant prime],4[variant prime]- ^{BNA NC} . The synthesis of 2[variant prime],4[variant prime]- ^{BNA NC} with pyrimidine bases was previously reported [ 16, 17]; the synthesis of 2[variant prime],4[variant prime]- ^{BNA NC} with purine bases is currently being optimized and will be reported elsewhere. All the modified oligonucleotides were synthesized by Gene Design, Inc. (Ibaraki, Osaka, Japan) using standard phosphoramidite procedures and purified using HPLC.

2.2. Thermal Melting Study of Duplexes

UV melting experiments were carried out using a SHIMAZU UV-1650 spectrometer equipped with a _{T m} analysis accessory. Equimolar amounts of two single-stranded oligonucleotides were dissolved in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl to give a final strand concentration of 4.0 μ M. The mixture was annealed by heating at 90°C followed by slow cooling to room temperature. The melting profile was recorded at 260 nm in the forward and reverse direction from 5 to 90°C at a scan rate of 0.5°C/min.

2.3. In Vitro Transfection Procedures

For AON transfection experiments, Huh-7 cells were seeded at 15 × ^{10 4} cells per well in 12-well plates. AONs were transfected by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's procedures. After a 4-hour transfection, cells were washed with PBS, fresh medium was added, and the cells were incubated for an additional 20 hours at 37°C. After incubation, cells were collected and subjected to analyses.

2.4. mRNA Quantification Procedures

Total RNA was isolated from cultured cells using an RNeasy Mini Kit (Qiagen) according to the manufacturer's procedure. Gene expression was evaluated by a two-step quantitative reverse transcription-PCR method. Reverse transcription of RNA samples was performed by using a High Capacity cDNA Reverse-Transcription Kit (Applied Biosystems, Foster City, CA), and quantitative PCR was performed using a Fast TaqMan Gene Expression Assay (Applied Biosystems). The mRNA levels of target genes were normalized to the GAPDH mRNA level. The following primer sets were used for quantitative PCR. For human apoB and GAPDH, assay IDs of Hs01071209_m1 and Hs02758991_g1 were used, respectively.

2.5. Western Blotting

Two days after transfection, the cultures were subjected to centrifugation at 4°C, 10,000 rpm for 15 min. Each supernatant was collected into an Amicon Ultra-4 Centrifugal Filter Ultracel PL-10k (Millipore) and centrifuged at 4°C at 3,000 rpm for 1 h, and then each supernatant was added to individual Vivaspin 500 units (Sartorius Stedim Biotech) and centrifuged at 4°C at 3,000 rpm for 0.5 h. Each sample (9 μ L) was added to 9 μ L of Novex Tris-Glycine SDS Sample Buffer (2x) (Invitrogen) and applied to a 3-8% NuPAGE Tris-Acetate Gel (Invitrogen). Electrophoresis was performed at 180 V for 130 min. The separated proteins were transferred to a PVDF membrane (Millipore) at 220 mA for 120 min. Membranes were then incubated with 10 mL of blocking buffer (Blocking One; Nacalai Tesque) for 12 h at 4°C. Membranes were successively incubated with primary antibody of anti-human ApoB antibody (R&D Systems) for 80 min at room temperature. Then, each membrane was washed with PBS containing 0.1% tween (PBST) 4 times. Membranes were incubated with goat anti-mouse IgG-HRP antibody (Santa Cruz Biotechnology) for 80 min at room temperature. Chemiluminescent detection was performed using an ECL Advance Western Blot Detection Kit (Amersham Biosciences) according to the manufacture's procedure. Bands were visualized using an LAS-4000 mini (Fujifilm).

2.6. Statistics

Application of linear regression techniques to plots of the expression of apoB mRNA levels versus the logarithm of transfection concentration allows estimation of the coefficient (slope) and intercept values using the following equation: apoB mRNA = Coefficient*log (Concentration) + Intercept. These estimates can be useful guides for the efficacies of the AONs. Linear regression analysis was applied to 500 bootstrap sample sets obtained from three independent cellular assays. The coefficient and intercept of each regression line were compared using Tukey's test or one-sample t -test.

3. Results and Discussion

To better understand the effect of strand shortening on 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs, a series of 2[variant prime],4[variant prime]- ^{BNA NC} -based antisense oligonucleotides of 10- to 20-nt-long phosphorothioate gapmers and 2[variant prime],4[variant prime]-BNA/LNA-counterparts were synthesized (Table 1). Formation of a stable duplex with the target mRNA is a minimum essential step of the onset of an antisense effect. We first evaluated the thermal stability of the duplex formed between each modified AON and the single-stranded 20-nt-long oligoribonucleotide complementary to ApoB-LNA-20 and ApoB-NC-20. As expected, the longer the strand or the greater the number of modifications, the higher the _{T m} value. Moreover, the _{T m} values of 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs surpassed those of their 2[variant prime],4[variant prime]-BNA/LNA-based counterparts for any given length (Table 1), in good agreement with previous reports [ 11, 17]. Note that the exact _{T m} values of LNAs in Table 1are different from those given by Straarup et al., due to differences in composition of the measurement buffer solutions and in the length of the complementary RNAs between the two studies.

Table 1: Oligonucleotides used in this study.

ID	Sequence	T m (°C)	IC50 (nM)
ApoB-LNA-20	5[variant prime]-TTCAGcattggtattCAGTG-3[variant prime]	75 ± 0.8	7.9 ± 1.7
ApoB-LNA-16	5[variant prime]-CAGcattggtatTCAG-3[variant prime]	64 ± 0.8	2.9 ± 0.4a
ApoB-LNA-14	5[variant prime]-AGCattggtatTCA-3[variant prime]	61 ± 0.2	1.4 ± 0.5b
ApoB-LNA-13	5[variant prime]-GCattggtatTCA-3[variant prime]	58 ± 0.2	2.8 ± 0.8a
ApoB-LNA-10	5[variant prime]-CattggtatT-3[variant prime]	25 ± 1.4	N.D.
ApoB-NC-20	5[variant prime]- TTCAG cattggtatt CAGTG -3[variant prime]	79 ± 1.0	11.4 ± 3.0
ApoB-NC-16	5[variant prime]- CAG cattggtat TCAG -3[variant prime]	69 ± 0.8	1.3 ± 0.3b
ApoB-NC-14	5[variant prime]- AGC attggtat TCA -3[variant prime]	64 ± 0.3	0.6 ± 0.1
ApoB-NC-13	5[variant prime]- GC attggtat TCA -3[variant prime]	61 ± 0.1	0.9 ± 0.1
ApoB-NC-10	5[variant prime]- C attggtat T -3[variant prime]	32 ± 0.4	N.D.

2[variant prime],4[variant prime]-BNA/LNA was shown in uppercase and 2[variant prime],4[variant prime]-BNA^NC was in italic. Natural DNA was shown in lowercase. All the linkages are phosphorothioated. We measured _{T m} and IC₅₀ values of all entries. _{T m} values were determined in three independent experiments (±SD). Nondetectable IC₅₀ values, due to low potency, were marked ND. ^a,b Pairs of two IC₅₀ values with superscript letters are NOT statistically significant.

We next used in vitro mRNA silencing assays to estimate the potency of 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs and to compare their potency directly to the corresponding 2[variant prime],4[variant prime]-BNA/LNA-based AONs. We used the Huh-7 human hepatoma cell line, which expresses high levels of apoB mRNA in cells and secrets its protein into the medium. Each AON was introduced using standard lipofection procedures. All the AONs, except the 10-mers, ApoB-LNA-10, and ApoB-NC-10, reduced apoB mRNA and protein expression (and hence secreted protein) levels in a dose-dependent manner in the cells and culture medium, respectively, (Figures 2(a)and 2(b)). ApoB-LNA-10 did not reduce apoB mRNA levels even at concentrations above 64 nM. This may be because ApoB-LNA-10 did not bind target mRNA at 37°C due to lack of affinity. ApoB-NC-10 also did not reduce apoB mRNA expression, despite its higher _{T m} value compared to that of ApoB-LNA-10. Application of linear regression techniques to plots of the expression of apoB mRNA levels versus the logarithm of transfection concentration allowed estimation of the coefficient (slope) and intercept values. Statistical comparison of these parameters between arms with the identical length would provide useful guides for the efficacies of the AONs. One-sample t -tests revealed that the coefficients (slopes) of the 10-mer AONs had statistically insignificant downward slopes, suggesting that these 10-mers have little or no silencing effect (Figure 3).

In vitro silencing properties of BNA-based AONs. (a) Various concentrations (15.6 pM-64 nM) of AONs were introduced into Huh-7 cells using Lipofectamine 2000. After 24-h incubation, the cells were collected, and the expression levels of apoB mRNA were determined. Data represent means ± SD. (b) Reduction of Apo B protein levels in the culture medium following transfection was confirmed by western blotting.

(a) [figure omitted; refer to PDF]

(b) [figure omitted; refer to PDF]

Boxplots of coefficients and intercepts of regression lines obtained from cellular assay data. Bold lines in the box indicate medians. Top and bottom lines of the box indicate upper and lower quartile points, respectively. Top and bottom lines in contact with a dotted line indicate the highest and lowest data point, respectively. Open circles indicate outliers.

(a) [figure omitted; refer to PDF]

(b) [figure omitted; refer to PDF]

A length-dependent decrease in potency in 14- to 20-nt-long 2[variant prime],4[variant prime]-BNA/LNA and 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs was observed (Table 1and Figure 1). In each series of AONs, the ApoB-LNA-14 and ApoB-NC-14 were the most potent, whereas the ApoB-LNA-13 was the most potent AON reported by Straarup et al. [ 20]. IC₅₀ values of the 15-, 14-, 13-, and 12-mer AONs were so close to each other ( [approximate] 0.5 nM) that Straarup et al. confirmed the order by conducting an in vivo silencing study and showed that 12- and 13-mer AONs are the most potent. In contrast, we confirmed the order of in vitro silencing effects of all the entries in Table 1by using larger 12-well culture dishes. These differences in experimental conditions ( in vivo versus in vitro ) may explain the differences in the IC₅₀ values of 2[variant prime],4[variant prime]-BNA/LNA-based AONs and their order of potency. An in vivo study might be necessary to estimate the true order. Nevertheless, we observed in both chemistries that shorter AONs (16-, 14-, and 13-mer) are statistically significantly more potent than 20-mer AONs. Because the 10-mers did not show any activity, activity is positively correlated with binding affinity and indicates the presence of a "threshold affinity." However, longer AONs with higher affinities did not exhibit higher activities. This suggests the presence of a "length penalty" [ 20, 22] and the presence of an "optimal affinity," which might be a more appropriate description than threshold affinity. The variables that independently govern "length penalty" and "optimal affinity" remain largely unknown. However, plasma and intracellular proteins preferentially bind to the phosphorothioate internucleotide linkages, and these linkages are known to control RNase H activity [ 23- 26]. Thus, the number of phosphorothioate linkages is partly related to the "length penalty."

Surprisingly, when compared to the identical length 2[variant prime],4[variant prime]-BNA/LNA-based counterpart, the corresponding 2[variant prime],4[variant prime]- ^{BNA NC} -based AON showed stronger inhibitory activity. The differential in inhibitory activity is more pronounced in shorter (13-, 14-, and 16-mers) AONs than in the 20-mer (Table 1and Figure 4). Indeed, as shown in Figure 3, statistical comparison using the Tukey test of the coefficients and intercepts of the regression lines, which indicate how efficiently and strongly AONs reduce apoB mRNA, revealed that 2[variant prime],4[variant prime]- ^{BNA NC} -based 13-, 14-, and 16-mers exhibit significantly stronger inhibitory activity than their 2[variant prime],4[variant prime]-BNA/LNA counterparts. These potency differences could not be explained simply by the higher _{T m} of the 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs compared to their 2[variant prime],4[variant prime]-BNA/LNA-based counterpart. 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs might exhibit less "length penalty" than their 2[variant prime],4[variant prime]-BNA/LNA-based counterpart. A weaker affinity for protein binding or lower RNase H inhibitory activity of 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs could cause this potency difference. On the other hand, Figure 4shows a plot of IC₅₀ versus _{T m} , in which ApoB-NC-20, which has the highest affinity, exhibited worse IC₅₀ value than ApoB-LNA-20. These findings suggest that very high affinity possibly undermines antisense potency, although binding affinity to the target generally correlates positively with potency in the case of traditional small molecule drugs. The precise onset mechanism giving rise to this phenomenon remains unclear due to the lack of experimental data. However, the U- or V-shaped curves in Figure 4clearly indicate that the potency of AONs is the result of a delicate balance of reward term and penalty term. Thus, fine adjustment to the optimal affinity and elimination of the penalties such as excess protein binding and RNase H inhibitory activity could result in superior efficacy, represented as the lowest point on the U- or V-shaped curve.

Figure 4: Relationship between target affinity and antisense potency. IC₅₀ as a function of _{T m} values of 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs (solid line) and 2[variant prime],4[variant prime]-BNA/LNA-based AONs (dotted line).

[figure omitted; refer to PDF]

In conclusion, we have shown that 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs targeting apoB mRNA have higher binding affinities to the target RNA than do 2[variant prime],4[variant prime]-BNA/LNA-based AONs. Additionally, in vitro transfection studies revealed the superior silencing effect of short 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs (<20-nt-long), indicating that 2[variant prime],4[variant prime]- ^{BNA NC} may have advantageous properties as short antisense drugs. We are currently investigating the potential and safety of 2[variant prime],4[variant prime]- ^{BNA NC} -based AONs as therapeutic drugs.

Acknowledgments

This work was supported by the advanced research for medical products mining program of the National Institute of Biomedical Innovation (NIBIO) and a research grant from the Ministry of Health, Labor, and Welfare (H23-seisakutansaku-ippan-004) of Japan. T. Yamamoto acknowledges the Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science (JSPS).

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