Design of Bifunctional Antisense Oligonucleotides for Exon Inclusion

Abstract

Bifunctional antisense oligonucleotide (AON) is a specially designed AON to regulate pre-messenger RNA (pre-mRNA) splicing of a target gene. It is composed of two domains. The antisense domain contains sequences complementary to the target gene. The tail domain includes RNA sequences that recruit RNA binding proteins which may act positively or negatively in pre-mRNA splicing. This approach can be designed as targeted oligonucleotide enhancers of splicing, named TOES, for exon inclusion; or as targeted oligonucleotide silencers of splicing, named TOSS, for exon skipping. Here, we provide detailed methods for the design of TOES for exon inclusion, using SMN2 exon 7 splicing as an example. A number of annealing sites and the tail sequences previously published are listed. We also present methodology of assessing the effects of TOES on exon inclusion in fibroblasts cultured from a SMA patient. The effects of TOES on SMN2 exon 7 splicing were validated at RNA level by PCR and quantitative real-time PCR, and at protein level by western blotting.

1 Introduction

Harnessing antisense oligonucleotides (AONs) to redirect the altered pre-messenger RNA (pre-mRNA) splicing and modulate target gene expression is an efficient therapeutic strategy for genetic disorders associated with alternative splicing. A number of AON approaches have been investigated on redirecting pre-mRNA splicing. The original strategy is to use AONs complementary to a cryptic splice site to prevent its use and favored selection of the authentic site [1]. This approach has been used regularly to alter the proportion of splice isoforms produced from mutated genes or alternative splicing units. In addition to blocking the splice sites, alternative splicing events are often controlled by regulatory proteins bound to exonic and intronic elements located beyond the alternative splice sites. A valid approach is to use AONs to directly target exonic or intronic elements by blocking the binding of regulatory proteins to these elements that are involved in pre-mRNA splicing. This strategy has been successfully used to augment the exon 7 inclusion in SMN2 gene by using a short AON to target an intronic splicing silencer (ISS) within the gene [2,3,4,5]. Nusinersen, an 18-mer AON annealing to the ISS-N1 element in SMN2 intron 7 is the first antisense drug approved by the US Food and Drug Administration (FDA) for treatment of any types of spinal muscular atrophy (SMA) [6,7,8]. This strategy has also been proved to be very effective in Duchene muscular dystrophy (DMD) by promoting the skipping of an exon in the DMD gene to restore the interrupted reading frame hence partial rescue of the functional dystrophin protein [9, 10]. Three AON drugs, eteplirsen for exon 51 skipping and golodirsen and viltolarsen for exon 53 skipping in the DMD gene, have been approved by the FDA for treatment of DMD [11,12,13].

The other splice switching approach is the use of bifunctional oligonucleotides to increase the number of positively or negatively acting signals in an exon or intron and to regulate the alternative splicing. The oligonucleotides were designed with one domain (the antisense domain) annealing to the target exon or intron, and another domain (the tail domain) containing a sequence that either recruits RNA binding proteins involved in pre-mRNA splicing [14] or is made of a synthetic protein domain covalently linked to the antisense domain [15]. This approach may be designed as targeted oligonucleotide enhancers of splicing (TOES) for exon inclusion [14, 16], or as targeted oligonucleotide silencers of splicing (TOSS) for exon skipping [17].

The effectiveness of TOES as a potential therapy for SMA by augmenting exon 7 splicing in SMN2 gene has been approved both in vitro in cellular model [14, 16] and in vivo in mouse model [18,19,20]. A bifunctional oligonucleotide targeted to SMN2 exon 7 was expressed in transgenic mice within a modified U7 snRNA gene. Expression of the TOES-U7 RNA in a mouse model of SMA produced a substantial improvement in function and lifespan [20]. Two other bifunctional oligonucleotides targeting the intronic splicing silencers in SMN2 intron 6 and intron 7, which have the dual effects of blocking the silencer and recruiting activator proteins, also showed the potential therapeutic effects in the transgenic mouse models of SMA [18, 19].

We describe here the details in design of bifunctional oligonucleotide for exon inclusion by correcting SMN2 exon 7 splicing as an example (Fig. 1). TOES oligonucleotides are designed to contain two domains, an antisense domain complementary to sequences of SMN2 gene and a tail domain comprising sequences known as binding moieties for splicing activator proteins. The following design principles for TOES oligonucleotides are followed: (1) the antisense sequence may anneal to the potential splicing silencer binding sites in either intron 6, exon 7 or intron 7, and should avoid any splicing enhancer binding sites; (2) a number of splicing enhancer motifs (e.g. SF2/ASF, SRSF1, and hTra2β1) may be included in the tail domain to improve the effectiveness of the oligonucleotides; (3) chemical modification can be applied to the antisense sequence, but not to the tail domain, which may inactive protein binding to the tail domain. The effects on exon inclusion are evaluated at RNA and protein levels in fibroblasts cultured from a patient with type II SMA carrying three copies of SMN2 gene.

Fig. 1

Design of TOES to promote SMN2 exon 7 inclusion. The sequence of SMN2 exon 7 is in upper case and the flanking introns in lower case. Nucleotide 6 in SMN2 exon 7 is T (in red). Two shaded sequences are the binding sites of Tra2β and SF2/ASF, respectively. TOES is designed with two functional parts, the antisense domain to anneal to nucleotides 2–16 in SMN2 exon 7, the tail domain containing 3 repeats of “GGAGGAC” motifs to recruit the SR protein SRSF1. Cap contains five nucleotides at the 5′-end of the tail (in green, which is chemically modified)

2 Materials

2.1 AON Design

• 1. Online software to identify splicing motifs, e.g., Human Splicing Finder (http://www.umd.be/HSF/HSF.shtml).

• 2. Online software to predict the secondary structures of the target gene and AONs (http://rna.urmc.rochester.edu/RNAstructure.html).

• 3. Online software to calculate oligonucleotide properties on annealing temperature, GC content, and self-complementary (http://biotools.nubic.northwestern.edu/OligoCalc.html).

2.2 Synthesis and Preparation of Bifunctional Oligonucleotides

• 1. Oligonucleotides are synthesized commercially by Eurogentec Ltd. (www.eurogentec.com).

• 2. RNase and DNase-free distilled water (see Note 1).

• 3. RNase and DNasefree 1.5 mL Eppendorf tubes.

2.3 Culture of Skin Fibroblasts from SMA Patient

• 1. Growth medium: Dulbecco’s modified eagle medium (DMEM), 10% Fetal Bovine Serum (FBS), 1% Glutamax.

• 2. Trypsin-EDTA.

• 3. Phosphate-buffered saline (PBS).

• 4. Incubator set at 37 °C and 5% CO₂.

2.4 Fibroblast Transfection

• 1. Transfection reagent (e.g. Lipofectamine 2000).

• 2. Reduced serum medium for transfection (e.g. Opti-MEM).

• 3. 6 well plate or 35 mm diameter culture dish.

• 4. Sterile 1.5 mL Eppendorf tubes.

2.5 RNA Extraction

• 1. RNA isolation kit.

• 2. β-mercaptoethanol.

• 3. 70% ethanol (molecular grade).

• 4. RNase-free water.

• 5. 1.5 mL RNase-free Eppendorf tubes.

• 6. NanoDrop spectrophotometer.

2.6 cDNA Synthesis

• 1. cDNA synthesis kit.

• 2. Thermocycler.

• 3. 0.2 mL PCR tubes.

2.7 Polymerase Chain Reaction (PCR)

• 1. cDNA template from Subheading 2.6.

• 2. Taq Polymerase.

• 3. Primers (10 μM forward primer and 10 μM reverse primer). Primers sequences are shown in Table 1.

• 4. PCR buffer: 10× PCR buffer, 10 mM dNTPs, 50 mM MgCl₂, Taq DNA polymerase (5 U/μL), and nuclease-free water.

• 5. 0.2 mL PCR tubes.

• 6. Thermocycler.

• 7. Tris–Borate–EDTA 1× (TBE) buffer.

• 8. Agarose.

• 9. DNA gel stain.

• 10. Loading buffer.

• 11. DNA ladder.

• 12. Gel imaging system.

Table 1 Sequences of primers used for exon 7 inclusion quantification by PCR and quantitative real-time PCR

2.8 Quantitative Real-Time PCR

• 1. cDNA template from Subheading 2.6.

• 2. qPCR primers (10 pmol/μL each). Sequences are shown in Table 1.

• 3. Universal SYBR Green Master Mix.

• 4. 96-well real-time PCR plate.

• 5. Sealing film.

• 6. Real-Time PCR Thermal Cycler.

2.9 Western Blotting

• 1. Protein extraction buffer: 0.25% SDS, 75 mM Tris–HCl (pH 6.8), or RIPA buffer.

• 2. Protease inhibitor cocktail tablets.

• 3. Pierce BCA Protein Assay Kit.

• 4. PBST washing buffer (PBS, pH 7.4, 0.1% Tween 20).

• 5. Mini gel tank and blot transfer set.

• 6. NuPAGE 10% Bis-Tris precast gels, LDS sample buffer (4×), SDS running buffer (20×), antioxidant, sample reducing buffer, transfer buffer (20×), methanol.

• 7. Protein molecular weight ladder.

• 8. PVDF membrane.

• 9. Odyssey blocking buffer for PVDF membrane blocking.

• 10. Antibodies: mouse anti-SMN monoclonal antibody (BD Transduction Laboratories), mouse anti-β-tubulin monoclonal antibody (Sigma), IRDye 800CW-conjugated goat anti-mouse secondary antibody (Li-Cor).

• 11. Odyssey imaging instrument to quantify western blot signals.

3 Methods

3.1 Design of Bifunctional Oligonucleotides

• 1. Predict the potential binding motifs of the negative splicing regulator heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in the target intron or exon sequences, using Human Splicing Finder online software.

• 2. Other splicing repressors, such as intronic splicing silencers and exonic splicing silencers, may also be identified in the literature. A number of annealing sites in intron 6, exon 7, and intron 7 have been reported to augment SMN2 exon 7 splicing by bifunctional AONs (Table 2) (see Note 2).

• 3. AONs, of 15–20 mer in length , are designed to anneal to the potential binding sites of hnRNP A1 or other splicing silencers.

• 4. The GC content of each AON sequence should be 40–65%, with an ideal content of approximately 60%.

• 5. Avoid four consecutive “G,” strong secondary structure or self-complementary sequences, and self-dimers.

• 6. Chemical modifications, e.g., 2′-O-methyl and locked nucleic acid (LNA), may be applied to the antisense sequence to improve stability and increase binding affinity.

• 7. Select the tail domain. Examples are listed in Table 2.

• 8. No chemical modifications are recommended in the tail domain except the cap sequence (Fig. 1) [16] (see Note 3).

Table 2 The reported bi-functional AONs designed for SMN2 exon 7 inclusion

3.2 Transfection of SMA Fibroblasts

• 1. Seed the cells in a 6-well plate at a concentration of 2 × 10⁵ cells per well, which gives 80% confluence on the next day.

• 2. Cells are cultured in 2 mL of growth medium for 24 h.

• 3. 24 h later, change the growth medium to 1 mL Opti-MEM and leave the cells in the incubator during the preparation of transfection mixes.

• 4. Prepare the transfection reagent mixes in sterile 1.5 mL tubes. For each sample, prepare two mixes: the first mix (Mix A) contains 100 μL Opti-MEM and 1 μL AON at desired concentration (e.g. 1 μL AON at 100 μM to get a 100 nM final concentration). While for the mock control add only 100 μL Opti-MEM. The second mix (Mix B) contains 100 μL Opti-MEM and 5 μL Lipofectamine 2000.

• 5. Mix the AON-containing tube (Mix A) with the lipofectamine-containing tube (Mix B) at a ratio of 1:1 (100 μL + 100 μL).

• 6. Incubate the transfection mix for 20 min at room temperature (RT).

• 7. Add 800 μL Opti-MEM in the transfection mix to top it up to a final volume of 1 mL.

• 8. Remove Opti-MEM from the 6 well plate and replace with 1 mL transfection mix in each well.

• 9. Incubate the plate for at least 6 h at 37 °C with 5% CO₂ (see Note 4).

3.3 Splicing Assay of Bifunctional AONs on SMN2 Exon 7 Inclusion at RNA Level

• 1. Extract RNA from SMA fibroblasts using RNeasy Mini Kit according to manufacturer’s instruction.

• 2. Reverse transcription: the cDNA is synthesized from 500 ng RNA using cDNA Synthesis kit according to manufacturer’s instruction.

• 3. PCR of SMN2 transcripts: Use 1 μL cDNA in a 25 μL PCR reaction with 500 pmol of each primer (Table 1), 200 μM of dNTPs, 1.5 mM MgCl₂, 2.5 units of Taq polymerase and 1× PCR buffer. The PCR amplification program is as follows: 1 cycle with 3 min at 94 °C (initial denaturation), 25–30 subsequent cycles of 30 s at 94 °C (denaturation), 30 s at 55 °C (annealing), and 30 s at 72 °C (extension), followed by a final 10-min extension at 72 °C. Check an aliquot of the PCR product (5–10 μL) in 1.5% agarose gel electrophoresis and SYBR safe DNA stain using an UV transilluminator. The top band is the full-length SMN2 product (505 bp). The lower band is the product without exon 7 (Δ7 SMN2, 451 bp).

• 4. Quantitative real-time PCR of full-length and Δ7 SMN2 transcripts: product specific primers (Table 1), cDNA and 1× PCR Master mix are mixed in a 20 μL PCR reaction. The program includes activation at 95 °C for 3 min, 40 cycles of 95 °C for 10 s, and 60 °C for 1 min. The cycle at which the amount of fluorescence is above the threshold (Ct) is detected. For quantification, it is possible to use the standard curve method produced from serial dilutions of cDNA from untreated SMA fibroblasts, or the ΔΔCt method. Normalize the ratios of full-length SMN2 and Δ7 SMN2 to a housekeeping gene (e.g. HPRT1 or GAPDH) (see Note 5).

3.4 Bifunctional AONs on Restoring SMN Protein Measured by Western Blotting

• 1. Remove culture medium from the well. Add 100 μL ice-cold lysis buffer to the cells. Keep on ice for 5–10 min. Collect lysates using cell scrapers to fresh 1.5 mL Eppendorf tube and homogenize thoroughly with pipette.

• 2. Centrifuge at 12,000 × g and 4 °C for 10 min. Transfer the supernatant to a fresh tube.

• 3. Measure protein concentration by a NanoDrop spectrophotometer using the Pierce BCA Protein Assay Kit according to the manufacturer’s instructions.

• 4. Load 5 mg total protein into NuPAGE precast gels and then electrophorese.

• 5. Transfer electrophoretically separated proteins from the gel to a PVDF membrane.

• 6. Block the PVDF membrane for 1 h in blocking buffer.

• 7. Incubate the membrane with the primary antibodies at 4 °C overnight on a shaker.

• 8. Wash the PVDF membrane for 3 × 10 min in PBST buffer.

• 9. Incubate the PVDF membrane with fluorescence secondary antibody for 1 h at room temperature.

• 10. Wash for 3 × 10 min in PBST and detect bands using the Odyssey Imaging software (Image Studio).

4 Notes

• 1. DEPC-treated RNase-free water should be avoided to dissolve oligonucleotides. Dissolved AONs should be aliquoted and stored at −20 °C and avoid repeated freeze-thaw.

• 2. For TOES design, the most efficient binding sites of the antisense domain will be the validated exonic or intronic splicing silencers. For exonic silencers, the binding site is favorable to the upstream of the exon.

• 3. If the antisense domain anneals to an exon, it should avoid inducing any potential exon skipping of the binding exon.

• 4. Chemical modification of all the RNA nucleotides through the entire tail domain may reduce the binding affinity to protein. However, chemical modification may be only added to the last five nucleotides at the 5′-end of the tail domain (cap, as shown in Fig. 1) to improve the stability while still keep its binding affinity.

• 5. The duration of transfection can be prolonged to overnight or 24 h. For cells less tolerant to lipofectamine transfection, shorter incubation period, e.g. 6 h, is recommended.

• 6. It is recommended at least two housekeeping genes are used in the quantitative real-time PCR assay.