2′-O-(N-(Aminoethyl)carbamoyl)methyl Modification Allows for Lower Phosphorothioate Content in Splice-Switching Oligonucleotides with Retained Activity

2′-O-(N-(Aminoethyl)carbamoyl)methyl (2′-O-AECM)-modified oligonucleotides (ONs) and their mixmers with 2′-O-methyl oligonucleotides (2′-OMe ONs) with phosphodiester linkers as well as with partial and full phosphorothioate (PS) inclusion were synthesized and functionally evaluated as splice-switching oligonucleotides in several different reporter cell lines originating from different tissues. This was enabled by first preparing the AECM-modified A, C, G and U, which required a different strategy for each building block. The AECM modification has previously been shown to provide high resistance to enzymatic degradation, even without PS linkages. It is therefore particularly interesting and unprecedented that the 2′-O-AECM ONs are shown to have efficient splice-switching activity even without inclusion of PS linkages and found to be as effective as 2′-OMe PS ONs. Importantly, the PS linkages can be partially included, without any significant reduction in splice-switching efficacy. This suggests that AECM modification has the potential to be used in balancing the PS content of ONs. Furthermore, conjugation of 2′-O-AECM ONs to an endosomal escape peptide significantly increased splice-switching suggesting that this effect could possibly be due to an increase in uptake of ON to the site of action.


5′-O-(4-Methoxytrityl)-2′-O-[(N-(trifluoroacetamidoethyl)carbamoyl)methyl]uridine (10).
Compound 9 (2.22 g, 5.04 mmol) was dried by evaporation of added anhydrous pyridine and dissolved in 50 mL of anhydrous DMF-pyridine (3:2 v/v) mixture. To the resulting solution, 4-methoxytritylchloride (1.87 g, 6.05 mmol) was added under nitrogen atmosphere and the reaction mixture was stirred at ambient temperature for 24 h. Solvents were partially removed under reduced pressure, cold saturated aqueous NaHCO3 was added to the residue and the mixture was extracted with ethyl acetate. The organic phase was washed with water, dried over Na2SO4, filtered and concentrated under reduced pressure. Traces of pyridine were removed by evaporation of added toluene.
The crude product was subjected to column chromatography using 0 to 10% methanol in dichloromethane as eluent to give compound 10 (2.98 g, 4.18 mmol, 83%

3′-O-(N,N-Diisopropylamino-(2-cyanoethoxy)phosphinyl)-5′-O-(4-methoxytrityl)-2′-O-[(N-(trifluoroacetamidoethyl)carbamoyl)methyl]uridine (11).
Compound 10 (1.71 g, 2.4 mmol) was dried by evaporation of added anhydrous THF and dissolved in 24 mL of the same solvent. To the resulting chilled (ice bath) solution, N,N′-diisopropylethylamine (2.09 mL, 12 mmol) was added under nitrogen atmosphere followed by 2-cyanoethyl N,Ndiisopropylphosphoramidochloridite (1.07 mL, 4.8 mmol). After 30 min, the ice bath was removed and the reaction mixture was stirred for another 2 h. The reaction was quenched by the addition of methanol (1.2 mL) and solvent was partially removed under reduced pressure. The residue was partitioned between ethyl acetate and 10% aqueous solution of NaHCO3 and the aqueous phase was re-extracted with ethyl acetate. The combined organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified via flash column chromatography using 0 to 10% acetonitrile in ethyl acetate containing 0.1% of triethylamine as eluent to give 11 (1.77 g, 81%

N 4 -Acetyl-2′-O-[(N-(trifluoroacetamidoethyl)carbamoyl)methyl]cytidine (15).
Acetic anhydride (0.145 mL, 1.54 mmol) was added to a solution of 14 (0.21 g, 0.31 mmol) in anhydrous pyridine (4 mL) and the reaction mixture was stirred at room temperature for 36 h. The reaction was quenched by 10% aqueous NaHCO3 and the mixture was extracted with dichloromethane. The organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure.
The crude residue (0.195 g) was dissolved in anhydrous tetrahydrofuran-acetonitrile (6 mL, 1:1), triethylamine trihydrofluoride (0.105 mL, 0.65 mmol) was added and the reaction mixture was stirred at room temperature for 4 h. Volatiles were evaporated in vacuo, and added toluene and dichloromethane were S8 evaporated. The crude product was purified via flash chromatography using 0 to 15% methanol in dichloromethane as eluent to afford compound 15 (

Synthesis of azido functionalized peptide (ELL-peptide)
The peptide was synthesized on a Biotage® Initiator+ Alstra™ microwave peptide synthesizer using manufacturer's protocol. The sequence was assembled on Rink Amide ChemMatrix® resin (Biotage) using Fmoc chemistry under inert gas (N2). The resin (

Analysing splice correction by RT-PCR
HeLa705, U2OS-705 and U-2 OS-mBTKi4 cells were treated with ONs at indicated concentrations as described above. After 72 h cells were harvested, by adding 350 μL RLT-buffer from the RNeasy mini kit (Qiagen) and frozen at -80°. After thawing the lysate RNA was extracted according to the manufacturer's protocol. cDNA was prepared from 500 ng RNA using the high capacity cDNA kit from Applied Biosystems. In the 705 system the splice pattern was analyzed by PCR using primers Fw705: 5′-TTGATATGTGGATTTCGAGTCGTC-3′ and Rev705: 5′-TGTCAATCAGAGTGCTTTTGGCG-3′ (Cyber-Gene); with the PCR program 5 min at 95°C; (30 s 95°C, 30 s 55°C, 30 s 72°C) for 29 cycles and 10 min at 72°C for final extension. To analyze the splice correction for the BTK ONs a multiplex PCR was performed using primers binding in the EGFPLuc gene Fwd: 5′-CTGGTGCCAACCCTATTCTCCTTC, and Rev: 5′-CCAGATCCACAACCTTCGCTTCAA, together with primers for the HPRT gene Fwd: 5′-GACTTTGCTTTCCTTGGTCAG and Rev: 5′-GGCTTATATCCAACACTTCGTGGG as a quality control for the RNA, and a program with 27 cycles with the annealing temperature set to 57 °C. The PCRs were conducted using the HotStarTaq plus master mix (Qiagen). The PCR products were run on 2% and 1.2% agarose gels respectively, in 0.5xTBE and with SYBR-safe (Thermo Fisher Scientific) to visualize the DNA fragments. Results were detected in a VersaDoc imaging system (Bio-Rad) and analyzed using the QuantityOne software (Bio-Rad). The ratio of correctly spliced RNA fragment as percentage of the total amount of fragments from the reporter gene was then calculated for each sample.

Cytotoxic analysis with WST-1 reagent
Cells were seeded at a density of 8x10 3 (U-2 OS_705 and Neuro-2a_705) cells per well in a 96-well plate and treated with 4 or 10 µM of the respective ON as described for the splice-switching assay. After 72 h, medium was removed and new medium supplemented with WST-1 reagent (final dilution 1:10) was added to the wells. The cells were incubated for 2 h at 37 °C, 5% CO2 in 95% humidity, followed by absorbance measurements at 450 and 650 nm, for formazan product absorbance and reference wavelength, respectively.

Data analysis
Data sets were expressed as means ± SEM. Statistical significance was determined by one-way ANOVA followed by comparison of each treatment with the group control by Fisher's Least Significance Difference (LSD) test (Graph Pad Prism 6 Software, Graph Pad Software, Inc.). In all cases P<0.05 was considered significant.

S28
Data S1. Purification details and analytical data for the synthesized ONs.
The crude deprotected ONs1-6, ON12, ON17, ON19 and ON22 were purified using RP HPLC on a Supelco Discovery ® BIO Wide Pore C18-5 (250 × 10 mm) column with 4 mL/min flow rate with a linear gradient from 0 to 50% of buffer B in buffer A over 45 min at 50 °C. The oligonucleotide-ELL-peptide conjugates (ON18, ON20 and ON23) were purified using RP HPLC on a Supelco Discovery ® BIO Wide Pore   dissolved in of anhydrous methanol (3.5 mL) was treated with ethylenediamine (0.16 mL, 2.37 mmol) at room temperature for 20 h. Volatiles were evaporated in vacuo and the crude residue was further treated with 25% aqueous ammonia (8 mL) at ambient temperature for 20 h. Water was partially evaporated under reduced pressure and the residue was freeze-dried. Small amount (25 mg) of resulted crude product was redissolved in Milli-Q water (1 mL) and purified using RP HPLC on C18 column (Supelco Discovery ® BIO Wide Pore C18-5) with a linear gradient from 0 to 50% of CH3CN-water (1:1) mixture over 20 min at 25 °C. tR = 14 min. ES-MS, calcd. m/z [M+H] + 345.14, found 345.14.    Figure S85. Additional toxicity analysis (WST-1) in three different cell lines with ONs19-24. Cells were seeded at a density of 7x10 3 cells per well in a 96-well plate the day before ON treatment using 1.0 μM of the respective ON as described for the splice-switching assay. After 72 h, medium was removed and new medium supplemented with WST-1 reagent (final dilution 1:10) was added to the wells. The cells were incubated for 2 h at 37 °C, 5% CO2 in 95% humidity, followed by absorbance measurements at 450 and 650 nm, for formazan product absorbance and reference wavelength, respectively. Values are presented relative to untreated cells. All analyses were performed in triplicate. No statistically significant differences were found between the tested ONs using Oneway Annova with multiple comparisons. N=3.