Peptide Conjugates of a 2′-O-Methoxyethyl Phosphorothioate Splice-Switching Oligonucleotide Show Increased Entrapment in Endosomes

Antisense oligonucleotides (ASOs) are short, single-stranded nucleic acid molecules that alter gene expression. However, their transport into appropriate cellular compartments is a limiting factor in their potency. Here, we synthesized splice-switching oligonucleotides (SSOs) previously developed to treat the rare disease erythropoietic protoporphyria. Using chemical ligation-quantitative polymerase chain reaction (CL-qPCR), we quantified the SSOs in cells and subcellular compartments following free uptake. To drive nuclear localization, we covalently conjugated nuclear localization signal (NLS) peptides to a lead 2′-O-methoxyethyl phosphorothioate SSO using thiol–maleimide chemistry. The conjugates and parent SSO displayed similar RNA target-binding affinities. CL-qPCR quantification of the conjugates in cells and subcellular compartments following free uptake revealed one conjugate with better nuclear accumulation relative to the parent SSO. However, compared to the parent SSO, which altered the splicing of the target pre-mRNA, the conjugates were inactive at splice correction under free uptake conditions in vitro. Splice-switching activity could be conferred on the conjugates by delivering them into cells via cationic lipid-mediated transfection or by treating the cells into which the conjugates had been freely taken up with chloroquine, an endosome-disrupting agent. Our results identify the major barrier to the activity of the peptide–oligonucleotide conjugates as endosomal entrapment.

The PS ligator bears a 3' terminal PS group that enables dimerization.To prevent spontaneous sulfur-oxygen exchange, the PS ligator was stored as a dimer.Immediately prior to use, an aliquot of the PS ligator was reduced by adding Tris(carboxyethyl)phosphine hydrochloride (TCEP HCl; Fluorochem M02624) or Bond-Breaker TCEP Solution, Neutral pH (Thermo Scientific 77720) to a final concentration of 50 µM.(D) LC-MS chromatogram for the reduced PS ligator.In (A) through (D), masses were calculated using the Oligowizard Nucleic Acid Calculator, an online tool available at http://oligowizard.com/.UV purity is expressed as percent area under the peak.BPS, biphenylsulfonyl; PS, phosphorothioate.

S12
Table S1.Oligonucleotides detected in HEL cell lysate following free uptake for 24 h.Data are mean molecules ± SEM for three biological replicates (n=3) and the same as those presented in Figure S10.ND, not detected.Percent of total molecules in treatment was calculated using mean values and the Avogadro constant.Values were rounded to the nearest hundredth.Average molecules per cell was calculated using 115,000 cells, assuming one doubling.Values were rounded to the nearest thousand.S3.Oligonucleotides detected in HEL cytoplasm following free uptake for 24 h.Data are mean molecules ± SEM for three biological replicates (n=3) and the same as those presented in Figure S10.ND, not detected.Percent of total molecules in cells was calculated using mean values in Table S1.Values were rounded to the nearest percent.Average molecules per cell was calculated using 115,000 cells, assuming one doubling.Values were rounded to the nearest thousand.

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Table S4.Oligonucleotide conjugates detected in HEL cell lysate following free uptake at 2 µM for 24 h.Data are mean molecules ± SEM for three biological replicates (n=3) and the same as those presented in Figure S19.Percent of total molecules in treatment was calculated using mean values and the Avogadro constant.Values were rounded to the nearest hundredth.Average molecules per cell was calculated using 115,000 cells, assuming one doubling.Values were rounded to the nearest thousand.S6.Oligonucleotide conjugates detected in HEL cytoplasm following free uptake at 2 µM for 24 h.Data are mean molecules ± SEM for two or three biological replicates (n=2 or n=3) and the same as those presented in Figure S19.Percent of total molecules in cells was calculated using mean values in Table S4.Values were rounded to the nearest percent.Average molecules per cell was calculated using 115,000 cells, assuming one doubling.Values were rounded to the nearest thousand.

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Table S7.Lead oligonucleotide conjugate detected in HEL cell lysate following free uptake of the conjugate in HEL cells for 24 h.Data are mean molecules ± SEM for three biological replicates (n=3) and the same as those presented in Figure S23.Percent of total molecules in treatment was calculated using mean values and the Avogadro constant.
Values were rounded to the nearest hundredth.Average molecules per cell was calculated using 115,000 cells, assuming one doubling.Values were rounded to the nearest thousand.S8.Lead oligonucleotide conjugate detected in HEL nuclear lysate following free uptake of the conjugate in HEL cells for 24 h.Data are mean molecules ± SEM for three biological replicates (n=3) and the same as those presented in Figure S23.Percent of total molecules in cells was calculated using mean values in Table S7.Values were rounded to the nearest percent.Average molecules per nucleus was calculated using 115,000 cells, assuming one doubling.Values were rounded to the nearest thousand.S9.Lead oligonucleotide conjugate detected in HEL cytoplasm following free uptake of the conjugate in HEL cells for 24 h.Data are mean molecules ± SEM for three biological replicates (n=3) and the same as those presented in Figure S23.Percent of total molecules in cells was calculated using mean values in Table S7.Values were rounded to the nearest percent.Average molecules per cell was calculated using 115,000 cells, assuming one doubling.Values were rounded to the nearest thousand.

Figure S1 .
Figure S1.LC-MS chromatograms for oligonucleotides.ON, oligonucleotide.Masses were calculated using the Oligowizard Nucleic Acid Calculator, an online tool available at http://oligowizard.com/.UV purity is expressed as percent area under the peak.

Figure S2 .
Figure S2.Melting data for oligonucleotides.(A) Representative dataset for single strands in 100 mM NaCl, 10 mM phosphate, 0.1 mM Na 2 EDTA, pH 7.0.(B) Representative dataset for the oligonucleotides paired with a 22-nt RNA representing the c.315-48C FECH pre-mRNA in the same buffer.In (B), T m s were determined from first derivative analyses of nonlinear fit melting curves.Data are mean ± SD for three technical replicates.T m , melting temperature.

Figure S3 .
Figure S3.LC-MS chromatograms for chemical ligators.(A) LC-MS chromatogram for the BPS ligator (C).(B) LC-MS chromatogram for the BPS ligator (T).(C) LC-MS chromatogram for the PS ligator.The PS ligator bears a 3' terminal PS group that enables dimerization.To prevent spontaneous sulfur-oxygen exchange, the PS ligator was stored as a dimer.Immediately prior to use, an aliquot of the PS ligator was reduced by adding Tris(carboxyethyl)phosphine hydrochloride (TCEP HCl; Fluorochem M02624) or Bond-Breaker TCEP Solution, Neutral pH (Thermo Scientific 77720) to a final concentration of 50 µM.(D) LC-MS chromatogram for the reduced PS ligator.In (A) through (D), masses were calculated using the Oligowizard Nucleic Acid Calculator, an online tool available at http://oligowizard.com/.UV purity is expressed as percent area under the peak.BPS, biphenylsulfonyl; PS, phosphorothioate.

Figure S4 .
Figure S4.Linear fit CL-qPCR calibration curves for oligonucleotides in water using BPS ligator (C).Data are mean Cp values ± SD for three technical replicates.Efficiencies were calculated using the ThermoFisher Scientific qPCR Efficiency Calculator.

Figure S5 .
Figure S5.Linear fit CL-qPCR calibration curves for oligonucleotides in water using BPS ligator (T).Data are mean Cp values ± SD for three technical replicates.Efficiencies were calculated using the ThermoFisher Scientific qPCR Efficiency Calculator.

Figure S6 .
Figure S6.Comparison of linear fit CL-qPCR calibration curves for oligonucleotides in water using the different BPS ligators.(A) Base pairing interactions between the oligonucleotides and the different BPS ligators.(B) Linear fit CL-qPCR calibration curves for oligonucleotides in water using the different BPS ligators.In (A), lines are Watson-Crick base pairs and dots are wobble base pairs.WC, Watson-Crick.In (B), data are mean Cp values ± SD for three technical replicates and the same as those presented in Figures S4 and S5.

Figure S7 .
Figure S7.Linear fit CL-qPCR calibration curves for oligonucleotides in HEL cell lysate.The Cp values measured in HEL cell lysates following free uptake for 24 h are overlaid.Data are mean Cp values ± SD for three biological replicates (n=3).Efficiencies were calculated using the ThermoFisher Scientific qPCR Efficiency Calculator.

Figure S8 .
Figure S8.Linear fit CL-qPCR calibration curves for oligonucleotides in HEL nuclear lysate.The Cp values measured in HEL nuclear lysates following free uptake for 24 h are overlaid.Data are mean Cp values ± SD for three biological replicates (n=3).Efficiencies were calculated using the ThermoFisher Scientific qPCR Efficiency Calculator.

Figure S9 .
Figure S9.Linear fit CL-qPCR calibration curves for oligonucleotides in HEL cytoplasm.The Cp values measured in HEL cytoplasm following free uptake for 24 h are overlaid.Data are mean Cp values ± SD for three biological replicates (n=3).Efficiencies were calculated using the ThermoFisher Scientific qPCR Efficiency Calculator.

Figure S10 .
Figure S10.Oligonucleotides detected following free uptake for 24 h.Oligonucleotides detected in HEL cell lysate (top left), nuclear lysate (top right), and cytoplasm (bottom left).A stacked view (bottom right) shows the oligonucleotides detected in HEL cell lysate are approximately equal to the sum of the oligonucleotides detected in HEL nuclear lysate and the oligonucleotides detected in HEL cytoplasm.Data are mean molecules ± SEM for three biological replicates (n=3).ND, not detected.

Figure S11 .
Figure S11.Quantification of 18S gDNA and HPRT1 following free uptake of the oligonucleotides in HEL cells for 24 h.(A) Quantification of 18S gDNA in HEL cell lysate.(B) Quantification of 18S gDNA in HEL cytoplasm and nuclear lysate.(C) Quantification of HPRT1 in HEL cytoplasm and nuclear lysate.In panels (A) and (B), data are mean Cp values ± SD for three biological replicates (n=3).In panel (C), HEL cytoplasm data are mean Cp values ± SD for three biological replicates (n=3).HPRT1 was not always detected in nuclear lysate; therefore, data are single Cp values or mean Cp values ± SD for two or three biological replicates (n=2 or n=3).

Figure S12 .
Figure S12.Femtograms (fg) oligonucleotide per unit 18S gDNA detected following free uptake in HEL cells for 24 h.Data are mean fg per unit 18S gDNA ± SEM for three biological replicates (n=3).ND, not detected.

Figure S13 .
Figure S13.LC-MS chromatograms for conjugates.The mass for the 5'-capped-maleimide-modified oligonucleotide was calculated using the Oligowizard Nucleic Acid Calculator, an online tool available at http://oligowizard.com/.The mass of each peptide was provided by GenScript (Piscataway, New Jersey, U.S.).UV purity is expressed as percent area under the peak.

Figure S14 .
Figure S14.Melting data for conjugates.Representative datasets are shown for the conjugates paired with a 22-nt RNA representing the c.315-48C FECH pre-mRNA in 100 mM NaCl, 10 mM phosphate, 0.1 mM Na 2 EDTA, pH 7.0.T m s were determined from first derivative analyses of nonlinear fit melting curves.T m s are mean ± SD for three technical replicates.The upper baseline for NLS3-ON5 and the RNA target was too short to fit; therefore, a T m of > 80 °C was estimated from the maximum of the first derivative of the raw data.

Figure S15 .
Figure S15.Linear fit CL-qPCR calibration curves for conjugates in water.Data are mean Cp values ± SD for three technical replicates.Efficiencies were calculated using the ThermoFisher Scientific qPCR Efficiency Calculator.

Figure S16 .
Figure S16.Linear fit CL-qPCR calibration curves for conjugates in HEL cell lysate.The Cp values measured in HEL cell lysates following free uptake for 24 h are overlaid.Data are mean Cp values ± SD for three biological replicates (n=3).Efficiencies were calculated using the ThermoFisher Scientific qPCR Efficiency Calculator.

Figure S17 .
Figure S17.Linear fit CL-qPCR calibration curves for conjugates in HEL nuclear lysate.The Cp values measured in HEL nuclear lysates following free uptake for 24 h are overlaid.Data are mean Cp values ± SD for three biological replicates (n=3).Efficiencies were calculated using the ThermoFisher Scientific qPCR Efficiency Calculator.

Figure S18 .
Figure S18.Linear fit CL-qPCR calibration curves for conjugates in HEL cytoplasm.The Cp values measured in HEL cytoplasm following free uptake for 24 h are overlaid.Data are mean Cp values ± SD for two or three biological replicates (n=2 or n=3).Efficiencies were calculated using the ThermoFisher Scientific qPCR Efficiency Calculator.

Figure S20 .
Figure S20.Quantification of 18S gDNA and HPRT1 following free uptake of the conjugates in HEL cells at 2 µM for 24 h.(A) Quantification of 18S gDNA in HEL cell lysate.(B) Quantification of 18S gDNA in HEL cytoplasm and nuclear lysate.(C) Quantification of HPRT1 in HEL cytoplasm and nuclear lysate.In panels (A) and (B), data are mean Cp values ± SD for three biological replicates (n=3).In panel (C), HEL cytoplasm data are mean Cp values ± SD for three biological replicates (n=3).HPRT1 was not always detected in nuclear lysate; therefore, data are single Cp values or mean Cp values ± SD for two biological replicates (n=2).ND, not detected.

Figure S22 .Figure S23 .
Figure S22.Linear fit CL-qPCR calibration curves for the lead conjugate.(A) Linear fit CL-qPCR calibration curve for the lead conjugate in HEL cell lysate.(B) Linear fit CL-qPCR calibration curve for the lead conjugate in HEL nuclear lysate.(C) Linear fit CL-qPCR calibration curve for the lead conjugate in HEL cytoplasm.The Cp values measured in each compartment following free uptake of the conjugate in HEL cells for 24 h are overlaid.Data are mean Cp values ± SD for three biological replicates (n=3).Efficiencies were calculated using the ThermoFisher Scientific qPCR Efficiency Calculator.

Figure S24 .Figure S26 .
Figure S24.Quantification of 18S gDNA and HPRT1 following free uptake of the lead conjugate in HEL cells for 24 h.18S gDNA data are mean Cp values ± SD for three biological replicates (n=3).HPRT1 was not always detected in nuclear lysate; therefore, data are single Cp values or mean Cp values ± SD for two biological replicates (n=2).ND, not detected.

Figure S28 .
Figure S28.Splice-switching activities of all conjugates and parent SSO following free uptake.Panels (A) and (B) are replicate experiments.Each panel shows a semi-quantitation (left) and raw image (right) of an agarose gel separating reverse transcribed and PCR amplified FECH transcripts extracted from K562 FECH-3-C-5 cells.Cells were treated with conjugate or oligonucleotide under free uptake conditions at 6 µM for 48 h.Untreated parental cell lines were included for reference.Percent (%) refers to percentage of total (i.e., aberrant + correct) FECH transcript.ND, not determined.The semi-quantitation of bands for NLS1-ON5 was not possible owing to a limited amount of RNA recovered from the cells treated with this compound.The ladder was 5 µL Quick-Load Purple 100 bp DNA Ladder (New England BioLabs N0551S).

Table S2 . Oligonucleotides detected in HEL nuclear lysate following free uptake for 24 h. Data
are mean molecules ± SEM for three biological replicates (n=3) and the same as those presented in FigureS10.ND, not detected.Percent of total molecules in cells was calculated using mean values in TableS1.Values were rounded to the nearest percent.Average molecules per nucleus was calculated using 115,000 cells, assuming one doubling.Values were rounded to the nearest thousand.

Table S5 . Oligonucleotide conjugates detected in HEL nuclear lysate following free uptake at 2 µM for 24 h.
Data are mean molecules ± SEM for three biological replicates (n=3) and the same as those presented in FigureS19.Percent of total molecules in cells was calculated using mean values in TableS4.Values were rounded to the nearest percent.Average molecules per nucleus was calculated using 115,000 cells, assuming one doubling.Values were rounded to the nearest thousand.