Screening Splice-Switching Antisense Oligonucleotides in Pancreas-Cancer Organoids

Abstract

Aberrant alternative splicing is emerging as a cancer hallmark and a potential therapeutic target. It is the result of dysregulated or mutated splicing factors, or genetic alterations in splicing-regulatory cis-elements. Targeting individual altered splicing events associated with cancer-cell dependencies is a potential therapeutic strategy, but several technical limitations need to be addressed. Patient-derived organoids are a promising platform to recapitulate key aspects of disease states, and to facilitate drug development for precision medicine. Here, we report an efficient antisense-oligonucleotide (ASO) lipofection method to systematically evaluate and screen individual splicing events as therapeutic targets in pancreatic ductal adenocarcinoma organoids. This optimized delivery method allows fast and efficient screening of ASOs, e.g., those that reverse oncogenic alternative splicing. In combination with advances in chemical modifications of oligonucleotides and ASO-delivery strategies, this method has the potential to accelerate the discovery of antitumor ASO drugs that target pathological alternative splicing.

Keywords: alternative splicing; antisense oligonucleotide; organoid; pancreatic ductal adenocarcinoma.

FIG. 1.

Lead antisense oligonucleotides (ASOs) exhibit different splice-switching efficiency in HEK293T cells. (A) Schematic of ASO-mediated splice-switching in SMN2. (B) Schematic of ASO-mediated splice-switching in PKM. (C) Radioactive reverse transcription polymerase chain reaction (RT-PCR) of SMN1/2 mRNA in HEK293T cells treated by free uptake of ASO-S. (D) PSI quantification of (C) (n = 3). (E) Radioactive RT-PCR of PKM mRNA in HEK293T cells treated by free uptake of ASO-P. (F) PSI quantification of (E) (n = 3). SC, scrambled control. One-way analysis of variance (ANOVA), *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent ± standard error of mean (SEM).

FIG. 2.

Limitations of ASO free uptake in pancreatic ductal adenocarcinoma (PDAC) organoids. (A) Schematic of ASO free uptake in organoid culture. (B) Matrigel domes were treated with different concentrations of scramble control, ASO-P, and ASO-S ASOs for 5 days. (C) Radioactive RT-PCR of SMN1/2 mRNA in hF23 organoids treated by free uptake of ASO-S. (D) PSI quantification of (C) (n = 3). (E) Radioactive RT-PCR of PKM mRNA in hF23 organoids treated by free uptake of ASO-P. (F) PSI quantification of (E) (n = 3). One-way ANOVA, ***P < 0.0001. Error bars represent ± SEM.

FIG. 3.

Splice-switching efficiency of ASO free uptake in Matrigel-diluted organoid culture. (A) Schematic of ASO free uptake in Matrigel-diluted organoid culture. (B) Radioactive RT-PCR of SMN1/2 mRNA in Matrigel-diluted, cultured hF23 organoids treated by free uptake of ASO-S. (C) PSI quantification of (B) (n = 3). (D) Radioactive RT-PCR of PKM mRNA in Matrigel-diluted, cultured hF23 organoids treated by free uptake of ASO-P. (E) PSI quantification of (D) (n = 3). SC, scrambled control. One-way ANOVA, *P < 0.05. Error bars represent ± SEM.

FIG. 4.

Lipofection-based ASO delivery in PDAC organoids. (A) Schematic of lipofection-based ASO delivery in PDAC organoids. (B) Bright-field image of PDAC organoids after ASO lipofection. (C) qPCR of PDAC organoid markers in hF23 and hT60 organoids transfected with SC ASO. (D) Radioactive RT-PCR of SMN1/2 mRNA in hF23 organoids transfected with ASO-S. (E) PSI quantification of (D) (n = 3). (F) Radioactive RT-PCR of PKM mRNA in hF23 organoids transfected with ASO-P. ds, double-skipped PKM isoform lacking both exons 9 and 10. (G) PSI quantification of exon 9 in (F) (n = 3). NTC, no-treatment control; SC, scrambled control. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent ± SEM.

FIG. 5.

Functional characterization of lipofection-based ASO delivery in PDAC organoids. (A) Western blot of PKM1 and PKM2 in hF23 organoids transfected with 125 nM ASO. Fold change was calculated by normalizing band intensity to the vinculin loading control (n = 2). (B) Western blot of PKM1 and PKM2 in hT60 organoids transfected with 125 nM ASO. Fold change was calculated by normalizing band intensity to the vinculin loading control (n = 2). (C) Pyruvate kinase (PK) activity and relative PK activity in hF23 organoids transfected with 125 nM ASO-P. (D) PK activity and relative PK activity in hT60 organoids transfected with 125 nM ASO-P. (E) Cell viability assay of hF23 organoids after lipofection of 125 nM of the indicated ASOs or lipofectamine 3000 alone (NTC), n = 3. (F) Cell viability assay of hT60 organoids after lipofection of 125 nM of the indicated ASOs or lipofectamine 3000 alone (NTC), n = 3. NTC, no-treatment control; SC, scrambled control. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent ± SEM.

FIG. 6.

Lipofection-based delivery of ASOs with different chemical modifications. (A) Radioactive RT-PCR of PKM mRNA in hF23 organoids transfected with 100 nM of SC, ASO-P, or i9 ASO with the indicated chemical modifications. (B) PSI quantification of exon 9 in (A) (n = 3). (C) Radioactive RT-PCR of PKM mRNA in hT60 organoids transfected with 100 nM of SC, ASO-P, or i9 ASO with the indicated chemical modifications. (D PSI quantification of exon 9 in (C) (n = 3). SC, scrambled control; i9, intron 9; MOE, 2′-O-methoxyethyl; LNA, locked nucleic acid; TMO, thiomorpholino oligonucleotide. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent ± SEM.