High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides

Abstract

The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics.

6BIO Enhances Oligonucleotide Activity in Cells: A Potential Combinatorial Anti-androgen Receptor Therapy in Prostate Cancer Cells

“…The identification of small molecules that facilitate ASO activity is a step in this direction. 17,48 In this study, we used small-molecule highthroughput screening to identify the kinase inhibitor 6BIO as an enhancer of the activity of potentially therapeutic PS LNA ASOs delivered by gymnosis. Pharmacological concentrations of 6BIO augmented the activity of an ASO targeted to the endogenous oncogenic miR21 in HCT116-miR21 cells (Figures 1 and 2), augmented the activity of a pre-mRNA SSO used to rescue EGFP expression in HeLa-eGPF-654 cells (Figure 3), and augmented conventional RNase H-mediated gapmer ASO gene silencing of BCL2, b-catenin, and HER3 expression in prostate cancer cells (Figure 4 and data not shown).…”

“…16,33 To investigate whether 6BIO could facilitate the ability of ASOs delivered by gymnosis to modulate molecular events that occur solely in the nucleus, we used a splice-switching oligo (SSO) targeted to the EGFP pre-mRNA. 17,34 This system relies on cells (HeLa-EGFP-654) stably expressing an EGFP pre-mRNA whose coding sequence has been interrupted by the insertion of an additional exon. 17,34 The binding of an SSO to the first 3 0 splice site causes the skipping of this internal exon and restores the correct EGFP reading frame and EGFP protein expression.…”

“…17,34 This system relies on cells (HeLa-EGFP-654) stably expressing an EGFP pre-mRNA whose coding sequence has been interrupted by the insertion of an additional exon. 17,34 The binding of an SSO to the first 3 0 splice site causes the skipping of this internal exon and restores the correct EGFP reading frame and EGFP protein expression. 17,34 Thus, the resulting EGFP expression is a direct reflection of the potency of the SSO.…”

“…17,34 The binding of an SSO to the first 3 0 splice site causes the skipping of this internal exon and restores the correct EGFP reading frame and EGFP protein expression. 17,34 Thus, the resulting EGFP expression is a direct reflection of the potency of the SSO. To trigger exon skipping, we used an LNA PS mixmer with the identical LNA distribution as we used for the miR21 antagomir screen.…”

“…However, despite years of developmental work, ASOs have progressed slowly in the clinic. There are many reasons for this, but approaches that augment gymnotic delivery, such as use of small molecules, 17 might be useful to maximize the clinical potential of therapeutic ASOs.…”