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. 2016 Feb;34(2):164-6.
doi: 10.1038/nbt.3427. Epub 2015 Dec 14.

Antisense oligonucleotide-directed inhibition of nonsense-mediated mRNA decay

Tomoki T Nomakuchi  1   2 , Frank Rigo  3 , Isabel Aznarez  1 , Adrian R Krainer  1
Affiliations

Antisense oligonucleotide-directed inhibition of nonsense-mediated mRNA decay

Tomoki T Nomakuchi et al. Nat Biotechnol. 2016 Feb.

Abstract

Nonsense-mediated mRNA decay (NMD) is a cellular quality-control mechanism that is thought to exacerbate the phenotype of certain pathogenic nonsense mutations by preventing the expression of semi-functional proteins. NMD also limits the efficacy of read-through compound (RTC)-based therapies. Here, we report a gene-specific method of NMD inhibition using antisense oligonucleotides (ASOs) and combine this approach with an RTC to effectively restore the expression of full-length protein from a nonsense-mutant allele.

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Conflict of interest statement

Competing Financial Interests

F.R. is an employee of, and A.R.K. is a consultant for, Isis Pharmaceuticals. A.R.K., I.A., and T.T.N. are inventors in a related patent application, PCT/US2014/054151.

Figures

Figure 1
Figure 1
Design and testing of EJC-targeting ASOs. (a) Schematic of the antisense approach to inhibit NMD of a target mRNA. An ASO (red) complementary to the predicted EJC-deposition site downstream of the PTC interferes with EJC assembly, inhibiting NMD. (b) An example of the ASO screening strategy, using HBB with a PTC in codon 39, stably expressed in U2OS cells. A set of 19 uniformly modified MOE phosphorothioate 15-mer ASOs was designed to cover the presumptive EJC-deposition site at 1-nt resolution. (c) ASOs targeting the HBB exon 2 EJC region were individually transfected at a nominal concentration of 100 nM. HBB mRNA was measured by radioactive RT-PCR using primers in exons 1 and 3. Some of the ASOs caused skipping of exon 2. The fold change in full-length mRNA with the T39 mutation, relative to the no-ASO control in lane 2, is indicated below each lane. The uncropped autoradiograph is shown in Supplementary Figure 2. (d) The optimal ASO for HBB (H-24, based on three experiments as in (c)) was tested at the indicated nominal concentrations (n = 3, *P < 0.05, **P < 0.001 versus no-ASO control). (e) The same screening approach was used to identify an ASO (M-33) that effectively inhibits NMD of MECP2 mRNA with a PTC in its penultimate exon (n = 3). See also Supplementary Figure 3. (f) Constructs to test the feasibility of targeted antisense inhibition of NMD in the case of targets with more than one downstream EJC. (g) 50 nM of individual ASOs and 25 nM each of the combined ASOs were tested with each chimeric construct (n = 4). Error bars represent standard deviation (s.d.). The corresponding autoradiographs are shown in Supplementary Figure 7.
Figure 2
Figure 2
Validation of the mechanism of action of NMD inhibition. (a) In vitro splicing of a radiolabeled pre-mRNA comprising exon 2, intron 2, and exon 3 of HBB was carried out in HeLa nuclear extract in the presence of GST-eIF4A3 (or GST) and varying concentrations of control or targeting ASOs. Spliced mRNA was specifically pulled down by GST-eIF4A3 in the absence of ASO or in the presence of control ASO in the splicing reaction. H-24 ASO prevented the association of eIF4A3 with the spliced mRNA in a dose-dependent manner. The relative changes in mRNA pull-down efficiency, normalized to the spliced mRNA level of the corresponding input, are indicated below each lane. 5% of the input RNA is shown on the left lanes. [ASO] = 100 nM, 200 nM, or 500 nM. (b) Diagram of constructs to test the dependence of the ASO effect on the position of the EJC. Addition of extra sequence (24 nt) at the end of exon 2 shifts the ASO hybridization site farther upstream from the exon-exon junction and from the site of EJC deposition. (c) H-24 ASO at 50 nM did not affect the NMD efficiency of the modified construct. n = 3, *P < 0.01. (d) Antisense-inhibition of NMD increased expression of both the truncated protein and the full-length read-through product translated from a modified HBB-T39 mRNA that allows more efficient read-through. Expression of the full-length protein was highest when ASO (50 nM) and G-418 (1 mg/ml) were combined. The expression levels of truncated and full-length proteins are normalized to their levels in the ‘no ASO’ samples. See also Supplementary Figure 11 for a representative blot. Student’s t-test, * P < 0.05, ** P < 0.001; n = 3. Error bars represent ± s.d.
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