The objective of this two-year dissertation research project is to inhibit the degradation of mRNA triggered by nonsense mutations so as to restore gene function and ameliorate the pathology caused by the mutations. RNA-based therapeutic approaches have become increasingly promising, as demonstrated by recent remarkable progress in the development of antisense oligonucleotides (ASOs) and stop-codon read-through drugs as therapeutics for various genetic diseases. Nonsense mutations, which account for a large proportion of pathogenic genetic lesions, can be partially overcome by suppressing premature stop codons (PTCs) using read-through drugs, such as ataluren or aminoglycosides. Nonsense-mediated mRNA decay (NMD) is a cellular quality-control mechanism that degrades mRNAs with PTCs. In the context of genetic diseases caused by PTCs, NMD can worsen the clinical outcome by degrading transcripts that code for truncated but semi- functional proteins, or by reducing the efficacy of read-through therapy. NMD is highly dependent on the exon- junction complex (EJC), which assembles upstream of exon-exon junctions at the completion of pre-mRNA splicing. I have designed ASOs that target presumptive EJC sites and suppress NMD of ?-globin and MECP2 transcripts with PTCs. To develop and apply this technique more broadly, and to pursue therapeutic applications of NMD-inhibiting ASOs, I will systematically test EJC-targeting ASOs on transcripts with clinically- relevant PTCs, including nonsense mutations in IDUA and CFTR genes that cause type I mucopolysaccharidosis and cystic fibrosis, respectively. I will design and test ASOs that target the EJCs of these transcripts, and test the lead ASOs in patient fibroblasts and murine models for their therapeutic potential. My ASO designs will be guided by the predicted EJC positions, as well as by using public transcriptome-wide data on EJC crosslinking or binding to RNA. To validate the mechanism of action, I will monitor the effects of individual ASOs on EJC assembly using an RNA-immunoprecipitation-based assay. This project seeks to develop therapies potentially applicable to numerous genetic diseases, while also providing fundamental mechanistic insights into the precise roles and functions of EJCs in NMD.
Type 1 mucopolysaccharidosis and cystic fibrosis are just two of numerous genetic diseases that could benefit from the gene-targeted suppression of nonsense-mediated mRNA decay (NMD). The proposed project seeks to inhibit NMD for selected mutant genes to restore the gene's function and to improve the efficacy of an emerging RNA-based therapy.
| Nomakuchi, Tomoki T; Rigo, Frank; Aznarez, Isabel et al. (2016) Antisense oligonucleotide-directed inhibition of nonsense-mediated mRNA decay. Nat Biotechnol 34:164-6 |
