Readthrough of disease-causing nonsense mutations by targeted selenocysteine recoding

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TITLE: Readthrough of disease-causing nonsense mutations by targeted selenocysteine recoding PROJECT SUMMARY Nonsense (stop) mutations comprise about 10% of disease-causing mutations, and are common in Duchenne muscular dystrophy, cystic fibrosis, and cancer. These mutations cause early termination of translation and lead to non-functional proteins. Therapies that enable readthrough of these premature termination codons have been sought for many years with limited success. Existing approaches are limited by lack of specificity, low efficiency, or the need to deliver small genes. The amino acid selenocysteine (Sec), sometimes known as the 21st amino acid, is incorporated into human proteins via recoding of opal (UGA) stop codons. This recoding mechanism is activated by the presence of a Sec incorporation sequence element (SECIS) in the 3’ untranslated region (3’UTR). This element was shown to be sufficient to stimulate selenocysteine incorporation and is active even when located far from the UGA codon. Here we will develop a novel approach for inducing readthrough of opal nonsense mutations through Sec recoding. This will be achieved through the use of short hybridizing oligonucleotides that bring the targeted mRNA in proximity to a SECIS element, inducing readthrough in a gene-specific manner, and restoring a functional protein. In Aim 1, we will screen oligonucleotides that hybridize to both the target mRNA and an endogenous SECIS-containing transcript. In Aim 2, we will develop a high-throughput cell sorting-based assay, allowing the identification of optimal oligonucleotides among the combinatorially large number of possible designs. We will screen multiple possible designs, including oligonucleotides containing a hybridizing part and a (possibly abbreviated) SECIS element. We will demonstrate our approach using two disease-associated genes (DMD and CFTR) and validate the identified oligonucleotides in disease cell models. The proposed approach has several important advantages over currently available therapeutic approaches to nonsense mutations. First, the oligonucleotides are specific to the targeted gene, reducing concerns of off-target effects. Second, the same oligonucleotide can potentially be used for any nonsense mutation in the same gene, reducing development cost and addressing patients with very rare mutations. Finally, safe and efficient delivery of short oligonucleotides to several tissues has already been demonstrated. Together, the specificity, broad usability, and use of proven delivery technologies, make our approach particularly attractive for therapeutic purposes. Aligned with NIBIB’s interests, this project will develop a platform technology that is applicable to a broad spectrum of disorders and diseases. If successful, the project will have a tremendous impact on the quality of life of people suffering from genetic diseases caused by nonsense mutations and from cancer.