(635) Genome-Wide screen to uncover genes promoting Premature Termination Codon (PTC) readthrough.

Background: More than 2,000 variants of the CFTR gene have been described and at least several 100 cause Cystic Fibrosis (CF). Nonsense variants are the second most prevalent mutation; they affect ~10% of CF patients and are caused by a nucleotide change that creates a premature termination codon (PTC). A ribosome encountering a PTC terminates translation, fails to displace downstream exon junction complexes, and triggers nonsense-mediated decay (NMD) of mRNA, resulting in absence of functional protein and a severe CF phenotype. One of the therapeutic strategies for PTC mutations is the use of agents to suppress the proofreading function of ribosomes at PTCs and favor insertion of a near-cognate amino acid. Such “ReadThrough Agents” (RTAs) allow the translation completion (downstream to PTC), enabling the production of a CFTR protein. Despite multiple screening campaigns to identify new RTAs (e.g. Ataluren, Negamycin, Tylosin…), aminoglycosides remain the most potent; however, sustained exposure to aminoglycosides induces only low levels of PTC RT or elicits severe toxicity. Understanding of ribosome translation termination exposed the interplay between factors and revealed potential targets for pharmacological intervention, such as eRF1 and eRF3. We performed a genome-wide screen (GWS) on our readthrough reporter cell line (CFF-16HBEge CFTR G542X expressing a CFTR peptide surrounding G542X fused to GFP and an inducible Cas9) and have identified potential targets for readthrough therapy.

Methods: First, we used the pLVXS-sgRNA-mCherry-hyg plasmid based sgRNA library from TakaraBio (Genome-Wide sgRNA Library System consisting of 19,114 target genes, 4 sgRNA per gene, based on the Brunello Library) to produce a lentivirus (LTV) Genome-Wide (GW) sgRNA library. Second, we transduced our readthrough reporter cell line with the LTV GW sgRNA library at a low multiplicity of infection (MOI~0.4) to ensure that cells carry a single sgRNA. Cells were subjected to hygromycin selection and expansion prior to induction of Cas 9 expression (2 days), followed by treatment with G418 (100 uM, 4 days). Third, GFP+ cells (i.e., GFP intensity above baseline value) were FAC sorted and analyzed by Next Generation Sequencing (NGS) for sgRNA frequency. sgRNA enrichment threshold of >100-fold compared to reference (unsorted, no Cas9 induction) was used to identify genes promoting PTC readthrough.

Results: The frequency distributions of sgRNAs encoded in the TakaraBio plasmid library and the distribution observed in our LTV transduced cells were virtually identical. In the cell sorted population, our two positive control genes, eRF1/ETF1 and eRF3/GSTP1 were above the enrichment threshold of 100-fold (4 and 3 sgRNAs > 100X, respectively), thus validating our genomic screen system. A total of 140 different gene targets with 1 or more sgRNAs enriched and 49 with 2 or more sgRNAs enriched were uncovered. The main pathways identified include: ribosomal subunit formation and maturation, ribosomal RNA processing, NMD, translation initiation and termination, peptide elongation, and selenocysteine synthesis pathway.

Conclusions: The results demonstrate that a genome wide screen of our reporter cell system combined with phenotypic selection (FACS) identifies genes known to promote PTC readthrough. We have established a list of gene candidates to be individually evaluated and which may lead to the identification of novel therapeutic targets relevant to PTC readthrough.

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