Goyenvalle, A. | Search bioRxiv

bioRxiv2024

A Genome-wide CRISPR screen unveils WDR91 protein as a promoter of productive ASO activity

Antisense oligonucleotides (ASOs) belong to promising therapeutics for the treatment of neurologic, muscular and metabolic disorders. Several ASOs have been approved so far and more than a hundred clinical trials are currently underway covering a dozen therapeutic areas. Yet, the mechanisms of internalization and cell trafficking of these molecules remain poorly understood. Moreover, with only a small fraction of ASOs reaching the correct cellular compartment following systemic delivery, the majority of targeted diseases requires recurrent injections of ASOs. A deeper understanding of these mechanisms would guide the improvement of their potency and thus, reduce the amount of delivered ASOs and their potential side-effects. Here, using a CRISPR screen, we investigated intracellular proteins involved in ASOs efficiency using a whole genome approach and identified several potential regulators which could significantly impact ASOs potency in melanoma cells. We validated WD Repeat Domain 91 (WDR91), a regulator of endosomal maturation, as a modulator whose depletion significantly inhibits ASO productive activity. This study provides the first list of ASO modulators using a biologically relevant assay to estimate the role of these proteins. In conclusion, these data could lead to a better understanding of the mechanisms favoring productive uptake or improved endosomal escape of ASOs.

molecular biology↗

bioRxiv2022

Novel uAUG creating variants in the 5UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare vascular disorder causing abnormal vessel formation and characterized by autosomal dominant transmission. The associated considerable variability in symptoms and clinical severity complicate the management of the disease. In clinical routine, 3 main genes, ACVRL1 (also known as ALK1), ENG and SMAD4 are screened for pathogenic variants at the origin of HHT. About 80% of HHT cases are caused by pathogenic coding variants in ACVRL1 and ENG. However, at least 15% remain with no molecular explanations in the 3 main genes. We here report the identification of 2 never reported variants, c.-79C>T and c.-68G>A, in the 5UTR of ENG in 2 unrelated HHT patients. These 2 variants are predicted to create upstream AUGs (uAUGs) in the 5UTR, which are in frame with a stop codon located within the CoDing Sequence (CDS), thus generating Overlapping upstream Open reading frames (uoORFs). In other cases, uAUGs can lead to fully upstream ORFs (uORFs) when associated with stop codons located within the 5UTR or to elongated CDS (eCDS) when in frame with the CDS and associated with the main stop codon. In order to assess the pathogenicity of these ENG variants, we performed in vitro functional assays based on the expression of wild-type and mutant constructs in human cells. We found that these 5UTR ENG variants were associated with a decrease of protein levels in HeLa and HUVEC cells. They were also associated with a decreased ability to activate BMP9-stimulated ALK1 receptor in a BMP-response element (BRE) assay. This assay is a mandatory element before providing a definitive molecular diagnosis and has been so far applied only on coding ENG variants. We applied the same experimental workflow on 3 additional uoORF-creating variants (c.-142A>T, c.-127C>T and c.-10C>T) and one eCDS-creating variant (c.-9G>A) in the 5UTR of ENG previously reported in HHT patients. We found that all the analysed uoORF-creating variants alter endoglin levels and function. A comparison of our experimental results with patients clinical characteristics suggests that uoORF-creating variants leading to ENG protein levels [≤] 40% in vitro would be associated with HHT. Additional experiments relying on an artificial deletion in our mutated constructs show that created uAUGs predicted to create uoORFs are able to initiate the translation indicating that the associated effect is likely caused by an alteration of the translation mechanism. Overall, we here identified two never reported 5UTR ENG variations in HHT patients and shed new lights on the role of upstream ORFs on ENG regulation. Our findings contribute to the amelioration of molecular diagnosis in HHT.

molecular biology↗