Lipoprotein(a) (Lp(a)) is a genetically determined causal risk factor for cardiovascular disease, with approximately 20% of the population exhibiting elevated levels. While there are promising drugs i Show more
Lipoprotein(a) (Lp(a)) is a genetically determined causal risk factor for cardiovascular disease, with approximately 20% of the population exhibiting elevated levels. While there are promising drugs in development, there are currently no approved therapies specifically designed to lower Lp(a) levels. For high-risk individuals with extreme levels of Lp(a), liver-directed genome editing could be an effective one-time solution. Genome editing approaches such as CRISPR and TALENs can reduce Lp(a) in LPA-transgenic mouse models, but they frequently induce large and potentially harmful genomic deletions. Here, we report the first application of TadA-derived cytosine base editing (CBE), delivered via helper-dependent adenovirus (HDAdV) and adeno-associated virus (AAV) vectors, to introduce premature stop codons into LPA. This strategy produced robust and durable lowering of circulating apolipoprotein(a) (apo(a)) in LPA-transgenic mice. Using SMRT-seq with single-molecule unique molecular identifiers, we quantified deletion events and found that CBE did not induce large deletions when targeting a single LPA site and produced only a small fraction (<4%) of large deletions when editing across multiple sites. In contrast, CRISPR-Cas9 cutting of LPA resulted primarily in large deletions. These findings demonstrate that CBE enables sustained reduction of circulating apolipoprotein(a) in an LPA-transgenic mouse model while largely preserving genomic integrity. Show less
The transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the m Show more
The transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the molecular mechanisms regulating its functions are poorly understood. In the present study, we investigated the formation and the role of LINGO-1 cis-dimers in the regulation of its biological activity. LINGO-1 homodimers were identified in both HEK293 and SH-SY5Y cells using co-immunoprecipitation experiments and BRET saturation analysis. We performed a hypothesis-driven screen for identification of small-molecule protein-protein interaction modulators of LINGO-1 using a BRET-based assay, adapted for screening. The compound identified was further assessed for effects on LINGO-1 downstream signalling pathways using Western blotting analysis and AlphaScreen technology. LINGO-1 was present as homodimers in primary neuronal cultures. LINGO-1 interacted homotypically in cis-orientation and LINGO-1 cis-dimers were formed early during LINGO-1 biosynthesis. A BRET-based assay allowed us to identify phenoxybenzamine as the first conformational modulator of LINGO-1 dimers. In HEK-293 cells, phenoxybenzamine was a positive modulator of LINGO-1 function, increasing the LINGO-1-mediated inhibition of EGF receptor signalling and Erk phosphorylation. Our data suggest that LINGO-1 forms constitutive cis-dimers at the plasma membrane and that low MW compounds affecting the conformational state of these dimers can regulate LINGO-1 downstream signalling pathways. We propose that targeting the LINGO-1 dimerization interface opens a new pharmacological approach to the modulation of its function and provides a new strategy for drug discovery. Show less