Membrane Anchorage-Induced (MAGIC) Knockdown of Non-synonymous Point Mutations.

Abstract

The promise of personalized medicine for monogenic and complex polygenic diseases depends on the availability of strategies for targeted inhibition of disease-associated polymorphic protein variants. Loss of function variants, including non-synonymous single nucleotide variants (nsSNVs) and insertion/deletion producing a frameshift, account for the vast majority of disease-related genetic changes. Because it is challenging to interpret the functional consequences of nsSNVs, they are considered a big barrier for personalized medicine. A method for inhibiting the specific expression of nsSNVs without editing the human genome will facilitate the elucidation of the biology of nsSNVs, but such a method is currently lacking. Here, I describe the phenomenon of membrane anchorage-induced (MAGIC) knockdown of allele-specific inhibition of protein and mRNA expression upon inner membrane tethering of point mutation-specific monoclonal antibodies (mAb). This phenomenon is likely mediated by a mechanism distinct from the protein degradation pathways, as the epitope-specific knockdown is replicated upon intracellular expression of a membrane-anchored single domain intrabody that lacks the Fc domain of the mAb. By harnessing the MAGIC knockdown of epitope-containing protein targets, I report a novel approach for inhibiting the expression of amino-acid-altering germline and somatic nsSNVs. As a proof-of-concept, I show the inhibition of human disease-associated variants namely, FGFR4 p.G388R, KRAS p.G12D and BRAF p.V600E protein variants. This method opens up a new avenue for not just therapeutic suppression of undruggable protein variants, but also for functional interrogation of the nsSNVs of unknown significance.