Antisense oligonucleotides targeting miR‐29b binding site increase translation of progranulin protein: potential therapeutic strategy for progranulin‐deficient frontotemporal dementia

Abstract

Background GRN mutations cause frontotemporal dementia (FTD) due to haploinsufficiency of progranulin. Several microRNAs (miRs), including miR‐29b, have been reported to negatively regulate progranulin protein levels. Here, we tested if antisense oligonucleotides (ASOs) – which are versatile modulators of target mRNA/protein levels – can be used to increase progranulin levels by sterically blocking the miR‐29b binding site.MethodWe designed 48 ASOs targeting the miR‐29b binding site in the 3’ UTR of the human GRN mRNA. We treated H4 neuroglioma cells and iPSC‐derived neurons with these ASOs and subsequently measured progranulin protein levels by western blot and ELISA. We performed further studies to determine the mechanism of action of these ASOs using ribosomal profiling, metabolic labeling, and FRET assays.ResultsWe identified 16 ASOs that increased progranulin protein levels in a dose‐dependent manner. Ribosomal profiling experiments revealed that cells treated with ASOs had marked enrichment in GRN mRNA in heavy polyribosome fractions, compared to cells treated with a scrambled control ASO, suggesting that the ASOs increase the rate of progranulin translation. Consistent with this, ASO treatment resulted in increased levels of newly synthesized progranulin protein. FRET‐based assays showed that ASOs can effectively compete miR‐29b from its binding site in the GRN 3’ UTR RNA under in vitro conditions.ConclusionsTogether, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This strategy may be therapeutically feasible for progranulin‐deficient FTD as well as other conditions of haploinsufficiency.