Premature termination codon readthrough restores progranulin expression in preclinical models of frontotemporal dementia and neuronal ceroid lipofuscinosis

Frontotemporal dementia (FTD) is a devastating and progressive disorder and a common form of early-onset dementia. There are currently no disease-modifying therapies available, signifying the need for new therapeutic approaches. Progranulin (PGRN) haploinsufficiency due to autosomal dominant mutations in the progranulin gene (GRN) is a major cause of familial FTD (FTD-GRN), with nearly a quarter of these genetic cases resulting from a nonsense mutation. Nonsense mutations introduce premature termination codons (PTCs) that can be therapeutically targeted by compounds allowing readthrough, and aminoglycoside antibiotics are known to be potent PTC readthrough drugs. When this research project was initiated, restoring PGRN through PTC readthrough had not been explored as a therapeutic intervention in FTD-GRN. We used human induced pluripotent cell (hiPSC) lines bearing clinical nonsense mutations spanning the GRN coding region (S116X⁺/˗, R418X⁺/˗, R493X-/- KI) to evaluate G418 and novel PTC readthrough enhancer (CDX-series) combination treatments. Our aim was to demonstrate proof-of-concept GRN PTC readthrough and lower the required dose of G418 to address the known toxicity of traditional aminoglycoside PTC readthrough agents. Screening in HEK293 cells expressing nonsense mutant (S116X, R418X, R493X) GRN expression constructs found PTC readthrough combination treatment with G418 and CDX5-288 enhancer most potently induced GRN readthrough. We demonstrated in vivo proof-of-concept GRN PTC readthrough by performing single intracerebroventricular (ICV) injections of G418 with or without CDX5-288 enhancer in our GRN R493X adeno-associated virus-based mouse model. Combination treatment in hiPSC-derived isogenic R493X-/- KI cortical neurons significantly restored PGRN levels and normalized overexpression of the mature form of the lysosomal enzyme cathepsin D. We attempted to achieve in vivo PTC readthrough of GrnR493X through repeated ICV administrations of G418 in GrnR493X/R493X mice. However, G418 doses capable of eliciting PTC readthrough in these mice were associated with significant neurotoxicity. We next conducted further neuropathological characterization of lysosomal dysfunction, neuroinflammation, and neurodegeneration in GrnR493X/R493X mice, identifying several phenotypes recently reported in Grn-/- mice, including decreased thalamic excitatory neuronal density. Taken together, our findings suggest that PTC readthrough may be a potential therapeutic strategy for FTD caused by GRN nonsense mutations and support further investigations into novel readthrough drugs with improved tolerability.

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