Increased FAN1 expression by mRNA-LNP attenuates CAG repeat expansion in Huntington patients' iPSC-derived astrocytes

Cheng, Y.-C.; Nocula-Lugowska, G.; Ramirez, J. A.; Fan, X.; Jin, F.; Jiang, Z.; Bennett, E.; Li, J.; Hokanson, D.; Grandhi, S.; Chen, M.; Cheng, C.; Lin, G.-Y.; Lin, L.; Lepsy, C.; Chaparro-Riggers, J.; Bloom, L.; Morrissey, D.; Stewart, M.; Tadin-Strapps, M.; Chiang, S.-H.

doi:10.1101/2023.11.24.568451

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bioRxiv · 10.1101/2023.11.24.568451

Increased FAN1 expression by mRNA-LNP attenuates CAG repeat expansion in Huntington patients' iPSC-derived astrocytes

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Abstract

Expansion of repeat sequences within the human genome can lead to disease pathogenesis, such as Huntingtons Disease, primarily affecting the nervous system. Genome-wide association studies (GWAS) of age-at-onset in Huntingtons disease (HD) patients demonstrated DNA mismatch repair (MMR) genes are modifiers of somatic expansion and may be potential therapeutic targets for repeat expansion (RE) disorders. FAN1, a Fanconi anemia-associated nuclease, has been reported as an influencer of repeat expansion in the RE mouse models. Here, we show the first demonstration that FAN1 knock-out in HD patient-derived fibroblasts and results in increased CAG repeat length. We also develop a robust novel cell-based platform using stem cell technology to produce the HD patients iPSC-derived astrocytes (iAstro). This platform is a disease-relevant system and has a significantly wider assay window, making it more suitable to assess the effect of gene modulation on CAG repeats. A substantial and exponential increase in repeat instability was exhibited in this HD patients iPSC-derived astrocytes platform. Over-expression of FAN1 protein via FAN1 plasmid transfection in this platform reduced CAG repeat instability, suggesting that upregulation of FAN1 protein may have a potential protective effect in CAG repeat expansion for a therapeutic setting. We leveraged the mRNA-LNP modality to enhance FAN1 protein expression and revealed that codon-optimized FAN1 mRNA-LNP robustly prevented increased CAG repeat in HD patients iPSC-derived astrocytes platform. The data from these cell-based platforms highlight that FAN1 plays a protective role in attenuating expanded somatic HTT CAG repeats and shed light on new therapeutic directions against repeat expansion disorders.

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Cheng, Y.-C., Nocula-Lugowska, G., Ramirez, J. A., Fan, X., Jin, F., Jiang, Z., Bennett, E., Li, J., Hokanson, D., Grandhi, S., Chen, M., Cheng, C., Lin, G.-Y., Lin, L., Lepsy, C., Chaparro-Riggers, J., Bloom, L., Morrissey, D., Stewart, M., Tadin-Strapps, M., Chiang, S.-H.. 2023-11-24. Increased FAN1 expression by mRNA-LNP attenuates CAG repeat expansion in Huntington patients' iPSC-derived astrocytes. https://doi.org/10.1101/2023.11.24.568451

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