mRNA Treatment Rescues Niemann-Pick Disease Type C1 in Patient Fibroblasts.

Messenger RNA (mRNA) holds great potential as a disease-modifying treatment for a wide array of monogenic disorders. Niemann-Pick disease type C1 (NP-C1) is an ultrarare monogenic disease that arises due to loss-of-function mutations in the gene, resulting in the entrapment of unesterified cholesterol in the lysosomes of affected cells and a subsequent reduction in their capacity for cholesterol esterification. This causes severe damage to various organs including the brain, liver, and spleen. In this work, we describe the use of NPC1-encoded mRNA to rescue the protein insufficiency and pathogenic phenotype caused by biallelic mutations in cultured fibroblasts derived from an NP-C1 patient. We first evaluated engineering strategies for the generation of potent mRNAs capable of eliciting high protein expression across multiple cell types. We observed that "GC3" codon optimization, coupled with N1-methylpseudouridine base modification, yielded an mRNA that was approximately 1000-fold more potent than wild-type, unmodified mRNA in a luciferase reporter assay and consistently superior to other mRNA variants. Our data suggest that the improved expression associated with this design strategy was due in large part to the increased secondary structure of the designed mRNAs. Both codon optimization and base modification appear to contribute to increased secondary structure. Applying these principles to the engineering of NPC1-encoded mRNA, we observed a normalization in NPC1 protein levels after mRNA treatment, as well as a rescue of the mutant phenotype. Specifically, mRNA treatment restored the cholesterol esterification capacity of patient cells to wild-type levels and induced a significant reduction in both unesterified cholesterol levels (>57% reduction compared to Lipofectamine-treated control in a cholesterol esterification assay) and lysosome size (157 μm reduction compared to Lipofectamine-treated control). These findings show that engineered mRNA can correct the deficit caused by mutations. More broadly, they also serve to further validate the potential of this technology to correct diseases associated with loss-of-function mutations in genes coding for large, complex, intracellular proteins.