Evaluating ribosomal readthrough as a precision medicine strategy for restoring factor VIII expression in hemophilia a

Abstract Current prophylactic treatments for hemophilia A (HA) primarily rely on factor replacement therapies, which consist of intravenous infusions of various FVIII concentrates. Non-factor replacement therapies include bispecific antibodies and rebalancing agents under development. These treatments are all extremely costly and may require frequent hospital visits. Small-molecule drugs offer a promising alternative or supplemental treatment option due to their significantly lower costs, which could improve accessibility, and their ease of administration, which may enhance patient compliance. Nonsense mutations account for 11.3% of total HA cases and result in premature termination codons (PTCs) that lead to truncated or absent factor VIII (FVIII) protein, often causing severe bleeding symptoms. Drug-induced ribosomal readthrough over PTCs is an on-target strategy for genetic disorders caused by nonsense mutations. This approach enables translation beyond the PTC, producing a full-length protein and potentially achieving phenotypic rescue without interfering with normal stop codons. In theory, HA is ideally suited for drug-induced readthrough therapy because even a small increase in FVIII activity to over 1% could markedly improve bleeding symptoms. However, the success of this approach depends on identifying compounds with high readthrough efficiency and minimal toxicity, as well as pinpointing specific mutations that are amenable to readthrough therapy. To explore this therapeutic potential, we established HEK293 cell lines stably expressing Gaussia luciferase (Gluc)-tagged B-domain-deleted FVIII containing 26 distinct nonsense mutations, including those most commonly observed in HA patients. These cell models enabled a systematic evaluation of multiple readthrough compounds across a broad spectrum of nonsense mutations. We tested aminoglycoside antibiotics (gentamicin, G418, ELX02) and non-aminoglycoside agents (ataluren and 2,6-diaminopurine [DAP]), each employing distinct mechanisms to induce ribosomal readthrough. Drug efficacy was assessed by measuring secreted Gluc activity, FVIII antigen levels, and cofactor activity in conditioned media. Among aminoglycoside compounds, G418 and ELX02 demonstrated significant readthrough activity for a subset of mutations. Specifically, 13 of the 26 nonsense variants exhibited >2% of wild-type (WT) FVIII expression levels in response to G418 and/or ELX02, demonstrating that these compounds can restore detectable FVIII production for selected PTC contexts. Ataluren, originally developed for cystic fibrosis and Duchenne muscular dystrophy, did not exhibit significant readthrough effects for the FVIII PTCs tested. In contrast, DAP, a purine analog derived from edible mushrooms, demonstrated broad activity, rescuing 18 PTCs with higher efficacy than other compounds in many cases. DAP was especially effective at TGA stop codons, where it promotes tryptophan incorporation, leading to the synthesis of full-length FVIII, as confirmed by immunoprecipitation and immunoblotting. To validate the translational potential of readthrough therapy by DAP in vivo, we expressed full-length human FVIII variants (WT and p.R355X) in CD-1 mouse livers using hydrodynamic tail vein injection. Mice were treated with DAP, and plasma samples were collected 24 hours post-plasmid DNA delivery. Human FVIII levels in mouse plasma were quantified using a human FVIII-specific ELISA, revealing a significant rescue of the p.R355X mutant (1.5-6% of WT levels) in DAP-treated mice. In summary, our findings underscore ribosomal readthrough as a promising alternative or complementary treatment strategy for HA patients with nonsense mutations. Both aminoglycoside and non-aminoglycoside compounds can restore partial FVIII expression in a mutation-specific manner. These results support the further development of readthrough-based therapies as a cost-effective and accessible treatment option for a subset of severe HA patients, potentially reducing their dependence on conventional replacement therapies.