Developing hemophilia A therapeutics by targeting translational and posttranslational regulation of FVIII

Title of project: Developing hemophilia A therapeutics by targeting translational and posttranslational regulation of FVIII Abstract: Hemophilia A (HA) is an X-linked inherited disease due to mutations in the gene encoding the blood coagulation factor VIII (FVIII), which affects 1 in every 5,000 male births. The majority of HA mutations are nonsense mutations, which introduce premature stop codons leading to no or truncated FVIII protein expression, and missense mutations, which are marked by excessive misfolding and degradation of FVIII protein. Current HA treatments include FVIII concentrate or bispecific monoclonal antibody infusions. However, these treatments are extremely costly and may require frequent office visits. The high cost precludes 80% of the global hemophilia population from routine access to care. Alternative approaches utilizing small molecule drugs offer several advantages, including greatly reduced costs and ease of delivery, which can enhance accessibility and patient compliance. We hypothesize that specific pharmacological molecules can enhance the expression, folding, and trafficking of certain nonsense and missense mutants, leading to increased coagulation factor levels sufficient to restore hemostasis. Gene therapy holds promise for a long-term cure for HA. However, FVIII is intrinsically difficult to express, requiring a high dose of adeno-associated virus (AAV) to produce therapeutic levels of FVIII in HA patients. A high AAV dose can trigger an immune response against the vector, causing complications and limiting FVIII expression. We have identified FVIII variants with 6-fold enhanced expression over the currently used B-domain deleted FVIII. We hypothesize that incorporating these FVIII variants in gene therapy can result in higher plasma FVIII activity levels with lower viral vector doses, offering a superior alternative to the existing FVIII variants. In Aim 1, we recently identified compounds that effectively rescue certain nonsense mutations of FVIII by drug-induced ribosomal readthrough. We will systematically test the most common nonsense FVIII mutations to identify candidate mutations and sequence patterns that will predict candidate mutations for effective readthrough therapy. In Aim 2, after screening compound libraries, we identified proteostasis regulators (PRs) that enhance the secretion of FVIII missense mutants. We will further identify common HA missense mutations that can be rescued by PRs and their molecular mechanism. We will validate in vitro results of both aims in mouse models. Aim 3 will further characterize the high-expression FVIII variants and explore the synergistic effects of combining these variants with existing strategies to boost FVIII expression. The efficacy and safety of candidate variants will be tested in HA mice through AAV-based gene therapy applications. Taken together, these studies will validate the use of small-molecule approaches for HA treatment, tailoring them to each patient's specific mutation profile as effective and convenient treatment options. Our novel high-expression variants will increase plasma FVIII co-factor activity while requiring lower viral vector doses. This advancement is crucial in developing a safer and more effective next-generation HA gene therapy approach.