Development of a RNA-based therapeutic approach for Hemophilia B caused by exon-skipping mutations

We propose a novel therapeutic approach for the inherited deficiency of coagulation factor IX (FIX), Hemophilia B (HB), an X-linked disorder (1/35,000 males). The conventional replacement therapy is effective to treat and prevent life-threatening and disabling bleeding episodes in most cases but has still limitations. Our approach aims at restoring FIX levels in the severe forms caused by different types of mutations that induce "exon-skipping" during mRNA maturation, relatively frequent in HB and many other genetic diseases. The U1snRNA, a pivotal component of the mRNA splicing machinery, is the key therapeutic tool for the project. Recently, we developed Exon Specific U1snRNAs (ExSpeU1) that bind to intronic sequences downstream of the exon, and demonstrated in cellular models that a unique ExSpeU1 corrects several mutations causing exon-skipping. This strategy substantially expands the ExSpeU1 applicability to cohorts of patients sharing similar pathogenic mechanisms. We expect to provide optimized ExSpeU1s able to restore FIX levels and ameliorate the bleeding tendency in HB mouse models for a panel of exon-skipping mutations. We also expect to provide a novel efficient methodology for the creation of mouse models for different mutations, which could be exploited for other correction attempts. Testing this RNA-based approach in HB, in which gene replacement therapy and DNA editing have been extensively explored, will virtually complete the panel of correction approaches applied to a single genetic disorder and permit the comparison of advantages and drawbacks. It is worth noting that ExSpeU1s have the advantage of rescuing gene expression only in physiologic tissues while maintaining the gene regulation, and their small size make them easily packageable in any delivery vector. Therefore, ExSpeU1s might represent a valuable therapeutic option for other rare and/or orphan monogenic disorders in which replacement gene therapy is hardly approachable.