Background: The emerging type VI CRISPR-Cas systems Cas13 ortholog to perform RNA editing by driving catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to specific mutated transcripts would represent a new technology to correct G to A mutations, which represent a big portion of the Hemophilia A causing missense mutations (19%).

Aims: To correct the missense mutations in mature RNA of F8 increasing the coagulation FVIII protein production, secretion and activity.

Methods: Transient expression of F8 cDNA containing missense mutation to evaluate the secretion and activity in presence or in absence of the mutations. To assess the increase of protein production and corrected FVIII activity, Activated Partial Thromboplastin Time (aPTT) activity assays and ELISA on cells culture media was performed in presence and in absence of the specific guide RNA and the Cas13b-ADAR2 correction system.

Results: We selected three severe missense mutations with G to A change impairing the residual activity in patient’s plasma. The selection of the missense mutants was based on CHAMPS and EAHAD databases, taking in consideration the clinical severity and number of affected patients reported (˃30 patients). In vitro transient expression studies indicated that the selected mutations (p.R1800H; p.R2182H; p.R2228Q) impair both FVIII expression and activity (≤1% of pooled normal plasma). We assayed in transient transfection the designed single guide RNAs able to target the specific mutation. Among the guides used two out of three demonstrated to be effective for the p.R1800H (1.6-fold; p value: 0,0248) and p.R2182Q (2-fold; p value: 0,0129).

Conclusions: These preliminary results contributed to elucidate the molecular basis of FVIII deregulation in presence of missense mutations in Hemophilia A patients, providing the first proof of concept on the usage of RNA Editing system applied in Hemophilia that confirmed FVIII protein increase in vitro.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/exploring-rna-editing-system-as-innovative-correction-strategy-for-hemophilia-a/