Amelioration Of Hemophilia B In Mice Through AAV Delivery Of CRISPR/Cas9 System

Early-stage gene therapy is an effective way to treat genetic diseases by introducing the exogenous gene into cells and replacing the defective gene.In recent years,emerging gene editing technologies based on nucleases have been developed to mediate gene introduction,knockout,correction and other types of highly targeted genome modification in cells,therefore opening up new ways for gene therapy.Among them,CRISPR/Cas9 technology shows a great promise in the application of gene therapy due to its high efficiency and convenient operation.The choice of a safe in vivo gene therapy delivery carrier is important,and adeno-associated virus（AAV）vectors are widely used due to their advantages of low immunogenicity and strong tissue specificity.Hemophilia B is a type of X-linked blood coagulation disorder,and is an important disease target for gene therapy.In this project,we delivered CRISPR/Cas9 system and homologous recombination template through AAV8,mediating a site-specific integration of hF9CDS to ameliorate hemophilia B in F9³83STOP83STOP mice.Due to the packing capacity limit of AAV vectors,we used an intein-mediated split-SpCas9 system,which partially alleviated the coagulation function of F9^383STOP mice.However,the genome editing efficiency was low,which is unhelpful for gene site-specific integration.In order to improve the efficiency of genome editing and therapeutic effects,we optimized the laboratory viral packaging platform.As a result,SpCas9 and the promoter were successfully packaged into an AAV vector,significantly restoring the coagulation function of F9^383STOP mice without off-target effects and hepatic toxicity.In conclusion,this project has improved the efficacy and safety of gene therapy for hemophilia B,paving the way for its clinical transformation.