The Application Of CRISPR-Cas9 System In Animal Modeling And Gene Therapy Of Phenylketonuria

Recently,CRISPR/Cas9 system has been applied to gene therapy for monogenetic disease.As a universal tool for precise gene editing,CRISPR/Cas9 technology brings gene therapy to the central stage.Here,we focus on the gene therapy for Phenylketonuria(PKU),an antosomal recessive genetic disease that may cause mental disability.Through CRISPR/Cas9 system,we generated a novel PKU mouse model,PahR408W,which carried the most common mutation type in PKU patients.PahR408W showed the same phenotype as the PKU patients in serum Phenylalanine(Phe)concerntration,hair color and the content of Pah protein.We also carried out gene therapy via viral vectors such as Adenovirus(Adv)and Adeno-associated virus(AAV).Mice treated with Adv showed a decrease in Phe concemtration in blood,reversion in color of hair and an increase in content of PAH protein.We also applied AAV-CRISPR/Cas9 system to gene therapy for PKU with a strategy to insert cDNA into Pah Locus.For improving the efficiency of knockin strategy,we developed a method to co-expression AAV receptor(AAVR).We found co-expression AAV receptor improved the efficiency of knockin for 4 times.In the meantime,PahR408W mice treated with AAV-AAVR/CRISPR-Cas9/Donor also showed a decrease in Phe concerntration and reversion in hair color which is much better than mice treated with AAV-CRISPR-Cas9/Donor.This project is a proof of concept for gene therapy via CRISPR/Cas9 system and pave the way for clinical trials.