| A genetic disorder is a disease caused by changes in genetic material(genetic mutations,chromosomal variations,etc.).At present,most of the clinical treatment of genetic diseases adopts symptomatic treatment methods such as drug treatment,diet therapy and surgical treatment,which can delay the disease process and play a certain therapeutic effect,but it cannot fundamentally solve the genetic disease.In the past 30 years,gene therapy has risen rapidly in the treatment of genetic diseases,bringing new treatment ideas for the treatment of genetic diseases.Starting from the root cause of disease,gene therapy introduces foreign normal genes into target cells,corrects or compensates for disease-causing genes,and achieves the purpose of radical disease cure.At present,41 gene therapy drugs have been approved for marketing worldwide,bringing great hope to the treatment of genetic diseases.In recent years,with the rapid development of genome editing technology,gene therapy has also entered a stage of rapid development.Genome editing technology can modify a specified DNA sequence to achieve the insertion,deletion or mutation of the gene of interest.Genome editing technology is applied to gene therapy to modify the pathogenic sequence to achieve the purpose of disease treatment.In genome editing technology,CRISPR/Cas9 has become a hot spot in current research because of its simple design and strong operability,while base editing(BE)technology derived from CRISPR/Cas9 provides a new method for the treatment of single-gene genetic diseases with single-base mutations because of its advantages of editing at the individual base level.Precise gene editing therapy with BE in vivo requires the use of appropriate vector tools to effectively deliver BE to target tissues.At present,among the delivery vectors of gene therapy,adeno-associated virus vectors(AAVs)are the most widely used in clinical experiments due to their low immunogenicity and lack of integration into the host cell genome.Wilson disease(WD)is mainly a genetic disease of copper metabolism caused by ATP7 B gene mutations,and there is currently no cure.We tried to use the AAV delivery BE system to carry out gene therapy research in WD animal models,and the main research content and results are divided into the following three parts:Part 1: Screening and AAV delivery studies targeting ATP7B-P992 L base editing repair vectors.AAV delivery has the problem of packaging capacity limitation,delivery capacity needs to be < 4.7 Kb,at present,most BE systems are more than 4.7 Kb,in order to solve this problem,we have made some changes to the BE system to control its volume within 4.7 Kb.There are two main methods: one is to split the BE system,and the other is to find small Cas9 combined with small adenosine deaminase.First,we evaluated the off-target situation of NG-ABEmax and NG-ABE8 e systems,and found that NG-ABE8 e was higher than NG-ABEmax in both g RNAdependent off-target and non-g RNA-dependent off-target,and then we selected NGABEmax for splitting,selected 573-574 protein sites for fractionation,and after fractionation,the carrier was transfected to verify the P992 L 293A cell model in vitro.The results showed that the editing efficiency of 28.4% was produced on the cell genome,indicating that we successfully split the NG-ABEmax vector.Then,the fractionated vector was infected with the P992 L 293A cell model after AAV packaging,and the repair efficiency of 15.4% was achieved on the cell model genome.In the study of selecting small Cas9 to build an integrated carrier,we selected Sauri Cas9 developed by Chinese scholar Wang Yongming and combined with artificially evolved ABE8 e to construct an integrated carrier.In the promoter change,we remove the enhancer from the CMV(CMV enhancer + CMV promoter),leaving only the CMV promoter part.The n Sauri Cas9,ABE8 e and CMV promoter are integrated to form an integrated carrier.When the AAV-packed integrated vector infects the P992 L cell model,the repair efficiency is 5.7%.Part 2: AAV delivery of NG-ABEmax split vector to repair G>A mutations in tx-j mice.After obtaining the NG-ABEmax split vector,we verified whether it can also play a repair role in the complex environment in vivo.We selected a WD mouse model(txj)of the G712 D mutation(G>A)of exon 8 for in vivo delivery studies.In vivo gene therapy on tx-j mice using AAV8-packed NG-ABEmax split vector achieved a repair efficiency of(15.63±1.56)% in the liver genome of mice,and the liver damage of mice was also reduced,achieving a more ideal therapeutic effect.Part 3: Construction of ATP7B-P992 L mice with gene therapy.P992 L mutations accounted for 15.5% of WD patients in China,and we constructed P992 L mutation mice.Since the editing window of the BE4 max system is 4-8 bits,the obtained mice mutate CCC into CTT.Due to the degeneracy of codons,both CTT and CTC encode leucine,theoretically producing the same animal model phenotype.Then,in vivo treatment of P992 L mice using AAV-packaged NG-ABEmax vector,the liver genome repair efficiency was found to be 4.4%.Overall,we successfully split the NG-ABEmax vector,AAV-packaged NGABEmax vector infects the P992 L 293A cell model in vitro,achieving a repair efficiency of 15.4% on the cell genome.When n Sauri Cas9,ABE8 e,and CMV promoter were integrated into an all-in-one vector and packaged into AAV-infected cell models,the repair efficiency was 5.7%.In vivo treatment of WD animal models(tx-j mice)delivered by AAV8 delivery of NG-ABEmax split vector achieved(15.63±1.56)%repair efficiency in the liver genome and reduced the damage to the mouse liver.The mouse germline with the mutation of the P992 L site of ATP7 B gene was obtained,and the in vivo treatment of P992 L mice using AAV-packaged NG-ABEmax vector was performed,and the liver genome repair efficiency was 4.4%.This study provides technical means for the clinical transformation of P992 L WD gene therapy. |
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