Preliminary Study On Safety Evaluation Of AAV9-Mediated CRISPR/SaCas9 System

BackgroundDuchenne muscular dystrophy?DMD?is an X-linked recessive lethal muscle degeneration disease caused by mutations in the DMD gene which encoding dystrophin.At present,there is no effective treatment for this disease,mainly through clinical comprehensive treatment such as glucocorticoids and rehabilitation training to delay the disease process.Even with medical care,most DMD patients die of heart or respiratory failure before the age of 30.In recent years,CRISPR/Cas9 technology has been widely used for DMD gene therapy.CRISPR/Cas9-mediated gene editing can perform targeted editing of specific sequences,achieving permanent targeted exon skipping of DMD genes,and the dystrophin protein expression can be restored by correcting the reading frame which damaged by mutation.A large number of in vitro and in vivo studies have evaluated the efficacy of CRISPR/Cas9 in the treatment of DMD with varying success rates.Although CRISPR/Cas9-mediated gene editing has shown potential in the long-term treatment of DMD,its clinical transformation applications still face many safety issues such as off-target,in vivo gene delivery and activation of immune responses.Therefore,the safety evaluation of CRISPR technology is the focus of our research.This study designed and constructed the high-efficiency sgRNA targeting the upstream and downstream of the mouse Dmd gene exon23 in vitro.At the same time,the distribution and expression of AAV9 were evaluated by in vivo fluorescence imaging in mice to determine the appropriate injection method and dosage of AAV9viral vector.In addition,this study also evaluated the presence of SaCas9 protein antibody in humans,providing experimental basis for the subsequent safety study of DMD gene therapy.Objective1?To design and construct the sgRNA sequences targeting Dmd gene exon23in mdx mice,and the editing efficiency of single and double targets was verified in HePa1-6 mouse hepatic cells?C57BL mice?to determine the efficient targets.2?The distribution and expression of AAV9 virus vector in different injection methods and different virus titers were analyzed in mice,and the appropriate virus injection method and dosage were initially determined.3?Detecting the presence of SaCas9 protein antibodies in humans and fetuses to provide a basis for the possible humoral immunity of CRISPR-SaCas9 in the treatment of DMD patients.Methods1.Design the sgRNA sequence targeting Dmd gene exon23 in mdx mice,and verify the single and double targets cleavage efficiency in HePa1-6 mouse hepatic cells?C57BL mice?by T7E1 detection or electrophoresis.2.Constructing the ssAAV-Luciferase plasmid carrying Luciferase fluorescent reporter gene.After packaging with adeno-associated virus 9?AAV9?,the ssAAV-Luciferase was injected into female Balb/c nude mice for 4⁶ weeks by tail vein injection or intramuscular injection.After 3 days of normal feeding,the expression of ssAAV-Luciferase in mice was observed by in vivo biofluorescence imaging.3.The serum,amniotic fluid and fetal umbilical cord blood discarded by pregnant women in the prenatal testing of our center were collected.The antibodies of SaCas9 protein were detected by ELISA after obtaining the consent of the patients and their families.Results1.Successfully synthesis 2 recombinant plasmids targeting the 5'end of Dmd gene exon23 and 2 recombinant plasmids targeting the 3'end of Dmd gene exon23,The T7E1 and electrophoresis results show that the single target R1 has a high efficiency,and the combination of double sgRNAs?L1+R1?has the highest efficiency.2.When AAV9 is injected into the tail vein,the virus can be uniformly and widely distributed and expressed in mice.The virus was most strongly expressed at the injection site during intramuscular injection,and a small amount was expressed outside the injection site.As the AAV9 virus injection dose increases,the distribution of the virus in the body is more extensive,and the expression is more intense.When intramuscularly injected 1.261×10¹³ vg/kg,the expression of the virus was confined to the local injection site and the muscle tissues of the limbs,diaphragm,etc.No obvious AAV9 expression was observed in other organs..3.SaCas9 protein antibodies were detected in serum samples of 42 normal pregnant women,but not in amniotic fluid samples of 23 cases and cord blood samples of 10 cases.Conclusion1.In this study,we successfully constructed and screened highly efficient sgRNAs targeting the both ends of exon23 of Dmd gene in vitro based on the CRISPR/Cas9 system,proving the feasibility of exon23 targeted knockouts in mouse cells;2.Preliminary observation shows that intramuscular injection is a suitable treatment for AAV9 vector-mediated CRISPR/Cas9 treatment of DMD,and1.261×1013 vg/kg is a suitable injection dose;3.SaCas9 protein antibody is widely present in adults.No SaCas9 antibody was detected in amniotic fluid and umbilical cord blood.