Distribution And Safety Evaluation Of Adeno-associated Virus Type 9 In Mice

BackgroundDuchenne muscular dystrophy(DMD)is a rare,fatal,and degenerative neuromuscular disease associated with the X chromosome.One in about 5,000 newborns worldwide is affected The DMD gene is a disease-causing gene of DMD.The mutation of this gene will cause the anti-dystrophin(dystrophin)to fail to be synthesized and function normally,thereby causing muscle damage and causing the occurrence of DMD disease.In recent years,with the continuous deepening of gene research,the treatment of DMD through the treatment-of DMD genes has gradually produced and developed.On the one hand,DMD is a disease caused by mutation of a single gene,namely DMD gene,and only the pathogenic gene needs to be considered during treatment.On the other hand,because muscle cells do not divide and have a long life span,one-time treatment may bring long-term or even lifetime benefits.Gene therapy for DMD aims to treat DMD at the genetic level.Specifically,by introducing gene fragments to repair the mutant or deleted dystrophin gene,the function of the dystrophin gene is restored,and the muscle function of the skeletal muscle,diaphragm,and myocardium is restored to normal.A suitable viral delivery vector is a key step in gene therapy for DMD patients using CRISPR/Cas9 technology.Adeno-associated virus(AAV)is a non-pathogenic human defective virus that can mediate long-term stable expression of foreign genes.Adeno-associated virus serotype 9,AAV9,isolated from non-human primates,has very low immunogenicity and toxicity.AAV9 can mediate long-term effective expression of exogenous genes.It has high transduction efficiency in skeletal muscle,brain,liver,pancreas and other tissues,and has good transduction effect on myocardium.AAV9 has been used as a viral vector mediating Cas9 internationally,and successfully edited the Dmd gene of mdx mice.AAV-mediated CRISPR/Cas9 gene editing,as an important method of DMD treatment,also faces more unique challenges than other therapies.Although there have been some achievements in the application of AAV9 in DMD gene therapy,the long-term distribution and safety of AAV9 in animals still lack relevant research.Then study the long-term distribution of AAV9 virus in mice under the injection method and dose;finally,the safety of AAV9 in mice was analyzed by dissecting the mice to make pathological sections of organ tissues.Provide an experimental basis for the follow-up research on the safety of DMD gene therapy.Objective1.Analyze the distribution and expression of AAV9 virus vectors with different injection methods and different virus titers in mice,and initially determine the appropriate virus injection method and dosage.2.Analyze the long-term distribution and safety of the AAV9 virus vector in mice when the injection method and dosage are appropriate.Methods1.Male tail Balb/c mice were injected with different titers of AAV9 virus(AAV9-Luc)carrying Luciferase fluorescent reporter gene by tail vein injection and intramuscular injection.After 3 days of normal feeding,the biofluorescence imager Observe the expression in mice.2.Use the injection method and dose determined in the previous experiment to inject AAV9-Luc into male Balb/c mice.After 30 days of normal feeding,observe the expression of fluorescence in mice and the fluorescence of individual organs under a biofluorescence imager Distribution.3.Inject AAV9-Luc into male Balb/c mice using the injection method and dose determined in the previous experiment.After 14 days of normal feeding,observe the general morphological changes of the mouse organs and the microscopic morphological changes of the pathological sections.Results1.This study found that when AAV9 is injected into the tail vein,the virus can be evenly and widely distributed and expressed in mice.During intramuscular injection,the virus is most strongly expressed locally at the injection site,and a small amount is also expressed outside the injection site.As the injection dose of AAV9 virus increases,the wider the distribution of virus in the body,the stronger the expression.When intramuscular injection(4×1011VG)was used,the virus expression was limited to the injection site and muscle tissues such as the limbs,diaphragm,and other organs.2.Choose the intramuscular injection as the injection method and the appropriate dose.In the 30-day long-term distribution study,the expression of fluorescence gradually increases with time,and the expression site gradually expands from the injection site of the right hind limb,and There was no obvious fluorescence expression in other organs except muscle.The fluorescence expression in the right hind limb muscle and right forelimb muscle gradually increased with time.The fluorescence expression of intercostal muscle,myocardium,and diaphragm increased gradually from 1 to 14 days after injection,while the fluorescence expression of intercostal muscle and myocardium decreased slightly from 14 to 30 days,and the expression of diaphragm did not change significantly.3.After injection of AAV9 virus into mice,the main organs(liver,spleen,lung,kidney,pancreas,brain,testis)and main muscles(limb muscle,diaphragm,intercostal muscle,and myocardium)of the animal are all normal in appearance.There were no abnormal phenomena such as bleeding,necrosis,swelling,and atrophy.The pathological section showed no abnormal cell infiltration,muscle fiber necrosis,hyperplasia and hypertrophy.ConclusionWhen the intramuscular injection dose is 4×1011VG AAV9 virus,the virus is localized in the muscles of the mice,but it is almost not distributed in the non-muscular organs.It is a more appropriate injection method and dose.And it is safer for mice to be given intramuscular injection at this dose.