Effect Of Adeno-associated Virus Vector Mediated NF-κB/p65-shRNA And Mini-dystrophin Gene Transfer On Duchenne Muscular Dystrophin

Objective 1.To investigate therapeutic benefits from long-term expression of mini-dystrophin gene in mdx mice.2.To design a recombinant adeno-associated virus（r AAV）construct carrying both the mini-dystrophin gene and NF-κB/p65-shRNA.3.To investigate synergistically therapeutic benefits from gene replacement and anti-inflammation in homo mice.Method 1.Differernt genotypic mouse strains were obtained by breeding transgenic mdx（Tg-mdx）mice and gene knockout mice,genotypes and phenotypes were detected by polymerase chain reaction（PCR）and immunofluorescence（IF）.2.Western Blot and IF were used to detect the expression of dystrophin,sarcoglycans（α,β and γ）,dystrobrevin,α1-syntrophin and neuronal nitric oxide synthase（n NOS）in longissimus thoracis（LT）and gastrocnemius（GAS）muscles of Tg-mdx mice aged 6,10 and 20 months.3.The integrity of muscle membrane in the GAS of Tg-mdx mice was detected by Evans Blue Dye（EBD）staining.To evaluate whole-body exercise and endurance,treadmill test was used to detect muscle endurance capacity.The grip force test was used to measure the muscle strength of forelimbs.4.pAAV（ss）-U6-p65 shRNA-Mcken-Syn-hOptiDys3978 plasmid was designed and AAV9-U6-p65 shRNA-Mcken-Syn-hOptiDys3978 was packaged by co-transfection in 293 cells with 3 plasmids.AAV was purified by gradient ultracentrifugation and virus titer was determined by Dot-blot.5.Male mice aged 5 days were divided into 4 groups: normal group（C57BL/6J）,experimental group（homo,dual-cassette）,experimental control group（homo, single-cassette）and blank group（homo）.Each group contained 5 mice.According to the standard procedure of AAV injection,experimental group were intraperitoneally injected with AAV9-U6-p65 shRNA-Mcken-Syn-hOptiDys3978（50 μl,1×1011 viral particles）,experimental control group were intraperitoneally injected with AAV9-Mcken-Syn-hOptiDys3978（50 μl,1×1011 viral particles）,blank group were intraperitoneally injected with PBS（50 μl）.6.Western Blot and IF were used to detect dystrophin expression in mice with different genotypes.The relationship between dystrophin expression and the level of inflammation cell infiltration was analyzed.Masson’s Trichrome staining was used to detect muscle fibrosis.7.Western Blot and IF were used to detect dystrophin expression and the level of inflammation cell infiltration in different tissues of mice in normal group,experimental group,experimental control group and blank group.Muscle physiological function assay was used to evaluate the effect of AAV treatment on skeletal muscle function in homo mice.Result 1.According to the experimental design,we generated 5 strains of mouse: Tg-mdx(dys^-/-;utro^+/+;Tg⁺),Tg-homo(dys^-/-;utro^-/-;Tg⁺),homo(dys^-/-;utro^-/-),homo-p65(dys^-/-;utro^-/-;p65 +/-)and Tg-homo-p65(dys^-/-;utro^-/-;p65 +/-;Tg⁺).2.IF results showed efficient expression of mini-dystrophin gene in different skeletal muscles of Tg-mdx mice,but not in cardiac muscle.Western Blot and IF results showed that the expression of sarcoglycans（α,β and γ）,dystrobrevin,α1-syntrophin and n NOS in muscle tissues of Tg-mdx mice were significantly higher than mdx mice at the age of 6,10 and 20 months.3.IF and EBD results showed that the expression of mini-dystrophin gene in GAS of Tg-mdx mice could ameliorate central nucleation and improve membrane integrity.The results of treadmill test and grip assays showed that the running distance and forelimb grip strength of Tg-mdx mice at the age of 6 months were significantly higher than mdx mice at the same age.No significant difference between wild-type（WT）and Tg-mdx mice was observed.The absolute tetanic force and specific force of both WT and Tg-mdx mice were significantly higher than mdx mice at the same time points.4.We designed a dual-cassette gene vehicle containing both the mini-dystrophin gene and NF-κB/p65-shRNA.AAV9 were successfully packaged by 3 plasmids co-transfection in 293 cells.Meanwhile,we optimized such dual-cassette AAV vector via the design of a modified muscle creatine kinase（MCK）enhancer and a muscle specific synthetic（Syn）promoter driving mini-dystrophin gene and U6 promoter driving NF-κB/p65-shRNA.5.Western Blot and IF results showed that the level of inflammation cell infiltration in Tg-homo-p65 group was significantly lower than Tg-homo and homo-p65 groups.Masson’s Trichrome staining results showed that the fibrosis area in Tg-homo-p65 group was significantly reduced compared with Tg-homo and homo-p65 groups.6.Western Blot and IF results showed that the level of inflammation cell infiltration in experimental group was significantly decreased compared with experimental control and blank groups.Muscle physiological function assay results showed that the physiological function of tibialis anterior（TA）muscle in experimental group was significantly higher than experimental control and blank groups at 2 months after AAV treatment.Conclusion 1.Long-term expression of mini-dystrophin gene could ameliorate pathology and improve the functions of dystrophic muscles in the Tg-mdx mice.2.AAV could mediate the expression of mini-dystrophin gene and NF-κB/p65-shRNA in homo mice.3.AAV9-mediated gene replacement and anti-inflammation dual-target therapeutic approach could down-regulate the level of inflammation,alleviate muscle degeneration and improve muscle physiological function in homo mice.