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Applicable species: mice, rats, NHP, dogs [12-19,12-20,12-23]. Dose range: mouse single point 1×10^9-1×10^11 vg/muscle, multi-muscle strategy total 1×10^11-1×10^12 vg/mouse; rat single point approximately 1×10^11 vg/muscle; rhesus IM AAV8 3×10^12 GC/kg divided into bilateral vastus lateralis muscles [12-19,12-20]. Injection volume: 25-50 μL for a single point in mice; 0.5-1 mL/point for NHP, and the total volume can reach 4-135 mL[12-20,12-22]. Needles: Mouse 29-30 G Hamilton; NHP 22-25 G long needle [12-20]. Speed: Inject slowly for 30-60 s, and wait for 10-20 s after withdrawing the needle to prevent reflux [12-19]. Anesthesia: light anesthesia (isoflurane/Avertin); NHP sedation/general anesthesia. Operation points: After hair removal and disinfection of mice, insert a needle 3-5 mm along the direction of muscle fibers; NHP has multiple points on both quadriceps/gluteal muscles with a spacing of ≥1 cm, and the injection points are marked with ink [12-21,12-22]. Coverage: Injected muscle transduction (mainly muscle fibers), distal muscles are not transduced; secreted transgenes can enter the bloodstream; NHP IM AAV8 has unexpectedly significant liver targeting (the number of liver GCs is equivalent to that of local muscle) [12-20]; the peak expression is 2-6 weeks, and loss of NHP immune-mediated expression is common but can be restored [12-24]. Multi-muscle strategy: Multiple scattered injections cover more muscle fibers, and the upper limit of the total dose is limited by liver toxicity; regional limb perfusion (VLP/IAPD) can be used as an alternative for large animals [12-22,12-23].