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Applicable species: mice, rats, NHP, dogs, pigs[12-32,12-34,12-35]. Dose range: adult mice 5×10^10 vg/mouse (bolus 5 μL), up to 3.35×10^11 vg/mouse; rat lumbar puncture bolus injection 100-200 μL; NHP IT range 4.8×10^12-9.2×10^13 vg/mouse (1.0-2.5 mL); pig 7×10^12 GC/only[12-32,12-35]. Injection volume: mice 5-10 μL (standard 5 μL); NHP 1.0-2.5 mL; pigs 400-800 μL/segment [12-32]. Needle: mouse 30 G min with iohexol contrast agent CT for confirmation [12-31,12-32]. Anesthesia: mice are awake and fixed (Hylden-Wilcox modification) or mild isoflurane; NHP isoflurane 1-3% lateral flexion position [12-32] Operation points: mice L5-L6 intervertebral space (located along the iliac crest line), sudden bounce on the tail = entering the subarachnoid space; NHP CSF reflux + contrast agent double confirmation, Trendelenburg head down 5-10 after bolus injection min can significantly improve brain/cervical segment coverage [12-30,12-32]: The coverage is the thickest in the lumbar segment, followed by the thoracic segment, and the weakest in the cervical segment; AAV9 transduces motor neurons + DRG sensory neurons + oligodendrocytes; the peak expression is 3-6 weeks [1 2-31]. Safety: Degeneration of DRG sensory neurons (most consistent target toxicity, see Section 8.4); excessive/too rapid → acute neurological deficit; fatal meningitis has occurred with pig AAV8-IL-10 [12-26,12-34].