4.1 Muscle tissue structure
The transduction of muscle fibers, satellite cells, smooth muscles and vascular pericytes varies greatly, and the local intramuscular results cannot directly represent the whole body muscle group.
4.2 AAV serotype selection
AAV9 and AAVrh74 can be used as natural candidates; MyoAAV and AAVMYO represent engineering directions for cross-model screening.
4.3 Muscle-specific promoter
MHCK7, desmin and CK8 are used for different strength and size balances; myocardial and liver expression needs to be monitored.
4.4 Administration method and dosage reference
Intramuscular (IM): Injection into muscle. Mouse single point: 1×10^9-1×10^11 vg/muscle, 25-50 μL, 29-30 G Hamilton, slow bolus injection for 30-60 s; rat single point: about 1×10^11 vg/muscle, 50 μL; NHP rhesus IM AAV8 3×10^12 GC/kg divided into bilateral vastus lateralis muscles, 0.5-1 mL/point, 100 points can reach 135 mL; canine GRMD system IV 0.5-6.24×10^14 vg/kg[4-4,4-5].
System IV: Covered by skeletal muscles throughout the body. Mouse 1×10^11-1×10^12 vg/mouse; AAV9/MyoAAV type extensive skeletal muscle transduction. Multi-muscle strategy: Multiple points (TA, gastrocnemius, quadriceps) dispersed injection covers more muscle fibers, and the upper limit of the total dose is limited by liver toxicity; regional limb perfusion (VLP/IAPD) is used to replace multi-point IM in large animals [4-6].
Coverage and limitations: IM only injects muscle transduction, not distal muscle, and the secreted transgene can enter the bloodstream; NHP IM AAV8 shows unexpected significant liver targeting (the number of liver GCs is equivalent to that of local muscle); expression lasts for 2-6 weeks, and terminal differentiation of muscle fibers lasts for ≥6-12 months. Advantages of engineered capsids must be verified in target strains and production batches [4-4,4-6].
4.5 Key research evidence
Studies have shown that IM injection of AAV8 in mice and macaques is accompanied by significant liver targeting and transgene expression [4-4]; Kornegay et al. evaluated systemic AAV-microdystrophin gene therapy in GRMD canine models [4-5]; bridging studies optimized NHP regimens from IM to limb perfusion delivery [4-6]. Loss of IM AAV expression in NHP is immune-mediated but not irreversible and can assess immune regulation.
4.6 References for this chapter
[4-1] Tabebordbar M, et al. Directed evolution of muscle-directed AAV capsids across species. Cell. 2021;184:4919-4938.e22. [H]
[4-2] Weinmann J, et al. Identification of a myotropic AAV by barcoded in vivo evaluation. Nat Commun. 2020;11:5432. DOI:10.1038/s41467-020-19230-w. PMID:33116134. [H]
[4-3] Gushchina LV. Intramuscular and intravenous AAV delivery in mouse models. Methods Mol Biol. 2026;2975:251-261. [N]
[4-4] Intramuscular injection of AAV8 in mice and macaques is associated with substantial hepatic targeting and transgene expression. Gene Ther. 2014. PMID:25393537. [H]
[4-5] Kornegay JN, et al. Assessment of systemic AAV-microdystrophin gene therapy in the GRMD model of Duchenne muscular dystrophy. Sci Transl Med. 2023;15(679):eabo1815. DOI:10.1126/scitranslmed.abo1815. [N]
[4-6] Bridging from intramuscular to limb perfusion delivery of rAAV: optimization in a non-human primate study. Mol Ther Methods Clin Dev. 2019. PMID:30828586. [H]