Systemic IV high-dose hepatotoxicity/thrombotic microangiopathy (TMA): NHP and piglet IV 2×10^14 GC/kg AAVhu68. All 3 NHPs had elevated transaminases, and 1 had acute liver failure + shock and was euthanized on the 4th day. Severe toxicity was also seen with piglet; ≥1×10^14 Hepatic sinusoidal platelet microthrombi and endothelial injury (SOS) at GC/kg have nothing to do with transgene; thrombocytopenia, coagulation abnormalities, and complement activation occurred 1-2 weeks after administration, which are related to pre-existing anti-capsid antibodies [12-11,12-25].
CSF administration DRG lesions: DRG mononuclear cell infiltration, neuronal degeneration, and axonopathy after NHP administration through blood or CSF; 33 NHPs were collected, 83% of the CSF pathways were involved, and they were usually clinically silent. The dose threshold is approximately ≥1×10^13 vg/IT/ICM, and <5×10^12 vg/unit is usually asymptomatic; elevated plasma/CSF NfL indicates DRG damage; the mitigation strategy is to insert a miRNA target sequence into the expression cassette to target the DRG transgene [12-26, 12-27, 12-36].
Clinical death signal: DMD high-dose IV (approximately 2×10^14 vg/kg level) rAAV9 causes liver failure, thrombotic microangiopathy, and death (Lek 2023 NEJM) [12-28].
Restrictions on immunogenicity and repeated administration: AAV induces anti-capsid neutralizing antibodies (NAb), which strongly limits repeated administration. Systemic administration with multiple injection intervals of ≥2 weeks is still restricted by NAb; strong promoter + neoantigen can cause cellular immune clearance in immune-competent animals, which is more obvious in NHP/human, and immune regulation (such as rapamycin SVP) can be evaluated; NHP IM AAV8 shows a loss of immune-mediated expression but is not irreversible [12-12,12-24].
Mouse strain barrier and others: AAV-PHP.B strong BBB penetration is limited to C57BL/6J (Ly6a dependent), BALB/c and other strains do not have this phenotype [12-7]; high-dose ubiquitous promoter (CBA/CMV) can induce neuronal stress and microgliosis in the target area; pig AAV8-IL-10 IT has caused fatal meningitis, suggesting that the immunogenicity of the transgenic product itself needs to be evaluated separately [12-34].