Tissues and Cells - Decision Logic. Confirm the target organ first, and then confirm the target cell type within the organ; the tropism of hepatocytes vs Kupffer vs sinusoidal endothelium, neurons vs astrocytes vs endothelium in the same organ can be completely different. Process: First determine the target cells → check the cell receptor expression → select the matching serotype → verify cell level transduction.
Common Misconceptions. Equate "tissue vg copy number" to "target cell expression". High vg may simply be that the capsid is retained by vascular endothelial cells/macrophages and does not enter the target cell; it must be verified with single-cell resolution (scRNA-seq, spatial transcriptome, cell-specific reporter) and attention should be paid to whether AAVR expression is in place.
Example. Vg can be measured in the brain after IV administration of AAV9, but neuronal transduction in the adult brain parenchyma is limited, mainly in the endothelium/glia; PHP.eB really enters the neurons [10-21][10-25].
Species and strains
Decision logic. First check whether the receptor of the target cell (Ly6A? TfR1? Galactose?) exists and is accessible in the species/strain used; give priority to serotypes that are conserved among species (8/9/rh10/rh74/MyoAAV). Those that are not conserved must be verified in the target species. C57BL/6J≠BALB/c≠NHP≠people.
Common Misconceptions. Direct copy of mouse PHP.B to NHP/human. PHP.B is highly transduced in the whole brain of C57BL/6J, and NHP disappears completely[10-40][10-27]; BI-hTFR1 is ineffective in ordinary mice and requires human TfR1 KI[10-43].
Example. Liver targeting selection AAV8/rh10/rh74 (conserved across species); do not default to PHP.B for BBB crossing. Effective in NHP.
Method of administration
Decision logic. Theroute determines receptor accessibility (luminal vs basolateral, vitreous vs subretinal). Process: Select the optimal path according to the target tissue → Confirm the serotype affinity under this path (may be different from IV) → Match verification. The same capsid IV affinity can be reversed with topical administration.
Common Misconceptions. IV and topical administration are thought to be equally effective. AAV2 IV is extremely low in the brain but highly effective when injected into the brain parenchyma; AAV6 is optimal for airway luminal administration, while IV is mainly for the liver and heart; AAV4 ICV is partial to the ependyma, and IV is partial to the lungs/kidneys/pancreatic islets.
Example. AAV9 IV administration mainly targets the liver and heart, while local injection into the myocardium directly enriches the myocardium; retinal SR→RPE/photoreceptor, IVT→ganglion mainly requires penetrating types such as AAV2.7m8 to reach the outer layer [10-47].
Dose and time
Decision logic. The affinity is more "pure" at low doses (before receptor saturation). At high doses, hepatic non-specific interception increases and the proportion of positive cells increases, but off-target and innate immune burden increase. The time window determines the peak expression (AAV1/6/7/8/9 is fast, AAV2/3/4/5 is slow) [10-26]; the time window is different between neonates and adults [10-25].
Common Misconceptions. Thought higher doses must be better. High-dose AAV9 IV can cause elevated liver transaminases and dorsal root ganglion toxicity (NHP/person); be wary of dose-dependent tropism shifts.
Example. PHP.eB can reach 69% of cortical neurons at 1×10¹¹ vg, without blindly increasing the dosage [10-21].
Promoter
Decision logic. The capsid determines "whether it can enter the cell", and the promoter determines "where it is expressed after entering." The two work together: capsid enters muscle + MHCK7 = muscle specific; capsid enters broadly + synapsin/GFAP = neuron/glia restricted.
Common Misconceptions. It is believed that the promoter can completely make up for the lack of capsid tropism. If the capsid does not enter the target cell at all, no matter how strong the tissue-specific promoter is, there will be no substrate; on the contrary, if the capsid enters the liver non-specifically, the strong promoter will only amplify off-target expression.
Example. rh74+MHCK7 (Elevidys) achieves high muscle expression, but if the capsid does not effectively enter the muscle fiber, the promoter cannot save the day [10-37].
Study endpoint
Decision logic. Select molecules (vg/mRNA), cells, tissues, and functional multi-layered endpoints based on affinity and target cells, and match expression kinetics with sampling time. If the capsid is endothelial, the endpoint should measure vascular function rather than neuronal electrophysiology; if the capsid is endothelial, the endpoint should measure glial activation rather than behavioral improvement.
Common Misconceptions. Only look at fluorescence intensity without looking at quantitative expression and function, and ignore non-target tissue sampling. GFP positivity ≠ effective therapeutic protein expression ≠ functional improvement; vg copy number, mRNA, protein, and function should be verified step by step.
Example. In DMD studies, rh74 microdystrophin needs to simultaneously measure muscle histology, muscle strength and liver function (off-target toxicity), not just fluorescence [10-38].