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The concept of two-tiered events. AAV's preference (tropism) for a certain tissue/cell is not a single event, but is determined by two layers of molecular events: "attachment receptor" and "required entry receptor". Confusing these two layers is the source of misjudgment of tissue vg as target cell expression.

Attachment factor: Glycosaminoglycans/glycoproteins determine the initial binding affinity and tissue distribution tendency of the capsid and the cell surface. Most of them are glycans (HSPG, sialic acid, galactose).

Essential entry receptor: Protein factor that determines endocytosis, reverse vesicle transport (TGN orientation), and infection success or failure. AAVR (KIAA0319L) is the broadest spectrum essential entry receptor discovered so far [10-5].

Key corollary. Just because a cell has a certain serotype of attached glycan on its surface does not mean that the cell can be effectively transduced; it must also express AAVR (or its alternative receptor such as CPD) and subsequent trafficking factors. This explains why "high tissue vg" does not equal "high target cell expression".

Receptor/co-receptor × serotype correspondence summary table. The corresponding relationships between receptors/co-receptors for commonly used capsids are as follows.

The classic hemagglutination experiment of Kaludov et al. 2001 proved that AAV4 uses α2-3 O-linked sialic acid (not α2-6), and AAV5 uses α2-3 N-linked sialic acid (not α2-6); it is AAV6 that truly recognizes both α2-3 and α2-6 N-links, and AAV1 only recognizes α2-3 N-links [10-6][10-7]. This difference determines that AAV4/5 prefers epithelia with high O-glucose/N-glucose abundance, such as the airway/ependymum, while AAV6 has a wider double junction spectrum and stronger airway transduction.

AAVR(KIAA0319L):Type I transmembrane protein, extracellular contains MANSC domain and multiple Ig-like PKD domains. It binds to the capsid through the PKD2 domain, is rapidly endocytosed, and transported to the trans-Golgi network (TGN) [10-5]. Most natural and engineered capsids, such as AAV1/2/3B/5/6/8/9/rh.10, rely on AAVR; exceptions are AAV4 and the closely related Clade G that can bypass AAVR [10-12]. In 2025, CD164 was also identified as an essential receptor for divergent AAVs [10-23]. AAVR is widely expressed in most nucleated cells, so it does not constitute a major determinant of tissue tropism. The difference in tropism is mainly determined by the upper attachment glycans/co-receptors; forced expression of AAVR in a certain tissue can make originally low-transducing tissues transduced by broad-spectrum AAV [10-24].

HSPG (heparan sulfate proteoglycan):Summerford and Samulski established it as the main receptor for AAV2 in 1998, with key residues R585/R588 (and R484/R487/K532) [10-8]. Those with HSPG as the main attachment receptor are AAV2, AAV3B, AAV6, and AAV13 [10-9]. Recent evidence suggests that HSPG binding of native AAV2 clinical strains is not strong, and that HSPG binding may be a culture-adaptive trait acquired by in vitro passage. HSPG is widely present in vascular endothelium, liver sinusoids, and renal tubular basement membranes, making AAV2 enriched in the liver after systemic administration, but it is also easily intercepted by liver non-parenchymal cells [10-9].

Sialic acid (the connection method determines the subdivision):α2-3 N-linked is AAV1, AAV5[10-6][10-13]; α2-3 O-linked is AAV4 (mucin type) [10-6]; α2-3+α2-6 N-linked is AAV6[10-7]. The high abundance of sialic acid on the airway/alveolar epithelial surface makes AAV4/5/6 bias toward the lung; the difference in glycoforms between the cerebral ependymium and RPE determines that AAV4 is biased towards the membrane and AAV5 is biased towards the glial. Deletion of the CMAH enzyme brings similar sialic acid profiles between humans and mice, which is one of the reasons why SIA-dependent capsids are relatively conserved across species [10-9].

Galactose (the organizational key of AAV9/rh.10):AAV9 recognizes the terminal N-linked galactose exposed after desialylation, and the key residues N470/D271/N272/Y446/W503 constitute the base pocket of the tripodal protrusion [10-18][10-19]; AAVrh.10 recognizes sulfation LacNAc (also contains terminal galactose) [10-9]. Galactose exposure on the surface of neonates and muscle/myocardial/pulmonary epithelial cells is high, which is the glycosyl base for AAV9 to cross muscle fibers and the BBB (neonatal); adult mice have low BBB endothelial galactose exposure, so AAV9 has weak adult BBB crossing [10-25].

Other receptors/co-receptors:PDGFRα/β are AAV5 co-receptors and are expressed in oligodendrocyte precursors, vascular pericytes, and some neurons [10-14]; EGFR is an AAV6 co-receptor [10-16]. Although there were early reports of HGFR(c-Met)/FGFR1/LamR and integrin αVβ5/α5β1, multiple genome-wide CRISPR screens did not confirm that they are essential and should be regarded as “reported but not confirmed” [10-9]. CPD (carboxypeptidase D, also known as AAVR2) is an alternative essential receptor for AAV discovered in 2025 and is important for AAV8 and AAV11 [10-17].

Mechanism summary. Receptor expression differences determine affinity at four levels: ① Cell type level - hepatocytes vs Kupffer cells vs liver sinusoidal endothelium in the same organ have different AAVR, HSPG, and galactose exposures, resulting in "organ enrichment ≠ target cell enrichment"; ② Developmental level - newborn mice have higher exposure to galactose on the BBB and muscle cell surfaces than adults, and AAV9 neonatal administration CNS/muscle transduction is much stronger than in adults [10-25]; ③ Species/strain level - glycosyl modifying enzyme (CMAH), GPI-anchored protein (Ly6A), TfR1 sequence differences lead to cross-species tropism drift (see 6.3); ④ Epithelial polarity level - airway luminal surface vs basolateral surface, subretinal space vs vitreous cavity, receptor accessibility is completely different, and the route of administration therefore reshapes tropism (see 6.3) 6.4).