Adeno-Associated Virus (AAV) vectors are widely used in gene therapy to deliver functional genetic material to patient cells. Successful gene delivery depends on the virus’s ability to recognize and attach to the target cell surface. This initial interaction is governed by specific structures called receptors, which act as the first point of contact for the viral particle. The AAV’s outer protein shell, the capsid, must successfully engage these receptors to initiate therapeutic gene expression, making the precise molecular fit fundamental to treatment success.
The Gateway: Understanding AAV Receptors
The cell surface contains complex molecules, including proteins and carbohydrates, that act as docking stations for external signals and particles. An AAV receptor is a molecule, typically a protein or a glycan (sugar chain), to which the AAV capsid attaches specifically. This interaction is often described as a lock-and-key mechanism, where the AAV capsid is the “key” fitting the complementary cell surface “lock.”
Successful receptor binding is the necessary first step for the entire gene delivery process and cellular entry. Many AAV serotypes first rely on a low-affinity attachment to a glycan molecule, termed the primary receptor, which helps concentrate viral particles on the cell surface. The particle then typically engages a proteinaceous molecule, often called a co-receptor or secondary receptor, which triggers the subsequent steps of cellular entry.
A protein known as AAV receptor (AAVR), or KIAA0319L, is a broadly used proteinaceous receptor for many AAV serotypes. AAVR is a type I membrane protein required for productive transduction by multiple, evolutionarily distant AAV variants.
Serotypes and Tissue Specificity
The natural variation in AAVs is classified into different serotypes (e.g., AAV1, AAV2, AAV8, and AAV9), each possessing a distinct capsid structure. This diversity dictates which receptors a serotype will recognize and bind to. Since different cell types express different surface receptors, the choice of AAV serotype directly determines its tropism, or its natural preference for a specific tissue.
AAV2 relies on Heparan Sulfate Proteoglycan (HSPG) as its primary attachment factor, which is abundantly expressed on many cell types, giving AAV2 a relatively broad tropism. In contrast, AAV9 uses terminal N-linked galactose. This difference in receptor engagement contributes to AAV9’s ability to effectively transduce heart, liver, and central nervous system cells after systemic administration.
Other serotypes display specific preferences based on receptor usage. For instance, AAV8 is effective at transducing liver cells, a trait exploited in hemophilia therapies. The unique receptor footprint allows scientists to select the appropriate vector to target the desired organ, such as choosing AAV5 for central nervous system applications or AAV2 for retinal delivery, as used in the approved therapy Luxturna.
Engineering Receptor Targeting
The natural tropism of AAV serotypes is often imperfect for therapeutic applications, leading scientists to engineer the vector’s capsid to control receptor targeting. This engineering has a twofold goal: to enhance binding affinity to a target tissue and to detarget the vector from non-target organs like the liver. Detargeting reduces the required therapeutic dose and limits potential toxicity. One approach is directed evolution, where AAV libraries are generated with random mutations and screened for variants that display superior targeting.
This process can yield new variants that recognize entirely new receptors or bind with higher efficiency to existing ones. For example, AAV variants have been engineered to efficiently cross the blood-brain barrier, a significant hurdle for treating neurological disorders. The engineered PHP.eB capsid, a derivative of AAV9, was developed using directed evolution to gain enhanced access to the central nervous system by binding to the Low-Density Lipoprotein Receptor-Related Protein 6 (LRP6).
Another method is rational design, which involves making specific, calculated changes to the capsid structure to introduce a new binding motif. Researchers can insert small, receptor-specific peptides or antibody fragments directly into the AAV capsid, creating a new binding domain. This redirects the vector to a specific cell type that expresses the corresponding receptor, such as a tumor cell or a neuronal subtype. Manipulating the amino acid sequences allows scientists to increase affinity for a specific receptor or eliminate binding to native, broad-distribution receptors, improving tissue specificity and safety.
Beyond Binding: Cellular Internalization
Once the AAV particle has bound to its primary and secondary receptors, the cell must internalize it, which is the next major step in gene delivery. The most common pathway is endocytosis, where the cell membrane pinches inward to engulf the vector, trapping it inside a membrane-bound compartment called an endosome. Different AAV serotypes may exploit slightly different endocytic routes, though clathrin-mediated endocytosis is a well-characterized mechanism for AAV2.
The AAV particle must escape the endosome before being trafficked to the lysosome and degraded. The progressively acidic environment inside the endosome triggers a conformational change in the AAV capsid structure. This structural shift exposes parts of the capsid, including a phospholipase A2 (PLA2) domain, which helps the viral particle break free from the endosomal membrane into the cytoplasm.
The vector then travels through the cytoplasm toward the nucleus, which houses the cell’s genetic material. Upon reaching the nucleus, the AAV particle must pass through the nuclear pore complex. Once inside, the final step is uncoating, where the capsid disassembles to release the single-stranded DNA payload. This allows the therapeutic gene to be converted into a functional double-stranded form and expressed by the cell.