Adeno-Associated Virus (AAV) is an important tool in the field of gene therapy. This small virus, naturally occurring and not known to cause human diseases, has a unique ability to deliver genetic material into cells. The outer protein shell of the AAV, known as its capsid, plays a central role in this process. Its structure and properties influence how therapeutic genes are delivered to target cells, making it a focal point in gene therapy development.
Understanding the AAV Capsid: Structure and Purpose
The AAV capsid is a non-enveloped, icosahedral protein shell. It is composed of 60 protein subunits: VP1, VP2, and VP3. These proteins assemble to form the complete capsid structure.
The VP1, VP2, and VP3 proteins share common core domains but differ in their N-terminal extensions. The VP3 common region forms the continuous shell of the virus. This arrangement protects the virus’s single-stranded DNA genome from degradation by cellular nucleases. Beyond protection, the capsid’s surface contains specific domains that determine its initial interaction with host cells.
The Capsid’s Role in Gene Delivery
The AAV capsid facilitates gene delivery, beginning with cellular entry. The capsid surface binds to specific receptors on the target cell membrane. This binding determines which cell types the virus can infect.
Following attachment, the virus is internalized into the cell through endocytosis. The capsid then navigates within the cell, undergoing intracellular trafficking to reach the perinuclear region. During this journey, the capsid undergoes conformational changes, triggering changes that aid in the virus’s escape from the endosome.
The final stage involves the release of the genetic payload into the nucleus, a process known as uncoating. Nuclear localization signals are important for guiding the capsid to the nucleus. Once inside the nucleus, the viral genome, which contains the therapeutic gene, is released from the capsid. This allows the introduced gene to be expressed using the cell’s machinery, typically persisting as an episome rather than integrating into the host genome. The capsid, being a foreign protein, can also be recognized by the body’s immune system, potentially eliciting an immune response that can affect the effectiveness of gene therapy.
Exploring AAV Capsid Variations
Naturally occurring AAVs are classified into different “serotypes,” such as AAV1, AAV2, AAV5, and AAV9, with over 100 variants identified. These serotypes possess distinct capsid proteins, leading to variations in their biological properties. A significant difference among serotypes is their “tropism,” which refers to their natural preference for infecting specific tissues or cell types. For instance, AAV9 is known for its ability to cross the blood-brain barrier and target brain tissues, while AAV8 exhibits strong tropism for liver cells. AAV2 has been used in clinical trials for retinal diseases and Parkinson’s disease.
This diversity in tropism is due to differences in how each serotype interacts with specific cellular receptors and co-receptors on the cell surface. For example, AAV2 primarily uses heparan sulfate proteoglycan as a receptor, while AAV5 utilizes α2-3 N-linked sialic acid. The variation in capsid proteins also affects their immunogenicity, meaning how strongly they might trigger an immune response in the body. Pre-existing antibodies to certain AAV serotypes in patients, often developed from prior natural exposure, can neutralize the administered vector, reducing treatment efficacy. Therefore, selecting the appropriate AAV serotype with the desired tissue tropism and a favorable immunogenic profile is an important consideration for successful gene therapy applications.
Innovations in AAV Capsid Design
Scientists are working to engineer AAV capsids beyond their natural forms to enhance their use in gene therapy. This innovation is driven by the need to improve targeting specificity, aiming to deliver genes precisely to desired tissues while minimizing off-target effects. By modifying the capsid, researchers seek to make gene delivery more efficient, allowing for lower therapeutic doses and potentially reducing side effects.
Another motivation for capsid engineering is to evade or reduce the body’s immune response. Natural AAV capsids can be recognized by the immune system, leading to neutralization and limiting the effectiveness of treatment, especially for repeated dosing. Engineered capsids are designed to be less immunogenic, potentially allowing for broader patient eligibility and more sustained therapeutic effects. Researchers also aim to enable gene delivery to tissues that are difficult to reach with natural serotypes, such as the lung, heart, or kidneys, expanding the range of diseases that can be treated with AAV-based therapies. This involves strategies like directed evolution and machine-guided design to create novel capsids with improved properties for therapeutic applications.