AAV Capsids_AAVnerGene

AAV Capsid Structure

AAV Based Gene Therapy

Adeno-associated virus (AAV), a non-pathogenic parvovirus, has emerged as the leading vector for gene therapy. AAV-based gene therapies have demonstrated significant clinical success, with eight treatments receiving regulatory approval for human use. These include revolutionary therapies for spinal muscular atrophy (SMA), inherited retinal disorders, and hemophilia, highlighting the profound clinical potential of this innovative technology. These therapies utilize distinct AAV capsids to target specific tissues.

Drugs	Company	Diseases	AAVs	Injection Routes	Targets	Approval	Price
Glybera	uniQure	LPLD	AAV1	Intramuscular injection	Muscle	EMA 2012	$1.2M
Luxturna	Spark	RPE65-LCA	AAV2	Subretinal Injection	Retina	FDA, 2017	$0.86M
Zolgensma	Novartis	SMA	AAV9	Intravenous infusion	Brain	FDA, 2019	$2.2M
Upstaza	PTC	AADC	AAV2	Bilateral intraputaminal infusion	Brain	EMA, 2022	$3.7M
Roctavian	BioMarin	Hemophia A	AAV5	Intravenous infusion	Liver	EMA,2022	$2.9M
EtranaDez	uniQure	Hemophia B	AAV5	Intravenous infusion	Liver	FDA, 2022	$3.5M
ELEVIDYS	Sarepta	DMD	AAVrh.74	Intravenous infusion	Muscle	FDA, 2023	$3.2M

While they have shown remarkable success, several critical challenges remain, including:

High costs, which hinder broad patient accessibility.
High vector dosages, which not only increases expenses but also raises safety concerns.
Anti-AAV immune responses, which can lead to the destruction of transduced cells, compromising treatment outcomes.
Neutralizing antibodies (NAbs), which limits the applicability of AAV vectors for many individuals.

To address these challenges, researchers are advancing next-generation AAV vectors through innovative capsid engineering strategies. This approach focuses on:

Enhancing AAV efficiency to reduce the required vector doses, thereby improving safety and minimizing the risk of immune responses.
Increasing AAV specificity to decrease off-target effects and enhance therapeutic precision.
Designing vectors that are effective in patients with preexisting immunity to natural AAV serotypes, expanding the potential patient populations

AAV capsid structure

The structure of the AAV capsid is crucial for the virus’s ability to deliver its genetic material, interact with host cells, and evade the immune system. The capsid consists of 60 protein subunits arranged in a 20-sided icosahedral shape with a diameter of about 25 nanometers. Capsid Proteins (VP1, VP2, VP3) is the AAV capsid is composed of three viral proteins: VP1, VP2, and VP3, all encoded by the cap gene. These proteins are produced from overlapping reading frames and assembled in a 1:1:10 ratio (VP1:VP2:VP3), forming the complete capsid structure.

VP1: The largest subunit, containing a phospholipase A2 (PLA2) domain essential for viral entry and release of the viral genome into the host cell.
VP2: Intermediate in size, its function is less understood but is believed to stabilize the capsid and assist in assembly.
VP3: The most abundant subunit, comprising 80-90% of the capsid, forming its structural framework and interacting with host cell receptors.

Icosahedral Symmetry: The AAV capsid has 60 identical subunits arranged in a highly ordered icosahedral symmetry, providing structural stability and enabling it to encapsulate the viral genome. Surface Topology: The capsid surface features unique structural landmarks:

- Peaks and Depressions: High peaks at the 3-fold axes and depressions at the 2-fold axes.
- 5-Fold Pore: A small pore at the 5-fold axis that helps release the viral genome during infection.
- 3-Fold Protrusions: Protrusions at the 3-fold axis involved in binding to host cell receptors, influencing tissue tropism.

Host Cell Receptor Binding Sites: The AAV capsid has specific regions that bind to host cell receptors, which differ by serotype:

- AAV2 binds to heparan sulfate proteoglycan (HSPG).
- AAV9 binds to galactose-containing glycans.

These receptor-binding regions, located in the variable loops of the capsid, can be modified through capsid engineering to alter tissue targeting. Phospholipase A2 (PLA2) Domain: The PLA2 domain, found in the VP1 subunit, is hidden inside the capsid in its mature form but is exposed during cell entry. This domain is crucial for helping the virus escape from the endosome after entering the host cell, allowing the viral genome to be released into the cytoplasm.

AAV Inner Capsid

The inner capsid of AAV refers to the internal environment within the capsid shell, which encapsulates and protects the viral genome. Although AAV lacks an inner protein structure or core, the inner capsid plays a critical role in maintaining the stability and proper packaging of the viral genetic material.

Encapsulation of Viral Genome: The AAV inner capsid houses a single-stranded DNA (ssDNA) genome of about 4.7 kilobases in length. The ssDNA is tightly packaged within the capsid shell and stabilized by interactions with the capsid proteins. These interactions ensure the genome remains intact during viral assembly, transit, and infection of host cells. This genome includes:
- Sequences for replication and packaging, such as the rep and cap genes.
- Therapeutic genes in gene therapy vectors.
Genome Packaging: AAV packaging efficiency is largely determined by the inner surface of the capsid, which interacts with the viral genome during assembly. Key components of this process include:
- Rep Proteins: Rep78/68 and Rep52/40 guide the DNA into the capsid during production.
- Inverted Terminal Repeats (ITRs): Located at both ends of the AAV genome, these sequences are crucial for recognizing and properly inserting the DNA into the capsid.
Role of the VP1 N-Terminal Region: The inner capsid also contains the N-terminal domain of the VP1 protein, which remains hidden in the mature virus. This domain:
- Contains phospholipase A2 (PLA2) activity, essential for viral entry.
- Is exposed under acidic conditions within the endosome, facilitating endosomal escape by degrading the host cell membrane and enabling the release of the viral genome into the cytoplasm.
Capsid Stability and Genome Protection: The inner capsid environment acts as a protective barrier for the viral genome, shielding it from: Nucleases, Environmental degradation, Immune detection. Capsid proteins interact with the DNA to ensure its stability, preventing premature release or damage before the virus reaches its target.
Conformational Changes During Infection: When AAV enters a host cell via endocytosis, the acidic conditions within the endosome trigger conformational changes in the capsid. Unlike many viruses, the AAV capsid generally does not fully disassemble. Instead, it forms a temporary pore-like structure through which the viral DNA is extruded. These changes:
- Expose the VP1 PLA2 domain.
- Partially uncoat the genome, enabling the DNA to exit the capsid and enter the nucleus for transcription.
Packaging Capacity Limitations: The compact nature of genome packaging within the inner capsid limits the genetic material that AAV vectors can deliver. The maximum genome size efficiently encapsulated is approximately 4.7 kilobases. Exceeding this size may result in:
- Incomplete packaging
- Inefficient production of functional AAV particles.

AAV Capsid Engineering

To overcome these limitations and expand the therapeutic potential of AAV, capsid engineering has become a critical area of research. By modifying the viral capsid—the protein shell that encapsulates the genetic material—scientists can enhance AAV’s targeting specificity, transduction efficiency, and immune evasion properties. Advances in capsid engineering, including rational design, directed evolution, and computational modeling, have enabled the development of novel AAV variants with tailored properties for diverse therapeutic applications. These engineered capsids hold the promise of unlocking new frontiers in gene therapy, enabling precise and effective treatments for a wide range of genetic disorders.

Rational Design: is a targeted approach to AAV capsid engineering that leverages detailed structural and functional insights to enhance the virus’s performance. By strategically introducing specific modifications—such as point mutations, insertions, or deletions in critical regions of the capsid—researchers can optimize its interactions with cellular receptors, improve tissue specificity, and reduce immune system recognition. This method relies on a deep understanding of the capsid’s atomic structure and its functional domains to guide precise alterations. One well-known application of rational design is the Y-to-F mutation, where tyrosine (Y) residues on the capsid surface are replaced with phenylalanine (F). This modification reduces the capsid’s susceptibility to proteasomal degradation, thereby improving its ability to deliver genetic material to target cells.
Directed Evolution: This approach involves creating extensive libraries of AAV capsid variants and iteratively screening them to identify those with desirable traits. Capsid libraries are generated using techniques such as:
- Insertional Mutagenesis: Incorporating peptides or sequences into specific regions of the capsid to alter its properties.
- Error-Prone PCR: Introducing random mutations into the capsid gene to create genetic diversity.
- Peptide Display (e.g., ATHENA-II): Inserting short peptides into surface-exposed loops to create new binding sites, enabling improved tissue targeting or receptor interactions. This method exploits peptide-receptor dynamics to design AAV vectors that selectively target specific cell types. It is mostly widely used method to create new capsid, such as AAV-PhP.eB, AAV-7M8.
- Loop Replacement: Similar to peptide display, loop replacement involves substituting specific loops (e.g., the RGD loop) or sequences in the AAV capsid with those from other AAV serotypes or engineered sequences. This modification can alter the capsid’s tropism, enabling it to target different tissues or cell types more effectively.
- DNA Shuffling (e.g., ATHENA-III): Recombining sequences from multiple AAV serotypes to produce chimeric capsids with hybrid functionalities, such as AAV-DJ and our AAV-ShD.
Chimeric Capsids: By combining gene segments from different AAV serotypes or co-transfecting plasmids encoding distinct AAV capsids (e.g., use AAV1 and AAV2 to create AAV1/2 hybrid), researchers can create chimeric capsids that integrate the most advantageous features of multiple variants. These engineered capsids frequently demonstrate enhanced tropism, reduced immunogenicity, and improved transduction efficiency compared to naturally occurring AAV serotypes. This approach allows for the customization of AAV vectors to better target specific tissues, evade immune responses, and deliver therapeutic genes more effectively.
Computational Modeling: The integration of artificial intelligence (AI) with AAV research has revolutionized capsid engineering. Advanced computational tools and machine learning algorithms enable the prediction and design of capsid modifications with unprecedented accuracy. By simulating interactions and optimizing designs in silico, researchers can accelerate the development of next-generation AAV vectors tailored for specific therapeutic applications.