As the field of gene therapy progresses, tiny particles known as adeno-associated viruses (AAVs) are making an outsized impact. These viral vectors, often referred to as AAV vectors, carry therapeutic genes to cells affected by genetic defects. While AAV gene therapy shows promise in treating inherited disorders such as spinal muscular atrophy and advancing vaccine development, ensuring the quality and consistency of these intricate products presents unique challenges.
For analytical chemists, working with AAVs might feel unfamiliar. Yet, these professionals play a key role in analyzing viral vectors and measuring critical quality attributes (CQAs) to ensure safety and efficacy.
This article introduces AAVs, highlighting their structure, function, and importance in AAV gene therapy from an analytical chemistry perspective. Insights are drawn from Andrea Krumm, Product Manager at Tosoh Bioscience, as featured in the video tutorial series (U)HPLC Analysis of Biopharmaceuticals: Quality Attributes and How to Analyze Them. Whether you’re a seasoned analyst or new to the field, understanding the fundamentals of AAVs is essential to keeping up with biopharmaceutical innovations.
What Are AAVs?
AAVs are small viruses, about 25 nanometers in diameter, with a single-stranded DNA genome approximately 4.7 kilobases long. AAVs are inherently stable due to their protective protein shell, or capsid, which compensates for the absence of an outer lipid membrane. This structure withstands harsh conditions, including temperature changes, drying, and exposure to detergents, simplifying storage, handling, and delivery during manufacturing.
Structurally, AAVs have two main components:
- AAV capsid: A protein shell made from three related proteins—VP1, VP2, and VP3—that form a symmetrical icosahedral shape. These proteins, produced from a single gene region, assemble in a typical ratio of 1:1:10, providing the stability and protection needed to deliver the genome to target cells. The capsid’s compact and robust design allows it to survive harsh conditions during manufacturing and delivery.
- The genome: A single strand of DNA, typically encoding the therapeutic gene, flanked by inverted terminal repeats (ITRs). ITRs are short, repeating sequences of DNA that fold into looped structures. These loops are essential for replication and packaging, as AAVs lack the necessary replication machinery and rely on proteins supplied by a helper virus during manufacturing.
Why Are AAVs Safe and Effective?
AAVs are considered safe viral vectors for gene therapy due to their unique biology. They do not integrate into the host genome because they lack the proteins and DNA signals needed for insertion, minimizing the risk of harmful mutations. Instead, AAVs deliver their DNA as extrachromosomal material, separate from the host’s genetic code, making them especially effective for targeting non-dividing cells.
In AAV gene therapy, vectors are engineered by removing native viral genes, leaving only the therapeutic gene and essential DNA for packaging and delivery. This streamlined version cannot replicate or cause infection but effectively delivers therapeutic DNA to target cells.
How Does AAV Gene Therapy Work?
AAV vectors deliver their therapeutic DNA through a stepwise process of cellular entry and cargo release. The journey begins as the capsid binds to specific receptors on the surface of the target cell, initiating entry. The cell engulfs the AAV through endocytosis, enclosing it in a vesicle called an endosome.
To complete delivery, the AAV escapes the endosome and travels to the nucleus. Once inside, the capsid uncoats, releasing its single-stranded DNA payload. The host cell’s enzymes convert this DNA into a double-stranded form, enabling the production of the therapeutic protein encoded by the inserted gene. This carefully controlled process ensures efficient and safe delivery without integrating into the host genome.
Analytical Challenges in AAV Characterization
The AAV production process doesn’t yield only the desired product. It generates a mix of full, partially filled, and empty capsids, along with aggregates. Full capsids contain the therapeutic genome and are the target product, while partially filled capsids have incomplete genomes, and empty capsids lack genetic material entirely. Aggregates can cause extra problems, including triggering immune responses.
Techniques including size-exclusion chromatography (SEC) and anion-exchange chromatography (AEX) are essential for separating and quantifying AAV vectors. These methods play a crucial role in ensuring AAV therapeutics meet safety and efficacy standards.
Conclusion
AAV capsids and their role in gene therapy offer transformative possibilities for medicine. For analytical chemists, understanding the fundamentals of AAVs lays the groundwork for ensuring their safety and efficacy. Learn more about chromatographic characterization of biotherapeutics in the Tosoh Bioscience video tutorial series, available exclusively on Separation Science.
