Adeno-associated virus (AAV) is a small virus used in gene therapy to deliver therapeutic genes into human cells. AAV does not cause disease in humans, making it a suitable gene delivery vehicle. Researchers engineer AAV by replacing its genetic material with desired genes, allowing introduction of genetic material to replace, silence, or edit problematic genes in specific cell types. AAV vectors can infect various cell types, including both dividing and non-dividing cells, leading to long-term expression of delivered genes.
Fundamentals of AAV Production
Adeno-associated virus is replication-defective, meaning it cannot multiply independently. It requires helper functions to replicate and package its genetic material into new viral particles. These functions are typically provided by other viruses, such as adenovirus or herpesvirus, but in laboratory settings, they are supplied by specific DNA molecules called plasmids.
A typical AAV production system involves at least three types of plasmids. The transfer plasmid carries the therapeutic gene of interest flanked by AAV inverted terminal repeats (ITRs). These ITRs are important for viral DNA replication and packaging.
The packaging plasmid, often called the Rep/Cap plasmid, encodes the AAV replication (Rep) and capsid (Cap) proteins. Rep proteins are involved in viral genome replication and packaging, while Cap proteins form the protective outer shell of the virus. The helper plasmid provides the necessary helper functions, typically derived from adenovirus genes, which are essential for AAV replication and assembly. To produce AAV, these plasmids are introduced into a host cell system, with human embryonic kidney (HEK293) cells being a commonly used choice due to their ease of culture and high transfection efficiency.
Key Production Methods
Several methods are used to produce AAV vectors, each with distinct characteristics regarding scalability, cost, and consistency.
Transient transfection is a widely used method, especially for preclinical research and early-stage clinical production. In this approach, the multiple plasmids (transfer, packaging, and helper) are simultaneously introduced into host cells, like HEK293 cells, to transiently express the components for AAV assembly. This method offers flexibility and can be adapted for different AAV serotypes. Chemical reagents like calcium phosphate, polyethylenimine (PEI), or lipid-based reagents are used to facilitate the uptake of plasmid DNA by the cells.
Stable cell lines are another production strategy where AAV components are stably integrated into the host cell’s genome. This creates cell lines that consistently produce AAV particles without repeated plasmid transfections. Stable cell lines offer improved scalability and batch-to-batch consistency compared to transient methods. However, developing them can be complex due to the potential toxicity of some AAV genes to the host cells.
The insect cell/baculovirus system provides an alternative for large-scale AAV production. In this method, insect cells (e.g., Sf9 cells) are infected with baculoviruses engineered to carry the AAV Rep and Cap genes, as well as the therapeutic gene. This system is known for its high-yield production capacity and efficient protein expression. It offers a cost-effective and scalable platform for manufacturing AAV vectors.
Purification and Quality Control
After AAV particles are produced within host cells, they undergo downstream processes to ensure suitability for therapeutic use. The first step involves harvesting cells and releasing AAV particles. This is typically achieved through cell lysis, a process that breaks open host cells to release the viral vectors. Lysis methods include chemical approaches (e.g., detergents) or physical methods (e.g., freeze-thaw cycles or mechanical disruption).
Following lysis, the crude lysate, containing AAV particles along with cellular debris and other contaminants, undergoes purification. Chromatography is a widely used technique for this, offering high specificity and scalability. Affinity chromatography, for example, uses specific binding interactions to capture AAV particles while allowing impurities to pass through. Ion exchange chromatography further separates AAV particles from remaining contaminants, including empty or partially filled capsids, based on charge differences. These chromatographic steps aim to achieve a highly pure viral vector product.
Quality control (QC) testing is performed on the purified AAV product to ensure its safety, purity, and potency before therapeutic use. Titer measures the concentration of viral particles. Purity assessments confirm minimal contaminants, which is important for patient safety. Potency or infectivity assays ensure AAV vectors effectively deliver their genetic cargo and express the therapeutic gene. Sterility testing checks for microbial contamination.