Adeno-associated viruses (AAVs) are small viruses that have become a powerful tool in gene therapy. These viruses are modified to deliver genetic material to target cells in the body, offering a way to address genetic disorders at their source. AAVs act as carriers, transporting therapeutic genes for medical treatments. This approach holds promise for various conditions by introducing functional genetic material.
Understanding AAVs as Gene Delivery Vehicles
AAVs are non-enveloped viruses belonging to the Parvoviridae family, not known to cause disease in humans. They naturally carry genetic material, making them suitable for gene therapy. Scientists modify AAVs by replacing their native viral genes with a desired therapeutic gene, transforming them into gene delivery vehicles.
AAVs are favored for gene therapy due to several characteristics. They exhibit low immunogenicity, provoking a minimal immune response that reduces adverse reactions. Their ability to infect a wide range of cell types, known as broad tropism, allows them to target various tissues and organs effectively. Genes delivered by AAVs can also lead to sustained expression in host cells, providing long-lasting therapeutic effects, sometimes for up to ten years in human muscle tissue.
Essential Components for AAV Manufacturing
AAV production relies on specific cell lines that serve as “factories” for viral production. HEK293 cells, human embryonic kidney cells, are commonly used due to their ability to efficiently take up and process genetic material. These cells are adapted to grow in suspension cultures, which allows for large-scale production in bioreactors.
Multiple types of plasmid DNA are introduced into these producer cells to initiate AAV assembly. One plasmid carries the therapeutic gene of interest, flanked by inverted terminal repeats (ITRs). These ITRs signal for the AAV genome to be packaged.
Other plasmids provide the necessary AAV genes: specifically rep genes for replication and cap genes, which encode capsid proteins that determine the AAV serotype and its ability to infect specific cell types. A third set of plasmids provides helper functions, often derived from adenovirus or herpes simplex virus. These are needed for AAV replication and assembly within host cells, even though a helper virus itself is not directly used in these “helper-virus free” systems.
The Core Stages of AAV Production
AAV vector manufacturing begins with the preparation of producer cells. Cells like HEK293 are grown and expanded in controlled environments such as shaker flasks or bioreactors. This cell culture ensures a sufficient quantity of healthy cells to support efficient viral production.
Following cell expansion, the next step is transfection, where DNA plasmids are introduced into the producer cells. This process can involve methods like calcium orthophosphate or polyethyleneimine (PEI), which facilitate plasmid entry into the cells. The optimal cell confluence for transfection can vary.
Once inside the cells, the introduced plasmids initiate viral replication and assembly. The rep and cap genes, along with helper functions, work together to replicate the therapeutic gene’s DNA and assemble new AAV particles, encapsulating the genetic material within the viral capsid. This assembly process occurs over several days, depending on the AAV serotype.
After AAV particles form within the cells, they are collected through harvesting. This often involves lysing, or breaking open, the producer cells to release the AAV vectors. Mechanical methods, such as repeated freezing and thawing or homogenization, can be used for cell lysis. Chemical methods involving detergents like Triton X-100 are also employed. The resulting mixture, containing the AAV particles, is then prepared for purification.
Purification and Quality Control
Once AAV particles are harvested, they undergo purification to separate the therapeutic vectors from cellular debris, host cell proteins, nucleic acids, and other contaminants. Chromatography is a common technique, including affinity chromatography and ion-exchange chromatography. Affinity chromatography uses resins that specifically bind to proteins on the AAV capsid, allowing selective separation of the vectors from impurities, including empty capsids that lack the therapeutic DNA. Ion-exchange chromatography separates particles based on their charge differences, distinguishing full AAV particles from empty or partially filled ones.
Ultracentrifugation is another method used to separate AAVs based on their buoyant density. This technique effectively separates full AAV virions from empty capsids. The purified AAVs are then concentrated and formulated into a stable solution for storage and delivery, ensuring their integrity and effectiveness.
Following purification, the AAV product undergoes quality control testing to confirm its safety, purity, and potency for clinical use. Purity assessments verify the absence of contaminants, including host cell proteins and residual DNA, often using techniques like SDS-PAGE and spectrophotometry. Potency and titer measurements quantify the concentration of functional viral particles capable of delivering the therapeutic gene, often determined by genomic titer assays like ddPCR or infectivity assays.
Sterility testing ensures the absence of microbial contamination. Identity testing confirms the correct AAV serotype and the integrity of the encapsulated genetic material through methods such as ELISA, Western blot, or next-generation sequencing. Safety evaluations include testing for endotoxins and verifying the absence of replication-competent virus, which could pose a risk to patients. These quality control measures are essential for meeting regulatory standards and ensuring the therapeutic efficacy and safety of the final AAV product.