Adeno-Associated Virus (AAV) is a small, naturally occurring virus that has found utility in biological research and, more recently, in advanced medical treatments. AAV packaging refers to the process of preparing these viruses to carry specific genetic material. This preparation transforms the AAV into a delivery system, allowing it to transport genes into target cells for therapeutic purposes.
Understanding the AAV Package
An AAV package consists of two parts: a protective outer shell and the genetic material enclosed within. The outer shell, known as the capsid, is made up of 60 protein subunits, forming an icosahedral structure with a diameter of about 20-26 nanometers. This protein capsid acts as a shield, protecting the genetic cargo from degradation and guiding the package to specific cells in the body.
Inside this capsid, the package carries a single-stranded DNA genome, around 4.7 kilobases in length. For therapeutic applications, the AAV’s original viral DNA is removed and replaced with a therapeutic gene, which is flanked by inverted terminal repeats (ITRs). Different AAV serotypes, variations in the capsid proteins, influence the package’s ability to target specific tissues, directing gene delivery to desired organs or cell types.
The Role of AAV Packaging in Gene Therapy
AAV packaging provides a safe and efficient means to deliver therapeutic genes into the body’s cells. AAV is suitable for this role due to its non-pathogenic nature, meaning it does not cause disease in humans. This safety profile makes it an attractive candidate for medical applications.
The virus also has the ability to infect both dividing and non-dividing cells, which broadens its potential for treating various conditions. The AAV package serves as a delivery vehicle for genetic material, allowing for the stable and long-term expression of therapeutic genes within target cells.
Creating an AAV Package
The process of creating an AAV package, often referred to as recombinant AAV (rAAV) production, involves multiple plasmids introduced into host cells. Human embryonic kidney (HEK293) cells are commonly used due to their rapid growth and high transfectability. These cells are engineered to express helper functions, such as those from adenovirus, necessary for AAV replication and packaging.
One plasmid carries the therapeutic gene of interest, flanked by inverted terminal repeats (ITRs), which are signals for packaging. A second plasmid provides the AAV rep and cap genes, which encode proteins needed for replication and capsid formation. A third plasmid supplies additional helper genes that facilitate the overall production process. These plasmids are introduced into the HEK293 cells through a process called triple transfection, where the cells produce the AAV packages. Following production, the AAV packages are harvested from the cell culture and purified to remove cellular debris and other impurities, ensuring a high-quality therapeutic product.
Medical Applications of AAV Packages
AAV packages are employed in gene therapy to treat a range of genetic disorders by delivering functional genes. One application is in inherited retinal diseases, such as Leber congenital amaurosis (LCA), where AAV vectors deliver a healthy copy of the RPE65 gene to retinal cells. This aims to restore the visual cycle and improve or stabilize vision for patients with mutations in this gene. Luxturna, an AAV2-based gene therapy, is an approved treatment for RPE65-mediated retinal dystrophy.
AAV packages also show promise for spinal muscular atrophy (SMA), a severe neuromuscular disorder. For SMA, an AAV9 vector delivers a functional SMN1 gene to motor neurons, which helps restore SMN protein production and can improve motor function. Zolgensma, an AAV9-based therapy, has received approval for treating SMA. In hemophilia, a bleeding disorder, AAV vectors can deliver genes for clotting factors, such as factor IX for hemophilia B, to the liver. This enables the liver to produce the missing clotting factor, helping to restore normal blood coagulation.
Ensuring Safe and Effective Delivery
The development and use of AAV packages in medicine involve several considerations to ensure safe and effective delivery. A challenge is the potential for immune responses against the AAV capsid, which can limit treatment effectiveness or lead to side effects. Pre-existing antibodies from prior exposure to wild-type AAVs can neutralize the therapeutic vector before it reaches target cells. This necessitates strategies such as screening patients for antibody levels or administering immunosuppressants like prednisone to manage these responses.
Another challenge involves delivering AAV packages to a wide range of tissues. While AAVs can target various cell types, achieving widespread transduction, especially in organs like the central nervous system, can be difficult. The small size of AAV particles limits the amount of genetic material they can carry, which can be a constraint for larger genes. Ongoing research focuses on developing new AAV serotypes or modifying existing ones to improve tissue targeting, reduce immunogenicity, and overcome these limitations for broader therapeutic applications.