September 18, 2023
The adeno-associated virus (AAV) is a very popular viral vector with great potential available for gene delivery. This article explores AAV vectors, their role in gene therapy, and their associated advantages and disadvantages.
The adeno-associated virus (AAV) is a small, non-pathogenic virus belonging to the parvovirus family. It gained prominence as a gene therapy vector due to its distinctive features, including low immunogenicity and the ability to provide long-term gene expression.
AAV vectors offer a safe and promising avenue for gene transfer, with a reduced probability of unwanted immune responses. At the heart of AAVs’ success in gene therapy lies their ability to serve as a Trojan horse, shuttling therapeutic genetic material, often in the form of a plasmid, into target cells with highest precision.
This process involves the manipulation of an AAV serotype, most commonly adeno-associated virus type AAV2, which can be engineered to transport specific genes under the control of a chosen promoter. AAV2's success in gene therapy also depends on its interaction with cellular receptors that facilitate its entry into the host cell.
1. Non-pathogenic nature
AAV vectors are considered non-pathogenic, meaning they do not cause significant harm or diseases in humans. This safety profile is a crucial advantage when considering their use as vectors in gene therapy.
2. Small size
The AAV is quite small, measuring only about 20 nanometers in diameter. This compact size makes it an ideal candidate for gene delivery because it can efficiently penetrate various tissues and cells.
3. Single-stranded DNA
An AAV carries its genetic material in the form of a single-stranded DNA genome. This genome can be modified and engineered to carry therapeutic genes, making it a versatile tool for delivering genetic payloads.
4. Lack of pathogenic genes
Unlike some other viruses, the AAV lacks genes that are known to cause diseases in humans. This further enhances its safety profile for use in gene therapy.
5. Existence of different serotypes
AAVs come in various serotypes, with AAV2 being one of the most commonly used in research and clinical applications. Each serotype has unique properties, such as tissue tropism (preference for specific tissues) and cell entry mechanisms, allowing for customization based on the target disease and tissue.
6. Integration into host genome
The AAV can integrate its genetic material into the host genome, but this occurs at a very low frequency. Most adeno-associated virus vectors predominantly exist as episomes in the nucleus, providing stable, long-term gene expression without the risk of disrupting essential host genes.
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The adeno-associated virus (AAV) has a unique capsid structure that is crucial for functionality. The AAV capsid acts as a protective shell for its genetic material, shielding it until it reaches the host cell’s target location.
The capsid proteins of AAVs are essential, as they influence the virus’s distinctive features, including its capacity to infect specific cell types. This complex interaction between capsid proteins is a key element in the efficacy of AAV as a tool for gene therapy.
The genome plays a multifaceted role in gene therapy, serving as the basis for the therapeutic genes delivered by AAV vectors.
The AAV genome carries the genetic payload directed towards target cells. This payload can include
The viral genome is essential for ensuring stable and controlled gene expression in target cells. This stability is vital for the long-term efficacy of gene therapy, especially in treating chronic conditions.
Although AAV vectors are predominantly non-integrative, it is important to understand the genome's potential to minimize any associated risks with integration into the host chromosome.
The AAV's adaptability is highlighted by its various serotypes, denoted by numbers such as AAV1, AAV5, AAV6, AAV7, AAV8, and AAV9. Each AAV serotype interacts differently with host cells, offering researchers a spectrum of options when designing gene therapy strategies.
For instance, when researchers aim to treat diseases affecting skeletal muscles, such as muscular dystrophy, selecting an AAV serotype like AAV6 or AAV9 with a propensity for skeletal muscle transduction can enhance the therapeutic impact.
The synergy between the AAV genome and its diverse serotypes empowers researchers to tailor gene therapy approaches with precision, optimizing outcomes for a wide range of genetic disorders and medical conditions.
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AAV-based gene therapy has shown significant progress in recent years, bringing hope to individuals with genetic disorders previously deemed untreatable.
One shining example is the use of AAV vectors in the treatment of hemophilia, a genetic disorder that hinders the blood's ability to clot. AAV-based therapies have displayed potential in providing a functional copy of the absent or deficient clotting factor, granting patients the possibility of a life unrestricted by this condition.
Additionally, AAV-based gene therapy has been used for characterization studies, providing valuable insights into the genetic underpinnings of various diseases. These studies help researchers better understand disease mechanisms and develop more targeted therapies.
Furthermore, primate models have played a crucial role in advancing AAV-based gene therapy research. These models offer a more accurate depiction of human biology and assist in enhancing therapeutic approaches prior to clinical trials.6
Adeno-associated virus (AAV) vectors, have received considerable attention and acclaim in the field of human gene therapy, as prominent therapeutic gene carriers.
One of the most notable advantages of AAV vectors is their outstanding safety profile. This safety record is a result of several factors:
AAV vectors can be engineered for tissue-specific targeting. This means that researchers can tailor AAV vectors to deliver therapeutic genes to specific tissues or organs, enhancing the precision and efficacy of gene therapy treatments.
While adeno-associated virus (AAV) vectors offer numerous advantages in gene therapy, they are not without their challenges and limitations. In this section, we will explore some of the key drawbacks and considerations associated with the use of AAV vectors for gene therapy applications.
One of the primary limitations of AAV vectors is their limited cargo capacity. An AAV can only accommodate a relatively small amount of genetic material, typically up to 4.7 kilobases (kb). This restriction poses challenges when dealing with large therapeutic genes or complex genetic constructs.7
While the AAV is known for its relatively low immunogenicity, it is not entirely immune to the body's defense mechanisms. Some patients may develop immune responses to AAV vectors, particularly if they have pre-existing immunity or have been exposed to AAVs in the past.
Patients with pre-existing immunity to specific AAV serotypes may not respond well to AAV-based gene therapy, limiting its applicability in some cases.
The formation of neutralizing antibodies can hinder the effectiveness of AAV vectors. These antibodies can neutralize the virus before it reaches the target cells, reducing the therapeutic impact.
The production of AAV vectors is a multistep process. It begins with the selection of a suitable host cell line, often HEK293 or CHO cells. These cells are genetically engineered to support AAV replication, effectively transforming them into dedicated factories for AAV production.
Next, the gene of interest, which may be a therapeutic gene, is cloned into a plasmid. This plasmid serves as a precise blueprint that guides the synthesis of the therapeutic gene during AAV replication.
The heart of AAV production is the generation of recombinant adeno-associated virus (rAAV). This feat is accomplished by co-transfecting the host cells with two key plasmids:
The host cells then carry out the intricate task of assembling the recombinant AAV particles. The process is further facilitated by the interplay of RNA, peptides, and cloning technologies. This multifaceted production process is a testament to the scientific ingenuity behind the creation of AAV vectors, which have become indispensable tools in the field of gene therapy.8
Yielding high-quality AAV vectors consistently is one of the primary challenges, and this includes monitoring the titer of AAV vectors produced. Factors such as maintaining the integrity of the AAV genome and optimizing transfection conditions require meticulous attention.
Another hurdle is ensuring that the production process adheres to strict Good Manufacturing Practices (GMP) standards. This ensures the safety and efficacy of AAV vectors for use in clinical settings.
Upscaling the production and purification of viral carriers – AAV vectors – also presents significant challenges, particularly for clinical applications. Manufacturing hurdles include:
Innovative solutions in the manufacturing process of AAVs help minimize these challenges to provide fast, affordable and safe therapies. Single Use Support contributes with cutting-edge single-use technologies to optimize scalability in manufacturing. 9
Single-use technology has accompanied the developments in AAV production for years. Single-use systems, including single-use bags and single-use shells, provide a sterile and controlled environment for AAV production. They do not require time-consuming cleaning and sterilization processes and offer a high level of flexibility.
Single-use technology not only enhances the efficiency of AAV production but also minimizes the risk of contamination, ensuring the safety and quality of AAV vectors.
Single Use Support provides solutions for filling and homogenization of viral vectors and freezing & thawing viral vectors based on single-use technologies to make the manufacturing process efficient and safe. This makes the production of AAVs more cost-efficient and safer, which ultimately pays off in the health of patients.
Researchers in virology are actively addressing challenges through ongoing innovation, including the development of novel AAV serotypes, improvements in cargo capacity, and enhanced manufacturing processes.
Advancements in immunomodulation and immune tolerance strategies aim to reduce immune responses to AAV vectors, increasing their effectiveness in a broader patient population.
Streamlined and scalable manufacturing processes will make AAV-based gene therapy more accessible and cost-effective, paving the way for wider clinical adoption.
Finally, AAV vectors continue to be investigated for their potential in treating a wide range of diseases, including neurodegenerative disorders, cardiovascular conditions, and rare genetic diseases.
An adeno-associated virus (AAV) is a small, non-pathogenic virus frequently used in gene therapy. It serves as a safe and efficient vector to deliver therapeutic genes into target cells for various medical applications.
Transgene expression in gene therapy refers to the process by which a therapeutic gene, known as a transgene, is introduced into a patient's cells and subsequently produces the desired functional protein. This expression of the transgene is the fundamental mechanism by which gene therapies aim to treat or alleviate genetic disorders or other medical conditions.
Examples of adeno-associated virus (AAV) vectors used in gene therapy are Luxturna®, Zolgensma®, Hemgenix®, Elevidys or Roctavian™. Zolgensma for example is a treatment for Spinal Muscular Atrophy (SMA). SMA is a severe genetic disorder that affects the motor neurons of the spinal cord, leading to muscle weakness and, in many cases, early mortality.
The adenovirus and the adeno-associated virus (AAV) differ in several ways: The adenovirus is larger, has a risk of causing illnesses in humans, and has its own genes. AAVs are smaller, usually non-pathogenic, and rely on a helper virus for replication. AAVs are often used as gene therapy vectors. Read more in the article: Adenovirus vs. Adeno-associated virus
Virions are individual, complete virus particles that are capable of infecting host cells. They represent the extracellular, infectious form of a virus. Virions are composed of genetic material (either DNA or RNA) surrounded by a protective protein coat known as a capsid. In some cases, virions may also have an outer lipid envelope.
A wild-type AAV, or adeno-associated virus, refers to the naturally occurring, unaltered form of AAV found in the environment. Wild-type AAVs are viruses that have not been genetically modified or engineered for specific purposes, such as gene therapy. They are typically isolated from various sources, including humans, animals, and the environment, and are characterized by their natural genetic makeup and properties.
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