Adeno-Associated Virus (AAV) for gene therapy

Khalil Essani - Single Use Support

Khalil Essani

September 18, 2023

Table of contents


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.

What is an AAV?

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.


Characteristics of an adeno-associated virus:

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.

AAV structure and capsid proteins

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.

AAV genome and serotypes

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

  • corrective genes to treat genetic mutations,
  • missing genes to replace defective ones,
  • or novel therapeutic genes for specific functions.

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.

AAV serotypes: diversity and targeting

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.

  • Tissue tropism: Different AAV serotypes exhibit varying tissue tropisms, meaning they have preferences for specific types of tissues or organs. This diversity facilitates precise targeting in gene therapy applications.
  • Optimizing therapeutic outcomes: Researchers can select the most suitable AAV serotype based on the disease they are targeting and the desired tissue specificity. This optimization enhances the therapeutic potential of AAV-based gene therapy.

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.
 2 3 

AAV in gene therapy

The adeno-associated virus (AAV) has become a revolutionary instrument in gene therapy by providing a flexible and secure way to deliver therapeutic genes precisely, both in laboratory studies (in vitro) and within living organisms (in vivo).4 5 

How AAV vectors are used in gene therapy

  1. Vector for gene delivery: AAVs serve as delivery vehicles, or vectors, for introducing therapeutic genes into target cells. These genes can be designed to correct genetic mutations, replace missing or defective genes, or introduce novel therapeutic functions
  2. Precision targeting: One of the strengths of AAVs is their ability to precisely target specific tissues and cell types. This targeting is typically accomplished by selecting the appropriate AAV serotype with natural tropism for the desired tissue.
  3. Safe and controlled gene expression: AAV vectors ensure controlled and sustained gene expression, which is particularly important in gene therapy for achieving long-term therapeutic gene expression and lasting treatment effects.

Examples of applications and successes in gene therapy

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 

Advantages of using AAV in gene therapy

Adeno-associated virus (AAV) vectors, have received considerable attention and acclaim in the field of human gene therapy, as prominent therapeutic gene carriers.

Safety profile

One of the most notable advantages of AAV vectors is their outstanding safety profile. This safety record is a result of several factors:

  • Low immunogenicity: The AAV has low immunogenicity, meaning it is less likely to trigger a harmful immune response when introduced into the body. This reduces the risk of adverse reactions commonly associated with other viral vectors.
  • Minimal pathogenicity: The AAV is not known to cause significant diseases in humans. It lacks pathogenic genes, making it a safer choice for gene therapy applications.

Tissue-specific targeting

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.

  • Customized serotypes: Different AAV serotypes have natural tropisms for specific tissues. By selecting the appropriate serotype, researchers can target the exact location where the therapy is needed.
  • Reduced off-target effects: Tissue-specific targeting reduces the likelihood of off-target effects, minimizing potential side effects and improving the overall safety of gene therapy.

Disadvantages of using AAV in gene therapy

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.

Limited cargo capacity

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 

Immune responses

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.

Neutralizing antibodies

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.

AAV production

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:

  1. Vector genome plasmid: This plasmid carries the therapeutic gene of interest, ensuring its precise integration into the rAAV particles. Co-infection with a helper virus may also be involved to facilitate AAV replication.
  2. Cap genes plasmid: The second plasmid encodes essential AAV genes, including the capsid genes (cap genes). These genes are responsible for forming the protective capsid that encases the vector genome.

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 

Challenges in the production of AAVs

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:

  • Logistics: Scaling up vector production to meet the demand for clinical trials and potential treatments can be logistically complex and costly.
  • Purification: Purifying vectors to high levels of purity is a critical step in the manufacturing process. Achieving consistent purity across large batches can be challenging.
  • Quality control: Ensuring the quality and consistency of vectors from batch to batch is essential for safety and efficacy. Stringent quality control measures are required.

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 

Cell and gene therapy filling system RoSS.FILL CGT

Single-use technology in AAV manufacturing

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. 

Future directions in adeno-associated virus research

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.

FAQs about adeno-associated virus

What is an adeno-associated virus (AAV)?

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.

What is transgene expression in the context of gene therapy?

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.

What are examples of adeno-associated virus vectors in gene therapy?

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.

What is the difference between adenovirus and adeno-associated virus?

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

What are virions?

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.

What is a wild-type AAV (adeno-associated virus)?

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.

Further articles about AAVs


AAV production & efficiency improvements with single-use technology

With adeno-associated virus being a popular viral vector, along with a growing interest in gene therapies, there is an increased need for cost-effective vector manufacturing technologies and viral vector production platforms for human gene therapy products.


3 Examples of FDA approved AAVs in gene therapy

In this blog post, we'll show examples of AAVs as gene therapy vectors and examine the tangible results of FDA-approved AAV gene therapies, which offer actual solutions and hope for patients affected by genetic disorders. 

Safe handling AAVs

Safe handling & Storage of Viral Vectors

Viral vectors like Adeno-associated viruses (AAVs) are popular vectors for gene therapy and vaccines. And yet, there is product loss due to leakages of primary packagings or other mishandling of fluids. In order to deliver high-quality and viable products efficiently, safe handling experience a growing interest.

AAV packaging services: Market overview

AAV packaging services: Market overview

Packaging AAVs is crucial as a method to encapsulate a gene of interest within a viral vector. Therefore, we will discuss related methods and services in this article.

  1. Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering,, Published 2021-02-04
  2. Gene Therapy Vectors Based on Adeno-Associated Virus Type 1,, Published 2019-12-31
  3. The progress of AAV-mediated gene therapy in neuromuscular disorders,, Published 2018-05-21
  4. Adeno-associated virus vector as a platform for gene therapy delivery,, Published 2019-02-01
  5. High-Throughput Dissection of AAV–Host Interactions: The Fast and the Curious,, Published 2018-05-19
  6. Adeno-Associated Virus at 50: A Golden Anniversary of Discovery, Research, and Gene Therapy Success—A Personal Perspective,, Published 2015-03-27
  7. Packaging Capacity of Adeno-Associated Virus Serotypes: Impact of Larger Genomes on Infectivity and Postentry Steps,, Published 2005-07-14
  8. Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins,, Published 2007-12-06
  9. Manufacturing of Adeno-Associated Viruses, for Example: AAV2,, Published 2011-05-16
Khalil Essani - Single Use Support

Khalil Essani

Product Line Management

Khalil Essani is Product Line Manager at Single Use Support. Scientist through and through he develops single-use technologies to further advance the biopharmaceutical industry during its shift towards single-use systems. Based on his extensive expertise and graduate in bioprocessing and biotechnology Khalil authors several publications. At Single Use Support he focuses on sterile products in the field of fluid and cold chain management.

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