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Gene Therapy: The Design and Development of AAV Vectors

Written by: Jamie Saito ‘25

Edited by: Melinda Li ‘22

Though scientists have identified the mutations for many neurological disorders, including Parkinson’s, Alzheimer’s, and spinal muscular atrophy, most remain untreatable. However, a new experimental form of treatment may be a solution to this problem – gene therapy. Gene therapy involves the introduction of genetic material into target cells and holds potential to target mutations that cause genetically-linked diseases. One approach to this treatment uses viral vehicles, referred to as viral vectors, to deliver genetic material to selected cells. Many types of viruses have been explored as possible vectors; however, adeno-associated viral (AAV) vectors are one of the most promising options at the forefront of current research.

Adeno-associated viruses belong to the Dependoparvovirus genus and require a helper virus, such as an adenovirus, to complete their life cycle (1). Because of this, it is believed that they do not directly cause any known human diseases. Discovered in the mid-1960s, many properties, such as their relatively simple genomes, make them an ideal candidate for gene therapy development (2). However, there is still work to be done to make these viruses a viable therapeutic option. To optimize the use of AAV vectors in gene therapy, scientists need to take two major factors into account: capsid design and genome.

Capsid Design

The capsid, or the protein shell of the virus, is a critical component of any viral vector. Capsid design influences the transduction of treatment and the immune response risk for patients (1). Since this portion of the virus is what is recognized by cells, its design impacts both the ability of the virus to infect a particular cell and antibody recognition (2). Different serotypes, or capsid variants, may be more effective at targeting particular cells. For neurological disorders, serotypes that maximize the efficacy rate in neurons are ideal choices (1). There are three main approaches that can be used to optimize capsid design: natural discovery, rational design, and directed evolution.

Natural discovery is rooted in the isolation of serotypes discovered in nature without modification (3). However, direct use of these capsids may lead to an increased risk of immune response. Because adeno-associated viruses are very common, many people have antibodies against them, which would cause the body to attack any AAV vector introduced with a therapeutic purpose and thus render the therapy ineffective. Nevertheless, natural discovery is not without its benefits. Scientists now have a large collection of known capsids, which they can use to engineer optimal capsids.

Another method of capsid design optimization is to take advantage of a rational design approach. Rational design is predicated on knowledge about structure-function relationships. Essentially, scientists can study information regarding “capsid stability, receptor binding or trafficking” and use this knowledge to design a capsid that will maximize transduction into specific cells (1). They do so by intentionally inserting known mutations into the virus’s genome, creating altered capsids. Though this approach may maximize the transduction of an AAV vector, it is challenging to create a functional capsid.

The final approach, directed evolution, has proved to be a successful technique in generating an ideal capsid, and takes advantage of findings on capsids based on previous studies. Scientists subject the viruses to selective pressure, such as error-prone PCR, which causes them to evolve a specific capsid design. This selective pressure can be altered so that the capsid will have an optimal design to target specific cells. Using this technique may provide scientists with the tools to create specific forms of gene therapy for a wide range of diseases.


Apart from capsid design, another factor that can be optimized for gene therapy is a vector genome. Upon introduction to the cell, the AAV transduction pathway is relatively simple. The viral vehicles are trafficked to the nucleus where its genome is released. Here, it converts its single-stranded DNA into double-stranded DNA, which is a crucial and rate-limiting step. The genetic material can then go through the transcription process to produce mRNA and eventually create proteins (4). This is where the vector genome comes into play.

Vector genome design relates directly to gene regulation and expression. Adeno-associated viruses have a very small genome, meaning their maximum capacity is around 5,000 base pairs long. This limits the amount of information that can be included in one viral vector, potentially restricting treatments in the future. Fortunately, about 96% of the AAV genome can be removed without affecting the transduction process, which may provide sufficient space for therapeutic genes to be encoded. This process however would require scientists to abbreviate the genes necessary to treat any given disease (2). Thus, choosing the optimal set of promoters and regulators are key in creating an effective AAV vector. These factors not only determine how much a transgene is expressed but may also restrict therapeutic expression of genes to a specific set of cells. This control is necessary when developing targeted treatments for specific diseases.

Additionally, the route of administration can heavily influence transduction efficacy and gene expression. Dosing options need to be determined dependent on the goal of a particular treatment. Because AAV vectors can be designed to target specific cells, direct delivery to the region of interest will optimize the effectiveness of the gene therapy.


Though AAV gene therapy holds the potential to change the treatment of many neurological diseases, scientists still need to develop techniques that can optimize capsid and genome design. While constructing the capsid, researchers just consider the immune response that an AAV treatment may cause in patients. Finding a design that can maximize the delivery of genes and minimize any potential risk of antibody recognition is a critical step in widespread AAV therapy use. Furthermore, genome design will determine how neurologically-linked mutations are being treated. Since adeno-associated viruses have a limited genome capacity, scientists must identify and choose what information is most important to include in a viral vector, which will allow them to target specific diseases. Advancements in both capsid genome design will increase the effectiveness of AAV gene therapy and may change the therapeutic landscape for patients with neurological diseases.


[1] Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019 May;18(5):358–78.

[2] Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther. 2021 Feb 8;6(1):1–24.

[3] Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet. 2020 Apr;21(4):255–72.

[4] Deverman BE, Ravina BM, Bankiewicz KS, Paul SM, Sah DWY. Gene therapy for neurological disorders: progress and prospects. Nat Rev Drug Discov. 2018 Sep;17(9):641–59.

[Image citation]: How Gene Therapy Works [Internet] [cited May 1 2022]. Available from https://www.sarepta.com/science/gene-therapy-engine

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