AAV - An Awesome Vehicle!


    As I promised last week, this will be the first in a series of blogs on gene therapy in which I will introduce what viral vectors are and how they have become a definitive weapon in the battle to cure genetic diseases. This week I will focus on AAV.

    Since its discovery in 1965, the Adeno-Associated Virus (AAV) has come a long way in its usefulness as a molecular tool. Originally found by Bob Atchison, M. David Hoggan and Wallace Rowe, AAV was known to be a parvovirus made of capsids containing single stranded DNA and inverted terminal repeats (ITRs). The ITRs are self-priming hairpins that enable genome replication. The capsid proteins encoded by the viral genome, VP1, VP2 and VP3 give the virus its characteristic icosahedral shape, its ~25 nm diameter size and its viral properties. AAV binds to cell surface receptors in order to enter the host cell, rather like any other virus. The most common receptors targeted by AAV include the Heparin Sulfate Proteoglycan receptors (HSPG) and integrin receptors.

    AAV structure:

    Left Panel: Computer simulated AAV model at 22 nm in diameter. Middle Panel: Electron Microscopy visualization of AAV particles. Right Panel: AAV rep / cap genes and the encoded VP1 / VP2 / VP3 proteins. Pictures courtesy of SignaGen Laboratories.

    AAV, pic

    AAV entrance into the cell - typically AAV binds to HSPG receptors, enters by endocytosis at the membrane, gets carried in the endosomes and escapes through into the nucleus to regulate gene expression:


    When the virus takes over cellular machinery, more than 100,000 particles can be produced by one cell. Moreover, AAV is not a pathogen. It can persist in the host cell for months without triggering any adverse symptoms. AAV can therefore be studied safely in Biosafety Level-2 (BSL-2) laboratories, with minimum sterilization, clean room specifications and decontamination procedures.

    AAV vectors are currently the most commonly used gene therapy based vectors. The first clinical trial to test recombinant AAV (rAAV) gene therapy happened in 1993, to test a treatment for cystic fibrosis, attempting to replace the monogenic mutation of CFTR protein with a healthy one. Despite early setbacks in the clinic the technology has now improved leading to better safety and efficacy. Today there are hundreds of rAAV based therapeutic products being developed. In 2012 the EMA in Europe approved Glybera, a rAAV-1 based gene therapy for familial lipoprotein lipase deficiency (LPLD), manufactured by the dutch company UniQure. Last year, the FDA approved Spark Therapeutics’s Luxturna, the AAV2 based gene therapy for certain forms of retinal dystrophy, including Leber Congenital Amaurosis.

    rAAV gene therapy products (picture courtesy of Drug Development and Delivery website:


    Some of the main advantages of the rAAV vector system include ease of producing large yields of virus, long-term robust transgene expression, its tropism for non-dividing and dividing cells and lack of adverse effects associated with its administration. Different rAAV vector serotypes occur naturally and target different cell receptors giving them specificity for target tissues. With the advent of directed evolution techniques, scientists have engineered hybrid and synthetically derived rAAV serotypes that have even higher target specificity for diseased tissues and can evade pre-existing immunity. This has greatly expanded the commercial potential for using rAAV-based gene therapy.

    rAAV Serotypes:

    AAV Serotypes

    Making the vehicle:

    The traditional procedure for making rAAV in the lab at small scale starts off by transfecting mammalian cells (usually HEK 293 cells) with three plasmids:
    Ad Helper plasmid - genes that help the virus to replicate
    Rep/Cap plasmid - the regulatory genes and structural genes that package the virus
    Transgene plasmid flanked by the ITRs - encode the therapeutic gene.

    When the cells start expressing these genes, rAAV particles are made. To harvest these particles, cells are lysed and the lysate is ultra-centrifuged through cesium chloride gradients in order to concentrate the virus. The purest virus fraction is collected from the gradient, dialyzed in PBS and stored for use.

    Traditional AAV production in the lab (Picture courtesy of SignaGen Laboratories):

    AAV making


    Now that clinical grade rAAV is in demand, increasingly Good Manufacturing Practices (GMP) production solutions are needed. GMPs are founded on Joseph Juran’s trilogy of principles, Quality Planning, Quality Control and Quality Improvement. A Quality by Design approach must be taken by manufacturers in order to build quality into the process as well as the product. This is taken seriously by the FDA, EMA and other global regulatory authorities when reviewing drugs for approval. GMP guidelines for pharmaceutical development, quality risk management, quality systems and development of drug substances are enshrined in the International Conference of Harmonization documents, named ICH Q8, Q9, Q10 and Q11. These documents require manufacturers to come up with Quality target product profile (qTPP) and Critical Quality Attributes (CQAs) that must be set up to determine key properties of the drug are within acceptable, safe limits at all times during the manufacturing process.

    What does this GMP jargon have to do with rAAV therapy? To produce rAAV for clinical use there are two general processes: the Upstream Process and the Downstream Process. The upstream process is geared towards transfecting cells with the viral plasmids, seeding cells, growing up the cells in a bioreactor and harvesting the virus. It is important for the Master Cell Bank to be characterized, quality checked and stored with meticulous notes since this is the source from which you will will derive the Working Cell Bank, which will be seeded. The downstream process aims to purify the harvested bulk through a series of purification and chromatography columns before retaining the virus, formulating it and freezing or lyophilizing it in a bottle. A combination of cation exchange, anion exchange and tangential flow filtration techniques are often employed for the purification process.

    Upstream Process:

    Downstream Process:

    In order to define CQAs a manufacturer must make challenging decisions on a variety of parameters during the production process. Chief among these questions involve:
    - What type of cell line do you use in order to make the virus? Mammalian cell line or insect cell line, such as baculovirus?
    - Do you transiently transfect the cell lines once they have been seeded from the cell bank? Or do you use stable cell lines that have been frozen with the Helper plasmid, Rep/Cap plasmid and Transgene plasmids? Do you use a helper adenovirus to infect the cells with the desired viral plasmids or do you transfect by traditional calcium phosphate or PEI method?
    - What type of cell growth medium and container do you use? Do you start with adherent cells in stacked flasks and build up to larger and larger bioreactor tanks or do you drop cells into a bioreactor to start with?
    - Do you choose single-use bioreactors and disposable culture systems or do you re-use culture vessels that require cleaning after each use?
    - What types of purification systems do you use? Cation Exchange Chromatography or Anion Exchange Chromatography?

    Linking manufacturing challenges to regulatory requirements is tricky. Once rAAV vector production begins it will be essential to test batch-to-batch variations for quality. This involves testing for identity, purity, stability and potency by sampling at different stages along the production cycle. Impurities are a great challenge as well. Between 1% and 8% of purified rAAV particles are known to contain incorrect nucleic acid sequences. That can be a huge number of incorrect particles in a single batch of virus.

    Along with the lack of knowledge base about rAAV (since the AAV structure is so complex relative to small molecule drugs), the other disadvantage of rAAV manufacturing is the impurity profile. There are Process-related impurities and Product-related impurities. Process-related impurities are derived from the manufacturing process. Raw components, cell substrate, cell culture medium and residual helper components could all contribute to these impurities. Product-related impurities result from the vector itself which interfere with efficacy and safety. These can include empty AAV capsids, non-infectious rAAV particles, aggregated, oxidized and degraded vectors, nucleic acid impurities, minor variations in stoichiometry of VP1, 2 and 3.

    Process-Related Impurities:

    Product-Related Impurities:

    Both genotoxicity (potential for causing cancer) and immunotoxicity (potential for causing an adverse immune reaction) are potential threats to the production of AAV. Therefore, as a Quality Control step, a manufacturer must set up a Risk Assessment plan for each and every one of the impurities in the tables above.

    As mentioned earlier, a single cell can generate many thousands of rAAV particles. However, in order to be useful in the clinic, rAAV-based viral vectors must be produced in even larger numbers, between 10e11 to 10e14 genomic particles (vector genomes / vg). Current GMP manufacturing scales for rAAV often fall short of this. Part of the reason is that smaller quantities of rAAV are satisfactory for the smaller scale clinical studies on rare “Ultra Orphan” diseases which only impact a few hundred individuals per hundred million people. However, as more common diseases are studied for rAAV therapy and as clinical trial phases advance from proof of concept to toxicity/Phase 1 studies onto Phase 2 / 3, more virus will need to be generated. This means scaling up from adherent cells to large bioreactors, providing a clean room environment within class 10,000 and having more stringent quality control parameters to ensure quality in production.

    Genomic particles vs Phase of Clinical Trial, a simplified graph of how many genomic particles are required for each phase of rAAV development:


    Progress of cGMP compliance at different stages of product development

    A vision for the future

    There are more than 7000 rare diseases that remain untreated. The use of rAAV as gene therapy is just one of a growing arsenal of weapons scientists are currently developing to fight these disorders. There are limitations to using rAAV, such as its packing size (usually no more than 5000 bp transgene can fit), the problem of neutralizing antibodies that stop the action of beneficial rAAV particles and problems with endosome escape once rAAV is inside the cell. Selecting novel rAAV capsids by directed evolution would be one way to overcome some of these deficiencies. However, it is possible that if rAAV is to be used in the future it will have to be done in combination with other drugs that mitigate these deficiencies.

    Next week I will do a mini-case study into FDA's newly approved Luxturna for the treatment of RPE65 mutation-associated retinal dystrophy. The therapy that has enabled visually impaired patients to see clearly for the first time.


    AAV pictures from:



    Production of AAV: Chandler RJ, Venditti CP, Gene therapy for metabolic diseases, Transl Sci Rare Dis. 2016;1(1):73-89.

    Daniel C. Smith, AAV Vector manufacturing - Challenges and Opportunities in the Manufacturing of AAV Vectors Used in Delivery of Gene Therapy Treatments, Drug Development and Delivery, 2/28/2017

    A-Mab Case Study: http://c.ymcdn.com/sites/www.casss.org/resource/resmgr/imported/A-Mab_Case_Study_Version_2-1.pdf, — upstream/downstream

    Impurities picture: Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment. J. Fraser Wright. Biomedicines, 2014:2, 80-97, — Impurities

    Hasti E, Samulski RJ, Adeno-Associated Virus at 50: A Golden Anniversary of Discovery, Research, and Gene Therapy Success—A Personal Perspective. 2015, 26:257-265

    GMP Compliance picture: Van Der Loo JCM, Wright JF, Progress and challenges in viral vector manufacturing. 2016: 25(1)42-52. — cGMP compliance phases figure

    Keeler AM, Flotte TR, Gene Therapy 2017: Progress and Future Directions. 2017; 10:242-248