AAV Gene Transfer Vectors

    Adeno-associated virus as a gene-transfer solution for high-throughput physiology

    To perform high-throughput physiological experiments and screens, it is essential to be able to express reporters or probes in the cells of interest. A gene transfer method is required to achieve this. The ideal method should

    • be both safe for the operator and feasible in a standard lab environment
    • be straightforward to implement even on large numbers of samples
    • offer the possibility of high efficiency or sparse labelling
    • allow expression in specific cell types in a physiologically relevant mixed population
    • express uniformly in target cells, minimising artefacts arising from variable expression
    • lead to long-lasting expression to permit longitudinal investigations
    • not be toxic to the target cells.

    Viral methods offer the highest efficiency and uniformity, but this may come with safety concerns, toxicity and complicated preparation requirements. However, the safety, specificity and efficiency of adeno-associated virus (AAV) has made them the favoured vector for gene therapy in both clinical trials and approved products (1). The wild-type AAV does not appear to cause any disease (2,3,4). The gutless recombinant forms (rAAV) have low immunogenicity and are unable to replicate (2,3). When generated without a helper virus and not expressing harmful inserts, they can be categorised in a research setting as GMO class I in many countries including Finland, as long as appropriate risk assessments, handling and inactivation procedures are implemented (4,5; note that ethanol does not inactivate AAVs).

    The capsids can be engineered for cell-specific or broad tropism while selection of promoters can contribute selective expression in infected cells (3). Expression from rAAVs is maintained for prolonged periods in dividing cells and at least several weeks in non-dividing cells.

    At the screening unit, we developed an efficient pipeline for generation of reporters, optogenetic actuators, modulator constructs and others in the form of rAAV plasmids. From these the recombinant AAV particles are easily generated at a scale suitable for miniaturised assays typical in high-throughput microscopy and physiology.

    We find that the rAAV system addresses all the needs that high-throughput physiolology and microscopy places on a gene transfer approach for routine use – safe for the operator, straightforward high-throughput preparation and implementation, non-toxic and uniform expression while also offering cell-specific infection and expression approaches.

    Please contact us if you would like our assistance with the AAV system for high-throughput physiology or other purposes.

    1. Rodrigues GA, Shalaev E, Karami TK, Cunningham J, Slater NKH, Rivers HM. Pharmaceutical Development of AAV-Based Gene Therapy Products for the Eye. Pharm Res. 2018 Dec 27;36(2):29. doi: 10.1007/s11095-018-2554-7. Review. PubMed PMID: 30591984; PubMed Central PMCID: PMC6308217.

    2. Chamberlain K, Riyad JM, Weber T. Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids. Hum Gene Ther Methods. 2016 Feb;27(1):1-12. doi: 10.1089/hgtb.2015.140. Review. PubMed PMID: 26757051; PubMed Central PMCID: PMC4761816.

    3. Mak KY, Rajapaksha IG, Angus PW, Herath CB. The Adeno-associated Virus – A Safe and Promising Vehicle for Liverspecific Gene Therapy of Inherited and Non-inherited Disorders. Curr Gene Ther. 2017;17(1):4-16. doi: 10.2174/1566523217666170314141931. PubMed PMID: 28292253.

    4. Ohje Muuntogeenisten Virusten Suljetun Käytön Luokituksesta Ja Eristystoimista, Geenitekniikan lautakunta 9/2011


    5. https://media.addgene.org/cms/filer_public/83/34/833467ca-de57-48f7-a930-f30b73395658/062618_addgene_adeno_associated_virus_sds.pdf