Adeno-Associated Virus (AAV) as a Vector for Gene Therapy

Abstract

There has been a resurgence in gene therapy efforts that is partly fueled by the identification and understanding of new gene delivery vectors. Adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver DNA to target cells, and has attracted a significant amount of attention in the field, especially in clinical-stage experimental therapeutic strategies. The ability to generate recombinant AAV particles lacking any viral genes and containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies. This review will provide an overview of some important factors to consider in the use of AAV as a vector for gene therapy.

Fig. 1

Schematic representation of the basic components of a gene insert packaged inside recombinant AAV gene transfer vector. AAV adeno-associated virus, ITR inverted terminal repeat

Fig. 2

Overview of AAV production/purification. Cell platform: HEK-293T, Sf9, or other suitable cell system can be grown on a small scale on 150 mm tissue culture-treated culture dish, hyperflasks, or shake flasks. Cells are then transfected with adenovirus helper virus, rep/cap, and ITR-transgene plasmids for 293T, or infected with baculovirus for Sf9. Producer lines with integrated expression of rep/cap and ITR-transgene can be infected with adenovirus and grown to scale. Scale-up: For larger-scale culture volumes, virus can be produced in roller bottles, continuous perfusion, or WAVE Bioreactor systems. Purification/polishing: Affinity or heparin chromatography are optimal for isolation of virus from culture supernatants with or without cell pellet harvesting. Benzonase/DNAse treatment of eluted virus is required for removal of extraviral DNA contamination, followed by anion-exchange chromatography to fractionate ‘empty’ vs. ‘full’ AAV particles. QC/release: Upper left of far right panel: image depicts a silver stain analysis of culture FT next to affinity/anion exchange purified AAV (pure). The three bands represent the viral capsid proteins VP1, VP2, and VP3. Upper right of far right panel: Dynamic light scattering analysis of purified AAV1 indicates a uniform particle distribution of approximately 25–30 nM. Bottom half of far right panel: Analytical ultracentrifugation can resolve the proportion of ‘empty’ vs, ‘full’ particles of purified material. Additional assays that should be employed are digital drop polymerase chain reaction for determining titer in GC/mL, cryo or transmission electron microscopy for visual representation of purified particles, endotoxin testing, and other assays to evaluate the presence of residual host-cell protein contamination. AAV adeno-associated virus, FT flow-through, GC genome copies, rep/cap replication/capsid, QC quality control