The role of the adeno-associated virus capsid in gene transfer

Abstract

Adeno-associated virus (AAV) is one of the most promising viral gene transfer vectors that has been shown to effect long-term gene expression and disease correction with low toxicity in animal models, and is well tolerated in human clinical trials. The surface of the AAV capsid is an essential component that is involved in cell binding, internalization, and trafficking within the targeted cell. Prior to developing a gene therapy strategy that utilizes AAV, the serotype should be carefully considered since each capsid exhibits a unique tissue tropism and transduction efficiency. Several approaches have been undertaken in an effort to target AAV vectors to specific cell types, including utilizing natural serotypes that target a desired cellular receptor, producing pseudotyped vectors, and engineering chimeric and mosaic AAV capsids. These capsid modifications are being incorporated into vector production and purification methods that provide for the ability to scale-up the manufacturing process to support human clinical trials. Protocols for small-scale and large-scale production of AAV, as well as assays to characterize the final vector product, are presented here. The structures of AAV2, AAV4, and AAV5 have been solved by X-ray crystallography or cryo-electron microscopy (cryo-EM), and provide a basis for rational vector design in developing customized capsids for specific targeting of AAV vectors. The capsid of AAV has been shown to be remarkably stable, which is a desirable characteristic for a gene therapy vector; however, recently it has been shown that the AAV serotypes exhibit differential susceptibility to proteases. The capsid fragmentation pattern when exposed to various proteases, as well as the susceptibility of the serotypes to a series of proteases, provides a unique fingerprint for each serotype that can be used for capsid identity validation. In addition to serotype identification, protease susceptibility can also be utilized to study dynamic structural changes that must occur for the AAV capsid to perform its various functions during the virus life cycle. The use of proteases for structural studies in solution complements the crystal structural studies of the virus. A generic protocol based on proteolysis for AAV serotype identification is provided here.

Fig. 2.1

The single-stranded DNA genome of AAV. The inverted terminal repeats (ITRs) flank the two open reading frames rep and cap. The rep gene encodes four nonstructural proteins – Rep78, Rep68, Rep52, and Rep40. The cap gene encodes three structural proteins – VP1, VP2, and VP3. The location of the promoters, p5, p19, and p40 are depicted by arrows

Fig. 2.2

The structure of a monomeric subunit of AAV2 as determined by Xie et al. [31]. This image was produced using the AAV2 coordinates from the Protein Databank, (PDB Accession no. 1lp3), with the molecular modeling software PyMOL (www.pymol.org) provided by DeLano Scientific, Palo Alto, CA [39]

Fig. 2.3

Viral vector production. The rAAV Vector plasmid contains the therapeutic gene flanked by the ITRs, usually of AAV2. The helper plasmid contains the rep and cap genes, as well as the adenoviral genes needed for replication. Both plasmids are transiently transfected into HEK293 cells that express the adenovirus E1A and E1B gene products

Fig. 2.4

Flowchart of the steps for rAAV production as described in Protocol 1. HEK293 cells are expanded, transfected, and harvested at 60 h posttransfection. The cells are lysed, and loaded onto either a cesium chloride gradient or an iodixanol gradient to separate infectious virions from empty capsids. Virus is purified using either heparin affinity chromatography or ion-exchange chromatography. Virus preparations are formulated and concentrated, and characterized

Fig. 2.5

Protease mapping of the AAV capsid for capsid serotype determination. A Samples are digested with a protease, in this case trypsin for one set of samples and chymotrypsin for the other set. A Western blot is performed using polyclonal antisera to AAV capsids, and based on the fragmentation pattern, a serotype determination can be made. Undigested sample (T₀) and samples digested for 12 h (T₁₂) for AAV2, AAV1, and AAV5 are shown. AAV5 is resistant to these proteases, while AAV1 and AAV2 exhibit differences in their fragmentation patterns. T₀ samples represent the undigested capsid proteins VP1, VP2, and VP3. B Different AAV serotypes demonstrate different susceptibilities to proteases due to the differences in their primary amino acid sequences. This differential susceptibility provides a unique signature for each serotype and allows for capsid serotype identification