LAUSR

AS MORE RECOMBINANT adeno-associated virus (rAAV) products are tested in or licensed for clinical use, the need for methods for large-scale, highquality manufacturing have increased proportionally. One of the recurring themes of recent advances in rAAV purification has been the desire to remove ‘‘empty capsids,’’ that is, icosahedral AAV pseudovirions that contain no viral DNA. Empty capsids are a natural product of AAV propagation, observed at ratios from 10% to 90% of total capsids, as visualized by transmission electron microscopy. Research grade and some investigational scale clinical grade vector purification methods may remove most empty capsids, if those protocols include any form of density gradient centrifugation (e.g., CsCl or iodixanol gradient purification). However, density gradient techniques are technically difficult to scale up for cGMP production. Recently, a number of new protocols to produce fewer empties without the need for density gradient purification were published. All of these efforts are based on the concept that empty AAV capsids are, and should be treated as, ‘‘contaminants’’ in rAAV preparations. In this issue, work from Aalbers et al. provides evidence of a case where that logic may not hold. This group—a clinical product development team from Arthrogen, the University of Amsterdam, and Leiden University working toward rAAV5-based gene therapy for rheumatoid arthritis (RA)—examined the ability of empty capsids to facilitate rAAV gene transfer into the synovial tissues of a murine RA model. They provide evidence that empty AAV capsids serve as decoys to overcome macrophage-mediated clearance of rAAV. Thus, the empty capsids serve not as a contaminant but as a ‘‘natural’’ facilitator of gene transfer. These data support earlier findings by a number of groups indicating that empty capsids may have a beneficial effect on the efficiency of gene transfer when rAAV is administered by systemic intravenous injection. Such studies call into question the wisdom or necessity of defining empty AAV capsids as a ‘‘contaminant’’ in clinical material. If one assumes that, developing methods for removing more and more empty capsids from a preparation would always be considered improvements in the purity of the product. In contrast, empty capsids could be defined as being a ‘‘component’’ of some products. Viral vectors are by their nature complex biologics, the product of abortive viral replication and packaging in eukaryotic cells. These approaches are not mutually exclusive. It is quite possible that certain rAAV products function better in the absence of empty capsids, perhaps in sites such as the retina, where neutralizing antibodies or synovial macrophages are not present. In these cases, ‘‘empty-free’’ rAAV products may be appropriate. In other circumstances, perhaps in arthritic joints, ‘‘empty-containing’’ rAAV products may be superior. In these instances, if an existing production and purification method produces a reproducible proportion of empties that is beneficial, there would seem to be no inherent advantage to dose the full and empty capsids as separate products. This could be viewed as being analogous to influenza vaccines, many of which contain ovalbumin and are contraindicated in patients with egg allergy, while one, FluBlok , is produced with a baculovirus system and is safe to use in patients with egg allergy. To some, these points may seem to be the purview of product regulators from the Food and Drug Administration and the European Medicines Agency rather than the research community. However, such considerations require ongoing basic and translational research investigation, with a particular interest in how these vectors may interact with diseased hosts. Only through such carefully designed studies can evidence emerge to support the assertions of both product developers and regulators.