Abstract
In a paper in Human Gene Therapy (Vandenberghe et al., 2010), a team led by James Wilson reported that during vector manufacturing a large portion of the viral particles, of many different serotypes, were found free in the cell culture medium. Furthermore, the free viruses were as infectious as those isolated from the cell pellets. The authors further uncovered a negative correlation between the virus concentration in the medium and its binding affinity to heparan sulfate proteoglycan (HSPG), a primary receptor for AAV2 (Summerford and Samulski, 1998) and possibly for AAV6. During vector harvest, for example, AAV2 was found mainly in the cell pellet, whereas AAV8 was found mainly in the medium. Genetic ablation of the heparin-binding capacity of AAV2 greatly facilitated its release from the cells, whereas acquisition of heparin-binding by AAV8 made it largely cell pellet-bound. Thus, the stronger the affinity for HSPG, the fewer viral particles found in the medium. This seemingly simple finding is intriguing for AAV virology and useful for vector production.
Let's look at AAV virology first. A tremendous body of work has defined AAV as a nonenveloped, nucleus-localized, single-stranded DNA virus. Like its helper, adenovirus, AAV particles are localized in the host cell nucleus and are not destined for secretion as are retroviruses (Flotte and Berns, 2005). This conclusion is unequivocally supported by immunofluorescence staining, cell apparatus fractionation, electronic microscopy, capsid gene mutagenesis, and so on. Although most of the early virology work was done with AAV2, it should also hold true for other AAV serotypes. As depicted in Fig. 1, newly synthesized capsid proteins in the cytoplasm assemble into empty particles and subsequently travel to the nucleus for viral DNA packaging (Fig. 1, step 1). The viral particles accumulate in the nucleus and can be seen as paracrystalline structures because of their high concentration.
Release of AAV particles into the cell culture medium. Step 1: Empty AAV particles are made in the cytoplasm and travel to the nucleus for DNA packaging. Full particles remain in the nucleus. Step 2: A portion of the full particles is freed into the medium by unknown mechanisms. Step 3: Depending on the binding capacity to the receptors (e.g., heparin sulfate proteoglycan, HSPG), the freed AAV particles either become bound to the cells (top), or remain largely free in the medium (bottom). Steps 4 and 5: The receptor-bound viruses reenter the cells and become uncoated or degraded. Thus, AAV serotypes that poorly bind and infect the host cells (e.g., 293 cells) will have a greater chance of being found free in the culture media.
It is intriguing but remains elusive how certain serotypes of AAV are capable of mass escape from the host cells into the culture medium (Fig. 1, step 2), as reported by Vandenberghe and colleagues and also observed by other laboratories. A plausible explanation is that the release of AAV particles is through cell membrane leakage, because of apoptosis and cell death in the late stage of vector production. The particles, both full and empty, are gradually leached out from the dead and dying cells into the culture medium. In the cases of AAV2, AAV6, and AAV8RQNR, a mutant that binds strongly to HSPG (Fig. 1, top, step 3), reabsorption onto cell membrane and reentry into the live cells could effectively reduce the concentration of free AAV in the medium, and even reduce the overall vector yield because of the uncoating and degradation of reentered viruses. However, in the cases of AAV8, AAV9, and AAV2HSPG mutant, which bind and infect 293 cells poorly (Fig. 1, bottom, step 3), the released viral particles remain free and accumulate in the medium. Our own experience and communication with other investigators also support this scenario. (1) High-salt or high-pH lysis buffer helps release much more AAV2 from the cell pellet; (2) consistent with tighter association with the cell pellet, AAV2, AAV3, and AAV6 vectors in general bind to and transduce host 293 cells much more efficiently than do AAV1, AAV4, AAV5, AAV7, AAV8, and AAV9; and (3) the ratio of empty particles to full particles in the medium fraction is usually much higher than the ratio in the cell pellet fraction, suggesting that empty particles synthesized in the cytoplasm more readily leak out than do full particles from the nucleus. Notwithstanding these observations, alternative, unconventional mechanisms continue to be of interest, for example, viral particle budding similar to retroviruses, secretion of AAV particles from the cytoplasm by exosomes, and so on. Regardless, a practical lesson learned here from AAV virology is that one can improve vector production by manipulating AAV and host cell interactions. For example, HSPG-deficient cells could potentially serve as better producers for AAV2 vectors.
The most valuable finding by Vandenberghe and colleagues is the fact that vectors released into the culture medium are just as infectious as the typical vectors isolated from the cell pellet. Given that serum-free media are now commonly used during the production phase of vector manufacturing (Lock et al., 2010), this important finding could streamline and reduce the costs of the purification process. Cell culture media as the starting materials are much cleaner than the “muddy” cell pellet lysates. Higher vector purity and infectivity could translate into better safety and lower immunogenicity (Ayuso et al., 2010). Finally, for high-throughput functional genomics studies using AAV vectors as the tool, direct use of high-titer vector-containing media will be much less labor intensive and more cost-effective than highly purified vectors, which involve multiple tedious purification steps and expensive equipment and reagents.