Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae

Abstract

The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.

A rAAV2‐producing yeast screening model to identify host proteins enhancing rAAV DNA replication and vector yield

“…ddPCR was performed as described previously . Yeast treated material was diluted 10‐to‐100‐fold to target the ddPCR dynamic range.…”

“…The aforementioned results suggested that rAAV yield improvement seen in selected yeast strains might be linked to host protein overexpression. To confirm that hypothesis, we overexpressed the best five candidate proteins from the screening library (PRE4, HEM4, TOP2, SDO1, and GPN3) using a different haploid yeast strain carrying four plasmids to produce rAAV as described in Barajas et al Analysis of triplicate pool of colonies of each YPH500 strain in 24 deep‐well plates corroborated the results seen during yTHC screening. HEM4, GPN3, and SDO1 overexpression strains showed 40% improvement in AAV DNA replication, whereas TOP2 overexpression strain exhibited 80% improvement.…”

“…The rAAV2‐GFP‐producing yeast system is a promising model for the evaluation of the impact of host cell factors on rAAV production. Results shown in Barajas et al as well as in this manuscript indicate that the yeast model is able to recapitulate key aspects of AAV formation such as DNA replication and full particle formation. Our results complement previous studies done in yeast and support the notion of the utility of this model.…”

“…Threefold dilutions of the purified material were analyzed with Progen AAV2 titration kit, following the manufacturer's instructions. Southern and western blot were performed to characterize rAAV‐GFP DNA and VP protein expression in yeast as previously described …”

“…Alvaro Galli's group successfully demonstrated AAV single‐stranded DNA replication and formation of AAV virus‐like particles in S. cerevisiae . In addition, we have further developed a rAAV model system in yeast that recapitulated key aspects of AAV biology leading to the formation of infectious rAAV particles . In the current work we use S. cerevisiae as a model to study the effect of host cell factors on rAAV2 DNA replication and vector formation, ultimately identifying host proteins that improve rAAV vector production.…”