Abstract
Large-scale manufacturing of rAAV is a bottleneck for the development of genetic disease treatments. The baculovirus/Sf9 cell system underpins the first rAAV treatment approved by EMA and remains one of the most advanced platforms for rAAV manufacturing. Despite early successes, rAAV is still a complex biomaterial to produce. Efficient production of the recombinant viral vector requires that AAV replicase and capsid genes be co-located with the recombinant AAV genome. Here, we present the Monobac system, a singular, modified baculovirus genome that contains all of these functions. To assess the relative yields between the dual baculovirus and Monobac systems, we prepared each system with a transgene encoding γSGC and evaluated vectors’ potency in vivo. Our results show that rAAV production using the Monobac system not only yields higher titers of rAAV vector but also a lower amount of DNA contamination from baculovirus.
Highlights
Adeno-associated virus is a member of the Dependovirus genus of the subfamily Parvovirinae and requires functions from helper viruses such as adenovirus [1], herpes simplex virus [2], vaccinia virus [3], or human bocavirus [4] in order to perform its replication cycle.The wild type AAV2 encodes an ssDNA genome of 4679 bases where both negative and positive strands are packaged in the icosahedral capsid [5]
The production of rAAV8-γSGC was performed by co-infecting Sf9 cells with a baculovirus encoding the AAV rep2 and cap8 genes, which are controlled by the very-late polh and p10 promoters, respectively, and with a baculovirus encoding the recombinant adeno-associated virus (rAAV)-γSGC
We successfully developed a single baculovirus for the production of rAAV, which fulfils the needs of industrial producers to have simplified large-scale and cost-effective production processes
Summary
The wild type (wt) AAV2 encodes an ssDNA genome of 4679 bases where both negative and positive strands are packaged in the icosahedral capsid [5]. The genome is flanked at both ends by 145 base T-shape structures called inverted terminal repeats (ITRs). These structures are necessary for AAV genome replication, second-strand synthesis [6,7], encapsidation [8], and insertion of the wt viral genome in human chromosome 19 [9]. In the recombinant AAV vector, the ITRs are flanking the recombinant transgene cassette, which allows for its replication and packaging. The wt-AAV2 genome encodes four replicase proteins, three capsid proteins, the membrane-associated accessory protein (MAAP) [11]
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