Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors

Abstract

Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Spodoptera frugiperda ( Sf9 ) insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM (transmission electron cryomicroscopy), denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments in vitro and in vivo , including in humanized liver mice. Using these approaches, we have made two major discoveries: (1) rAAV capsids have post-translational modifications (PTMs), including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms; and (2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data show that host cell protein impurities differ between platforms and can have their own PTMs, including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus- Sf9 vectors in various cell types in vitro (p < 0.05–0.0001), in various mouse tissues in vivo (p < 0.03–0.0001), and in human liver in vivo (p < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity, as well as cost considerations.

Viral vector platforms within the gene therapy landscape

“…Since the baculovirus provides helper function, 208 a single BEV system in conjunction with Sf9 cell lines that stably express rep , can be employed for flexible and high-titer large-scale vector production. 209 Interestingly, a report that vectors produced from Sf9 cells are differentially posttranslationally modified, as compared to those generated from human- or mammalian-derived cell lines raised question as to whether differences in production schemes may impact tropism and transduction efficacies, 210 although further work in this area is necessary to cross-validate these findings. Nonetheless, BEV/Sf9 systems exhibit reduced encapsidation of contaminating DNAs 205 and therefore remain an attractive production strategy for large-scale, clinical-grade vectors.…”

Popularizing recombinant baculovirus-derived OneBac system for scaling-up production of all recombinant adeno-associated virus vector serotypes

“…A recent study revealed that the rAAV capsid PTMs and rAAV genome methylation differ between two main platforms corresponding to the plasmid-transfected human HEK293 cells and the BEV-infected insect Sf9 cells. It also showed that rAAVs derived from the HEK293 cells are more potent than rAAVs derived from Sf9 cells [54]. However, this study only focused on rAAV1 and rAAV8, and did not illustrate the relevance between these differences and the activity of rAAVs.…”

Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform

“…The second question is related to the efficiency of vector genome packaging in insect cells. In general, insect cells have been reported to produce more AAV empty capsids [43,56,93] compared to mammalian cell platforms, [94][95][96] which counteracts any success achieved with genomic and functional particles of wtAAV in the presence of helper virus functions. [97] Moreover, in contrast to well-defined and wellcharacterized helper genes of adenovirus or HSV, [98,99] the baculovirus helper elements have not yet been fully identified, even though their helper function has been known for almost two decades.…”

“…The second question is related to the efficiency of vector genome packaging in insect cells. In general, insect cells have been reported to produce more AAV empty capsids [ 43,56,93 ] compared to mammalian cell platforms, [ 94–96 ] which counteracts any success achieved with increased cell‐specific AAV yield and necessitates active efforts towards engineering insect cells or BEVs to improve packaging efficiency and produce more functional particles. A head‐to‐head comparison of the mammalian cell versus insect cells for wild type AAV production may help identify any insect‐cell specific limitations for producing packaged AAV particles because mammalian cells, the natural host of AAV, have been reported to produce nearly 100% genomic and functional particles of wtAAV in the presence of helper virus functions.…”

“…With the demonstrated success of AAV production in the One‐Bac Sf9 packaging cell line, the generation of a similar Hi5 cell line remains open to further evaluation. Additionally, in the context of a recent report regarding the effect of post‐translational modifications (PTMs) of AAV capsids on vector transduction efficiency, [ 93 ] a side‐by‐side comparison of AAV produced in Sf9 and Hi5 cell lines might provide new insights into the vector quality attributes (PTMs, in vivo potency) imparted by different cell lines.…”