Optimization of rAAV Manufacturing at the Triple Transfection Step

Abstract

The recombinant adeno-associated virus (rAAV) is the vector of choice for the delivery of gene therapies for a wide range of monogenic disorders because of its track safety record in clinical trials and prolonged gene expression with relatively low immunogenicity. More recently, rAAVs have also gained attention as vaccine carriers against infectious agents because the rAAV particle is thermodynamically stable and durable under the right conditions at room temperature, a feature that facilitates vaccine distribution where it is most needed without expensive preservation measures. rAAVs are biological drugs, and therefore, their manufacturing relies on highly complex biological processes that are not completely understood. While several rAAV-based drugs are available in the market, the production of high-titer, high-quality vector remains a challenge despite the many chemical, biological and engineering advances implemented to date. Several studies show, for instance, that rAAV preparations are heterogenous in character, comprised of filled, partially filled or empty capsids of which the desired product may be represented only as a small sub-population of particles. This heterogeneity is not understood in all its potential implications. It is compounded by the observation that packaged recombinant genomes often contain deletions or insertions or are recombined via mechanisms that are not firmly established but that are being actively investigated. The nucleic acid material incorporated into viral particles may itself be derived from alternative sources arising from the biological reagents used in production, namely host cell DNA, RNA, or backbone plasmid sequences, the latter via a process called reverse packaging. The illegitimate virions arising from these processes are often accompanied by protein impurities that escape purification procedures and may elicit an immune response when administered. In addition, depending on the manufacturing platform chosen for vector production, the proteins making up the viral capsid and the non-structural factors responsible for their assembly may differ due to post translational modifications which may impact rAAV transduction efficiency, particle stability or vector tropism. Thus, heterogeneity and purity issues raise questions about the potency and safety of the final products, add uncertainty to experimental work from all stages of development to clinical testing, limits reproducibility, and complicates translational medicine where the clinical vector likely differs from the pre-clinical one. Considering recently documented adverse reactions and fatalities in gene therapy trials at very high doses, research studies aimed improving vector titer and quality are pressing. The main goal of this study was to optimize the manufacturing of rAAV by altering the ratio of segregated adenovirus helper genes provided during production. Adenovirus helper genes are involved in rAAV genome transcription, translation and packaging. For this, molar equivalents of the known canonical adenovirus helper genes were delivered separately or in combination to study their contribution to rAAV vector titers in a manner that matched current production conditions. It was found that the adenovirus DNA binding protein (DBP) mRNA is underexpressed when it is under the control of the early E2a gene promoter compared to DBP mRNA that is expressed from the more complex pF6 genome, the intact helper plasmid. The difference in DBP expression was not enough to explain lower rAAV production levels because rescue of early DBP mRNA expression by modulation of the early E2a plasmid input at transfection did not result in a comparable rescue of rAAV production. An error was discovered.