Abstract
Viral vectors have a great potential for gene delivery, but manufacturing is a big challenge for the industry. The baculovirus-insect cell is one of the most scalable platforms to produce recombinant adeno-associated virus (rAAV) vectors. The standard procedure to generate recombinant baculovirus is based on Tn7 transposition which is time-consuming and suffers technical constraints. Moreover, baculoviral sequences adjacent to the AAV ITRs are preferentially encapsidated into the rAAV vector particles. This observation raises concerns about safety due to the presence of bacterial and antibiotic resistance coding sequences with a Tn7-mediated system for the construction of baculoviruses reagents. Here, a faster and safer method based on homologous recombination (HR) is investigated. First, the functionality of the inserted cassette and the absence of undesirable genes into HR-derived baculoviral genomes are confirmed. Strikingly, it is found that the exogenous cassette showed increased stability over passages when using the HR system. Finally, both materials generated high rAAV vector genome titers, with the advantage of the HR system being exempted from undesirable bacterial genes which provides an additional level of safety for its manufacturing. Overall, this study highlights the importance of the upstream process and starting biologic materials to generate safer rAAV biotherapeutic products.
Highlights
Gene therapy gives rise to hopes for a large spectrum of genetic diseases that are mainly untreatable using conventional pharmacology
The circular baculoviral DNA derived from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) has been engineered to insert a bacterial artificial chromosome (BAC) into the polyhedrin locus[23] that contains the kanamycin selective gene, the mini-F bacterial replicon, and a mini-attTn7 site inserted into the LacZα region (Figure 1A).[10]
We show that recombinant baculovirus derived from the Tn7 system tends to lose the exogenous expression cassette after a few rounds of viral amplification while the homologous recombination (HR) system preserves baculoviral genome stability upon serial passages
Summary
Gene therapy gives rise to hopes for a large spectrum of genetic diseases that are mainly untreatable using conventional pharmacology. The regulatory agencies requested an assessment of the product for residual baculovirus DNA to the sponsor (uniQure), as a process impurity due to a potential side effect.[16] For this purpose, we have developed a protocol based on high-throughput sequencing to identify and quantify residual DNAs, called the single-stranded virus sequencing, and we demonstrated that baculoviral DNA is the major source of DNA contamination in the final rAAV product with up to 2.1% of total NGS reads.[17] The gentamycin resistance gene coding sequence that has been used to select recombinant baculovirus after Tn7 transposition, was still detectable by qPCR in research-grade rAAV lots It has been reported in the literature that the BEV genome derived from the Tn7-bacmid system is subject to instability and spontaneous deletion of the exogenous DNA cassette, leading to the generation of defective interfering viruses (DIs) which accumulate at the expense of the intact ones limiting scaleup of batch production.[18] Altogether, these technical concerns prompted us to consider options to overcome these barriers and define new standards to produce baculoviral reagents for rAAV vector manufacturing
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