Homologous Recombination Offers Advantages over Transposition‐Based Systems to Generate Recombinant Baculovirus for Adeno‐Associated Viral Vector Production

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.

Solutions against emerging infectious and noninfectious human diseases through the application of baculovirus technologies

“…What is interesting to us about this work is that these studies enabled the insect larval platform to produce rAAV, thus significantly lowering the overall costs. In another report, the genomic stability of rec-baculoviruses was evaluated in accordance with the genetic-engineering platforms to produce those recombinants, either through transposons or homologous recombination (Aurelién et al 2021). All these current contributions are enabling the insect-cell-rAAV-production platform to remain a viable alternative for the gene-therapy industry, thus underscoring that approach as a principal milestone after Glybera® was produced by a ThreeBac system.…”

An easy method to generate recombinant pseudorabies virus expressing the capsid protein of Porcine circovirus type 2d

“…Homologous recombination is a type of genetic recombination in which the genetic material of the virus, eukaryote, or bacteria is exchanged naturally between two molecules of DNA that contain similar recombinant arms. Over the past few decades, it has been used extensively in the construction of recombinant adeno-associated virus (Fisher et al, 1997 ; Jacob et al, 2020 ), poxvirus (Fisher et al, 1997 ; Wyatt et al, 2015 ), and herpesvirus (Wilkinson and Weller, 2003 ; Boscheinen et al, 2019 ). It is a powerful tool to precisely manipulate the genome for producing a new gene or virus according to the experimental need.…”

The Magic Staff: A Comprehensive Overview of Baculovirus-Based Technologies Applied to Human and Animal Health

“…More recently, Neuhold and colleagues [ 11 ] presented a golden-gate-based single-step cloning system called GoldenBac that is compatible with both Tn7 transposition and homologous recombination (HR) bacmids. Along the same lines, SmartBac [ 12 ], employs a polyprotein approach that is constructed by overlapping PCR and Gibson assembly coupled with Cre-LoxP recombination for the generation of large transfer plasmids that carry more than one GOI.…”