JP-2026076163-A - Production of recombinant viral vectors from plant hairy roots

Abstract

[Challenge] To enable the regular use of gene therapy, it is necessary to improve the productivity and/or quality of vectors. Therefore, alternative systems for producing recombinant viral vectors such as rAAV vectors are needed. [Solution] The present invention relates to a method for producing recombinant viral vectors from the hairy roots of plants, more particularly from the hairy roots of plants belonging to the Brassicaceae family. The present invention also relates to transgenic plants, hairy root cultures, and recombinant viral vectors obtainable by the method of the present invention. [Selection Diagram] None

Inventors

• セルジュ・ブラウン
• フロリアン・カドン
• ヨハン・ディクス
• マリナ・ギエ
• サミア・マルティン
• マリー・ランドー

Assignees

• ジェネトン

Dates

Publication Date

20260511

Application Date

20251224

Priority Date

20191218

Claims (1)

1. Methods described in the specification and drawings, hairy root cultures, recombinant mammalian virus vectors, or transgenic plants.

Description

This invention relates to a method for producing recombinant viral vectors from hairy roots, particularly from hairy roots of plants belonging to the Brassicaceae family. The increasing use of recombinant viral vectors for gene therapy and DNA vaccination necessitates efficient production systems. Examples of recombinant viral vectors for therapeutic purposes include lentiviral vectors and adeno-associated virus vectors. Among gene therapy products under development, recombinant adeno-associated virus (AAV) vectors are currently the most widely used and possess the greatest potential for in vivo delivery. The preference for rAAV vector systems stems from several factors: the absence of diseases associated with wild-type viruses, the ability of AAV to transduce not only non-dividing cells but also dividing cells, and the resulting observation of long-term, active transgene expression in several Phase I/II/III clinical trials. Furthermore, different rAAV vector serotypes can be utilized to specifically target different tissues, organs, and cells. Recent commercial approvals of recombinant adeno-associated virus gene therapies in Europe and the United States (e.g., Luxturna® and Zolgensma®) represent groundbreaking advances in the field of gene therapy. AAV is an envelopeless icosahedral particle that contains a single-stranded DNA genome. AAV belongs to the genus Dependoparvovirus (Weitzman and Linden, 2011), a genus that relies on helper viruses to provide essential genes in trans for proliferative infection. Its 4.7kb genome contains two main open reading frames, namely regulatory (Rep) and structural capsid (Cap) genes, which encode several proteins necessary for viral replication, capsid structure, and packing of the viral genome. From the cap gene, three proteins—VP1, VP2, and VP3—are spontaneously produced by a combination of alternative splicing and leaky scanning of transcripts from the p40 promoter. These all share the same common C-terminal sequence. The AAV capsid consists of 60 units of VP1, VP2, and VP3 in a ratio of approximately 1:1:10. Different open reading frames in the cap gene translate a protein called assembly activation protein (AAP), which is necessary for capsid assembly (Sonntag et al., 2010). The rep gene produces four proteins: the two largest, Rep78 and Rep68, arise from transcription initiated using the p5 promoter, while the other two, Rep52 and Rep40, are obtained from transcription using the p19 promoter. In addition to the Rep proteins, inverse end sequences (ITRs) are also required for AAV DNA replication and inclusion (Balakrishnan and Jayandharan, 2014; Robert et al., 2017). In the case of rAAV, several production strategies exist for generating viral vectors. Transient transduction of plasmid DNA into mammalian cells to produce AAV viral vectors is the most commonly used strategy in the clinical-grade production of such viral vectors. rAAV vectors are typically produced in human fetal kidney 293 cells (HEK293) after transduction of three DNA plasmids carrying the Rep and Cap genes, the rAAV transgene, and a specific gene providing helper adenovirus function. By introducing both the Rep and Cap genes and/or the rAAV genome, a modified, stable cell line is generated, thereby creating a packaging or production cell line. The insect cell/baculovirus system was developed by Urabe et al. (2006) for the production of AAV. The first generation was based on three different baculoviruses (BVs) inserted into the polyhedrin locus. The three recombinant BV vectors encoded the Rep, Cap, and rAAV genome transgenes, respectively. A second-generation BV was developed with a reduced number of BVs (Smith et al., 2009). In this method, the rep and cap sequences were inserted head-to-head into a single baculovirus. In addition, a system for AAV production using co-expression has been established in yeast by Barajas et al., 2017. WO2015013313WO2019/193119PCT/EP2019/076958WO16185122 This is a schematic diagram of a structure designed to produce AAV protein in hairy roots. This figure shows eight constructs designed to test the AAV protein production capacity of Brassica lapa hairy roots. Construct 1 comprises (i) a first expression cassette containing the CaMV35S promoter ("p35S"), the Cap gene ("CAP"), and the CaMV35S terminator ("t35S"), and (ii) a second expression cassette containing the CaMV35S promoter ("p35S"), the Rep gene ("Rep"), and the nos terminator ("tNOS"). Construct 2 comprises (i) a first expression cassette containing a nos promoter ("pNOS"), a VP1 gene ("VP1"), and a nos terminator ("tNOS"), (ii) a second expression cassette containing a nos promoter ("pNOS"), a VP2 gene ("VP2"), and a nos terminator ("tNOS"), (iii) a third expression cassette containing a variant of the CaMV35S promoter (referred to as "p2*35S"), a VP3 gene ("VP3"), and a CaMV35S terminator ("t35S"), and (iv) a fourth expression cassette containing a variant of the CaMV35S promoter (referred to as "p2*35S"),