Production of recombinant viral vectors from plant hairy roots

Abstract

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.

Description

Technical Field

[0001] The present invention relates to hairy roots, and more particularly, to a method for producing recombinant viral vectors from hairy roots of plants belonging to the Brassicaceae family.

Background Art

[0002] Due to the progress in the use of recombinant viral vectors for gene therapy and DNA vaccination applications, an efficient production system has been demanded. Examples of recombinant viral vectors for therapeutic purposes include lentiviral vectors and adeno-associated viral vectors.

[0003] Among the gene therapy products under development, recombinant adeno-associated virus (AAV)-based vectors are currently the most widely used and have the greatest potential for in vivo delivery. The use of the rAAV vector system is preferred in part because there are no diseases associated with the wild-type virus, AAV can transduce not only non-dividing cells but also dividing cells, and as a result, long-term active transgene expression has been observed in several Phase I / II / III clinical trials. Furthermore, different rAAV vector serotypes can be utilized to specifically target different tissues, organs, and cells. The recent commercial approvals of recombinant adeno-associated virus gene therapy drugs in Europe and the United States (e.g., Luxturna® and Zolgensma®) represent landmark achievements in the field of gene therapy.

[0004] AAV is an envelopeless icosahedral particle that contains a single-stranded DNA genome. AAV belongs to the genus dependoparvovirus, a genus that relies on helper viruses to transpair essential genes for proliferative infection (Weitzman and Linden, 2011). 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). Four proteins are produced from the rep gene; the two largest proteins, Rep78 and Rep68, arise from transcription initiated using the p5 promoter, while the other two proteins, 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).

[0005] In the case of rAAV, several production strategies exist for generating viral vectors.

[0006] Transient translocation 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 translocation of three DNA plasmids, each carrying the Rep and Cap genes, the rAAV transgene, and a specific gene providing helper adenovirus function.

[0007] 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.

[0008] An 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 of BV has been developed with the number of BVs reduced to two (Smith et al., 2009). In this method, the rep and cap sequences were inserted head-to-head into a single baculovirus.

[0009] In addition, a system for AAV production using co-expression has been established in yeast by Barajas et al., 2017. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] WO2015013313 [Patent Document 2] WO2019 / 193119 [Patent Document 3] PCT / EP2019 / 076958 [Patent Document 4] WO16185122 [Overview of the project] [Problems that the invention aims to solve]

[0011] Despite improvements in these systems in recent years, the productivity and / or quality of vectors need to be improved in order to enable the regular use of gene therapy.

[0012] Therefore, there is a need for alternative systems for producing recombinant viral vectors such as rAAV vectors. [Means for solving the problem]

[0013] A first aspect of the present invention is a method for producing a recombinant mammalian virus vector from the hairy roots of a plant, a) A step of inducing the formation of hairy roots from the plant, b) The plant is transformed with at least one vector comprising one or more expression cassettes, wherein one or more expression cassettes comprises genes encoding protein components necessary for the production of recombinant viral vectors. Includes, The aforementioned plant belongs to the Brassicaceae family. Regarding the method.

[0014] More specifically, the aforementioned plants belonging to the Brassicaceae family may be selected from the group consisting of Raphanus sativus, Raphanus sativus var. niger, Brassica oleracea L. convar, Brassica napus, Arabidopsis Thaliana, and Brassica rapa, with Brassica rapa being the plant in particular.

[0015] In one particular embodiment, step a) is carried out by transforming a plant with a bacterial strain containing the rol gene cluster, the bacterial strain being able to infect the plant.

[0016] In one particular embodiment, the bacterial strain is Rhizobium rhizogenes or Agrobacterium tumefaciens.

[0017] In one particular embodiment, the recombinant viral vector is a recombinant adeno-associated virus (AAV) viral vector.

[0018] In a particular embodiment, one or more expression cassettes include AAV rep and cap genes. In a particular embodiment, the AAV rep and cap genes are each under the control of a promoter derived from a virus that infects cruciferous plants, such as the cauliflower mosaic virus 35S (CaMV35S) promoter.

[0019] In another specific embodiment, one or more expression cassettes include genes encoding the VP1, VP2, VP3, AAP (assembly activation protein), Rep52, and Rep78 proteins. In another specific embodiment, each gene encoding the VP1, VP2, VP3, AAP, Rep52, or Rep78 proteins is controlled by a constitutive promoter such as the cauliflower mosaic virus 35S (CaMV35S) promoter or the nopalin synthase (nos) promoter, or by an inductive promoter such as the alcohol dehydrogenase (AlcA) promoter.

[0020] In one particular embodiment, the gene encoding VP1 is under the control of the nos promoter, the gene encoding VP2 is under the control of the nos promoter, the gene encoding VP3 is under the control of the CaMV35S promoter or a functional variant thereof, and the gene encoding AAP is under the control of the CaMV35S promoter or a functional variant thereof.

[0021] In a particular embodiment, the gene encoding VP3 is under the control of a functional variant of the CaMV35S promoter having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 13, and the gene encoding AAP is under the control of a functional variant of the CaMV35S promoter having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 13.

[0022] In a particular embodiment, each of the genes encoding VP1, VP2, VP3, and AAP is under the control of the AlcA promoter.

[0023] In a particular embodiment, the gene encoding VP3 is further under the control of an enhancer, specifically, the tobacco mosaic virus omega (TMVΩ) enhancer.

[0024] In a particular embodiment, each of the genes encoding Rep52 and Rep78 is under the control of the AlcA promoter.

[0025] In a particular embodiment, the gene encoding Rep52 is further under the control of an enhancer, specifically, the tobacco mosaic virus omega (TMVΩ) enhancer.

[0026] In a particular embodiment, the genes encoding VP1, VP2, VP3, AAP, Rep52, and / or Rep78 proteins are codon-optimized.

[0027] In a particular embodiment, the plant is further transformed with a vector encoding a viral helper function necessary for efficient replication of the virus, specifically, a vector encoding an adenovirus helper function.

[0028] In one particular embodiment, the plant is further transformed with a vector containing a viral genome that includes a gene encoding the product of interest, more specifically, a vector containing a gene encoding the product of interest adjacent to two AAV-ITR sequences.

[0029] Another aspect of the present invention is, a) A process for inducing the formation of hairy roots from plants belonging to the Brassicaceae family, and b) The plant is transformed with at least one vector comprising one or more expression cassettes, wherein one or more expression cassettes comprises genes encoding protein components necessary for the production of recombinant viral vectors. This relates to hairy root cultures obtainable by [method].

[0030] In one particular embodiment, hairy root cultures can be obtained by transforming plants belonging to the Brassicaceae family, the plants being selected from the group consisting of Raphanus sativus, Raphanus sativus var. niger, Brassica oleracea L. varietal group, Brassica napus, Arabidopsis thaliana, and Brassica rapa, the plant being Brassica rapa in particular.

[0031] In one particular embodiment, the recombinant mammalian virus vector is a recombinant adeno-associated virus (AAV) virus vector. In another particular embodiment, one or more expression cassettes are as defined above.

[0032] A further aspect of the present invention relates to recombinant mammalian virus vectors obtainable by the methods described above. In one particular embodiment, the recombinant mammalian virus vector is produced from the hairy roots of a plant belonging to the Brassicaceae family, the plant being selected from the group consisting of Raphanus sativus, Raphanus sativus niger, Brassica oleracea L. varietal group, Brassica napus, Arabidopsis thaliana, and Brassica rapa, the plant being Brassica rapa in particular. In one particular embodiment, the recombinant mammalian virus vector is a recombinant adeno-associated virus (AAV) virus vector. In another particular embodiment, the recombinant mammalian virus vector is produced from the hairy roots of a plant belonging to the Brassicaceae family, the plant being transformed with at least one vector comprising one or more expression cassettes, the one or more expression cassettes comprising genes encoding protein components necessary for the production of the recombinant virus vector, the one or more expression cassettes being as defined above.

[0033] Another aspect of the present invention relates to a transgenic plant transformed with at least one vector comprising one or more expression cassettes, the one or more expression cassettes comprising genes encoding protein components necessary for the production of recombinant mammalian viral vectors, and the plant belongs to the Brassicaceae family.

[0034] In one particular embodiment, the transgenic plant belonging to the Brassicaceae family is selected from the group consisting of Raphanus sativus, Raphanus sativus var. niger, Brassica oleracea L. varieties, Brassica napus, Arabidopsis thaliana, and Brassica rapa, the plant being Brassica rapa in particular.

[0035] In one particular embodiment, the recombinant mammalian virus vector is a recombinant adeno-associated virus (AAV) virus vector. In another particular embodiment, one or more expression cassettes are as defined above. [Brief explanation of the drawing]

[0036] [Figure 1] This is a schematic diagram of a structure designed to produce AAV protein in hairy roots.

[0037] This figure shows eight constructs designed to test the AAV protein production capacity of Brassica lapa hairy roots.

[0038] 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").

[0039] 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"), an AAP gene ("AAP"), and a CaMV35S terminator ("t35S"). The "p2*35S" promoter is a variant of the CaMV35S promoter that includes an overlap of -343 to -90 bp fragments, as described by Kay et al., 1987.

[0040] Construct 3 comprises (i) a first expression cassette containing an inductive promoter for alcohol dehydrogenase ("pAlcA"), the Rep52 gene ("Rep52"), and the CaMV35S terminator ("t35S"), (ii) a second expression cassette containing an inductive promoter for alcohol dehydrogenase ("pAlcA"), the Rep78 gene ("Rep78"), and the nos terminator ("tNOS"), and (iii) a third expression cassette containing the CaMV35S promoter ("p35S"), the gene encoding the ALCR protein required for activation of the alcohol dehydrogenase promoter ("AlcR"), and the CaMV35S terminator ("t35S").

[0041] Construct 4 comprises (i) a first expression cassette containing an inducible promoter for alcohol dehydrogenase ("pAlcA"), a tobacco mosaic virus omega enhancer ("TMVΩ"), a Rep52 gene ("Rep52"), and a CaMV35S terminator ("t35S"), (ii) a second expression cassette containing an inducible promoter for alcohol dehydrogenase ("pAlcA"), a Rep78 gene ("Rep78"), and a nos terminator ("tNOS"), and (iii) a third expression cassette containing a CaMV35S promoter ("p35S"), a gene encoding the ALCR protein required for activation of the alcohol dehydrogenase promoter ("AlcR"), and a CaMV35S terminator ("t35S").

[0042] Construct 5 comprises (i) a first expression cassette containing an inductive promoter for alcohol dehydrogenase ("pAlcA"), the VP1 gene ("VP1"), and the nos terminator ("tNOS"), (ii) a second expression cassette containing an inductive promoter for alcohol dehydrogenase ("pAlcA"), the VP2 gene ("VP2"), and the nos terminator ("tNOS"), and (iii) an inductive promoter for alcohol dehydrogenase ("pAlcA"), the tobacco mosaic virus omega enhancer ("TMVΩ"), and the VP3 gene ("V The expression cassette comprises (iv) a third expression cassette containing the P3 gene and the CaMV35S terminator ("t35S"), (iv) a fourth expression cassette containing the inducible promoter of alcohol dehydrogenase ("pAlcA"), the AAP gene ("AAP"), and the CaMV35S terminator ("t35S"), and (v) a fifth expression cassette containing the CaMV35S promoter ("p35S"), the gene encoding the ALCR protein required for activation of the alcohol dehydrogenase promoter ("AlcR"), and the CaMV35S terminator ("t35S"). [Modes for carrying out the invention]

[0043] A first aspect of the present invention is a method for producing a recombinant mammalian virus vector from the hairy roots of a plant, a) A step of inducing the formation of hairy roots from the plant, b) The plant is transformed with at least one vector comprising one or more expression cassettes, wherein one or more expression cassettes comprises genes encoding protein components necessary for the production of recombinant viral vectors. Includes, The aforementioned plant belongs to the Brassicaceae family. Regarding the method.

[0044] Recombinant mammalian viral vectors The term "viral vector," according to the present invention, refers to a carrier derived from a virus, i.e., a "vector." This term encompasses any viral particle, whether having or lacking a viral genome.

[0045] In the context of this invention, viral vectors lacking a viral genome are also called virus-like particles (VLPs). VLPs are highly organized structures that self-assemble from virus-derived structural proteins. These stable and versatile nanoparticles possess excellent adjuvant properties that can induce innate and adaptive immune responses. Over the past few years, VLPs have been applied in other sectors of biotechnology, leveraging their structural stability and resistance to manipulation, to transport and represent heterologous molecules or to serve as building blocks for novel nanomaterials. VLPs can be produced from components of a wide range of virological families, including Parvoviridae (e.g., adeno-associated viruses), Retroviridae (e.g., HIV), Flaviviridae (e.g., hepatitis C virus), Paramyxoviridae (e.g., Nipah), and bacteriophages.

[0046] The term "viral vector having a viral genome" specifically refers to an infectious viral particle. The genome of a recombinant viral vector is modified compared to the wild-type (wt) viral genome by replacing a portion of the wt genome with a target transgene. The term "target transgene" refers to a gene whose nucleic acid sequence does not naturally exist in the viral genome. The target transgene may be a coding sequence or a non-coding sequence. Recombinant viral vectors are particularly used in gene therapy. As used herein, the term "gene therapy" refers to the transfer of target genetic material (e.g., DNA or RNA) into a host for the treatment or prevention of a hereditary or acquired disease or condition. The target genetic material encodes a product (e.g., polypeptide or functional RNA) whose production in vivo is desired. For example, the target genetic material may encode a therapeutically valuable hormone, receptor, enzyme, or polypeptide. Alternatively, the target genetic material may encode a therapeutically valuable functional RNA, such as therapeutically valuable antisense RNA (e.g., antisense RNA suitable for exon skipping) or shRNA.

[0047] In the context of this invention, a viral vector is a mammalian viral vector, that is, a viral vector is derived from a virus capable of infecting mammals, particularly humans. Therefore, in the context of this invention, a viral vector is not derived from a virus capable of infecting plants.

[0048] Recombinant viral vectors may, in detail, be derived from adenoviruses, parvoviruses (especially adeno-associated viruses), retroviruses (especially lentiviruses or spumaviruses), herpes simplex viruses, alphaviruses, flaviviruses, rhabdoviruses, measles viruses, Newcastle disease viruses, picornaviruses, or poxviruses.

[0049] In one particular embodiment, the recombinant viral vector is a recombinant AAV (rAAV) vector.

[0050] In the present invention, the AAV vector capsid can be obtained from naturally occurring or non-naturally occurring serotypes. In one particular embodiment, the serotype of the AAV vector capsid is selected from the natural AAV serotypes. Instead of using the natural AAV serotype, artificial AAV capsids, including AAVs having non-naturally occurring capsid proteins, may be used in the context of the present invention, but are not limited to such artificial capsids. Such artificial capsids can be generated by any suitable technique of using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterologous sequence that may be obtained from a selected different AAV serotype, a non-proximate portion of the same AAV serotype, a non-AAV virus source, or a non-viral source. Capsids derived from artificial AAV serotypes may be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids.

[0051] According to one particular embodiment, the capsid of the AAV vector is AAV-1, -2, AAV-2 variant (e.g., AAV-2 optimized with a quadruple mutant capsid, including an engineered capsid having the Y44+500+730F+T491V modification, as disclosed by Ling et al., 2016), -3 and AAV-3 variant (e.g., AAV3-ST variant, including an engineered AAV3 capsid having two amino acid modifications S663V+T492V, as disclosed by Vercauteren et al., 2016), -3B and AAV-3B variant, -4, -5, -6 and AAV-6 variants (e.g., AAV6 variants including the triple mutant AAV6 capsid Y731F / Y705F / T492V, disclosed by Rosario et al., 2016), -7, -8, -9 and AAV-9 variants (e.g., AAVhu68), -2G9, -10 such as -cy10 and -rh10, -rh39, -rh43, -rh74, -dj, Anc80, LK03, AAV.PHP, AAV2i8, porcine AAVs such as AAVpo4 and AAVpo6, and tyrosine, lysine, and serine capsid variants of AAV serotypes. In addition, other manipulated non-natural variants (e.g., AAV-spark100), chimeric AAVs, or AAV serotype capsids obtained by shuffling, rationale design, error-prone PCR, and machine learning techniques may also be useful.

[0052] In one particular embodiment, the AAV vector is a chimeric vector, i.e., its capsid comprises VP capsid proteins derived from at least two different AAV serotypes, or comprises at least one chimeric VP protein having VP protein regions or domains derived from at least two AAV serotypes. For example, a chimeric AAV vector can be obtained by combining an AAV8 capsid sequence with a sequence from an AAV serotype different from the AAV8 serotype, such as any of those described in detail above. In another embodiment, the capsid of the AAV vector comprises one or more variant VP capsid proteins, such as those described in WO2015013313, more specifically, RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6 capsid variants. In one particular embodiment, the AAV vector capsid is a hybrid of AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins. For example, the AAV serotype may be the -rh74-9 serotype as disclosed in WO2019 / 193119 (e.g., the Hybrid Cap rh74-9 serotype described in the examples of WO2019 / 193119; also referred herein as the rh74-9 serotype "-rh74-9", "AAVrh74-9", or "AAV-rh74-9"), or the -9-rh74 serotype as disclosed in WO2019 / 193119 (e.g., the Hybrid Cap 9-rh74 serotype described in the examples of WO2019 / 193119; also referred herein as the -9-rh74 serotype "-9-rh74", "AAV9-rh74", "AAV-9-rh74", or "rh74-AAV9"). In a particular embodiment, the AAV vector capsid is a peptide-modified hybrid of AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins, such as an AAV9-rh74 hybrid capsid or an AAVrh74-9 hybrid capsid modified with a P1 peptide, as described in PCT / EP2019 / 076958.

[0053] In another embodiment, the modified capsid may also be obtained from capsid modifications inserted by error-prone PCR and / or peptide insertion (as described, for example, Bartel et al., 2011). In addition, capsid variants may include single amino acid changes such as tyrosine variants (as described, for example, Zhong et al., 2008).

[0054] In a particular embodiment, the AAV vector has naturally occurring capsids such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-cy10, and AAVrh10 capsids. In a particular embodiment, the recombinant AAV vector has the AAV8 capsid.

[0055] The genome of the AAV vector may include 5'- and 3'-AAV inverted terminal sequences (ITRs) adjacent to the genetic material of interest. The ITRs may be derived from any AAV genome, such as the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-cy10, or AAVrh10 genome. In one particular embodiment, the genome of the AAV vector includes 5'- and 3'-AAV2 ITRs.

[0056] In addition, the genome of an AAV vector can be either single-stranded or self-complementary double-stranded (McCarty et al., Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are generated by deleting a terminal resolution site (trs) from one of the AAV terminal repeats. These modified vectors, whose replicated genome is half the length of the wild-type AAV genome, tend to pack DNA dimers.

[0057] The AAV vector may contain a capsid and ITR derived from the same AAV serotype, or it may contain a capsid derived from a first serotype and an ITR derived from a serotype different from the first serotype. In other words, any combination of AAV serotype capsid and ITR can be realized in the context of the present invention. Such vectors having capsid ITRs derived from different serotypes are also called "pseudotyped vectors."

[0058] Plants belonging to the Brassicaceae family As used herein, the expression “plants belonging to the Brassicaceae family” has its general meaning in this art. This expression encompasses all plants of the Brassicaceae family, also known as the Cruciaceae, Mustardaceae, or Cabbageaceae families.

[0059] According to the Royal Botanic Gardens, Kew, the Brassicaceae family includes more than 330 genera and approximately 3,700 species. The largest genera are Draba (365 species), Cardamine (200 species), Erysimum (225 species), Lepidium (230 species), and Alyssum (195 species).

[0060] Well-known species include, but are not limited to, Brassica oleracea (cabbage, cauliflower, etc.), Brassica rapa (turnip, Chinese cabbage, etc.), Brassica napus (rapeseed, etc.), Raphanus sativus (common radish), Armoracia rusticana (horseradish), Matthiola (stock), Arabidopsis thaliana (model organism), and many others. Among these species, some produce edible roots (turnip, radish, etc.).

[0061] In one preferred embodiment, the plants belonging to the Brassicaceae family are selected from the group consisting of Brassica rapa, Raphanus sativus, Raphanus sativus var. niger, Brassica oleracea L. varieties, Brassica napus, and Arabidopsis thaliana.

[0062] More preferably, the plant belonging to the Brassicaceae family is Brassica rapa or Brassica napus.

[0063] More preferably, the plant belonging to the Brassicaceae family is Brassica rapa.

[0064] Step a) to induce the formation of hairy roots from the aforementioned plant. Any technique capable of inducing the formation of hairy roots in plants can be used in this invention.

[0065] Hairy roots are a type of proliferative root that appears at the site of plant injury after infection caused by the Gram-negative soil bacterium Agrobacterium rhizogenes. The hairy root phenotype is characterized by rapid hormone-independent growth, lateral branching, genetic stability, and a lack of geotropism.

[0066] It should be noted that Agrobacterium rhisogenes is named Rhizobium rhisogenes in accordance with taxonomic changes to the genus Agrobacterium and the family Rhizobiaceae. Rhizobium rhisogenes can be considered identical to the name Agrobacterium rhisogenes.

[0067] Hairy roots were first identified as a disease in plant extracts caused by Rhizobium rhisogenes, which can be isolated from soil. This Gram-negative bacterium transfers DNA from its rooting (Ri) plasmid to the genome of infected plant cells, which leads to root formation. Specifically, the rol gene group (FF White et al., 1983), which includes the genes rolA, rolB, and rolC, is present on the T-DNA of the Rhizobium rhisogenes Ri plasmid, and the expression of these genes induces hairy root formation.

[0068] In one particular embodiment, the formation of hairy roots is induced by transforming plants with a bacterial strain containing the rol gene cluster, and the bacterial strain can infect plants.

[0069] As used herein, the term "rol gene group" has its general meaning in this industry. This term refers to a group of bacterial genes capable of inducing hairy root formation (Schmulling et al., 1988; Bulgakov et al., 2008). Typically, the rol gene group is contained within plasmids such as pRi plasmids.

[0070] In one particular embodiment, the bacterial strain either naturally contains the rol gene group in its genome or has been modified by introducing a heterologous rol gene group.

[0071] In one particular embodiment, the bacterial strain belongs to the genus Rhizobium.

[0072] In one preferred embodiment, a strain of Rhizobium rhisogenes is used.

[0073] Several strains of Rhizobium rhisogenes can be used in the practice of the present invention. Suitable strains include, but are not limited to, Rhizobium rhisogenes TR7 strain, also known as ATCC 25818, as well as LBA 9402, A4T, A4, LBA1334, ATCC 11325, ATCC 15834, LMG 155, HRI, TR105, ATCC 39207, R1000, LBA 9422, strain 1072, BL311, R1600, R1601, C58C1, A4RS, MSU440, ARqua1, 8194, TR101, 2659, LBA8490, NIAES1724, C8 (MAFF03-10268), and DC-AR2 strain.

[0074] In one preferred embodiment, the strain of Rhizobium rhisogenes is strain ATCC 15834 or ATCC 25818.

[0075] The strain of Rhizobium rhisogenes is more preferably strain ATCC 15834.

[0076] In another embodiment, a strain of Agrobacterium tumefaciens is used. In a particular embodiment, the Agrobacterium tumefaciens strain used is modified so that a group of rol genes is introduced into its genome. In a particular embodiment, Agrobacterium tumefaciens is modified by transformation using a pRi plasmid containing the rol gene group.

[0077] In another embodiment, the Agrobacterium tumefaciens strain used does not contain the rol gene group. In this embodiment, plant transformation using Agrobacterium tumefaciens induces the formation of callus tissue. The callus tissue then differentiates into hairy roots after the addition of one or more hormonal substances. In a preferred embodiment, the hormonal substance is an auxin family hormone such as 1-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA).

[0078] Several strains of Agrobacterium tumefaciens can be used in the practice of this invention. Suitable strains include, but are not limited to, A. tumefaciens C58, C58C1, LBA4404, GV2260, GV3100, A136, GV3101, GV3850, EHA101, EHA105, and AGL-1.

[0079] Transformation with bacterial strains such as Rhizobium rhisogenes and / or Agrobacterium tumefaciens is a well-known technique in the art. Those skilled in the art are familiar with the different techniques commonly used to carry out the transformation process. Depending on the plant species to be transformed, different parts of the plant can be used for infection. Such plant parts may include, for example, seeds, stems, leaves, stalks, cotyledonous nodes, hypocotyls, or other plant parts or cells, but are not limited to these.

[0080] Typically, infection by Rhizobium rhisogenes and / or Agrobacterium tumefaciens is carried out by applying a Rhizobium rhisogenes and / or Agrobacterium tumefaciens inoculation source to a pre-injured plant.

[0081] It is preferable to use a semi-solid medium or liquid nutrient solution optimized for maintaining hairy roots, resulting in increased growth rate and productivity of hairy roots compared to uninfected plant cells. Many types of materials, solutions, and media are known and can be used in the present invention, but some preferred examples include Murashige and Skoog medium (MS) and Gamborg B5 medium. Several modified media can be used that are optimized to match the nutrient requirements of the host plant used in the production of sustainable hairy root cultures.

[0082] In one particular embodiment, step a) inducing the formation of hairy roots is performed prior to step b), in which the plant is transformed with at least one vector comprising one or more expression cassettes, the one or more expression cassettes comprising genes encoding protein components required for the production of recombinant viral vectors.

[0083] In another specific embodiment, step a) inducing the formation of hairy roots is performed after step b), in which the plant is transformed with at least one vector comprising one or more expression cassettes, the one or more expression cassettes comprising genes encoding protein components required for the production of recombinant viral vectors. In this embodiment, hairy roots can be obtained by transforming a transgenic plant expressing genes encoding protein components required for the production of recombinant viral vectors with a bacterial strain comprising a group of rol genes and capable of infecting the plant as defined above.

[0084] In one preferred embodiment, steps a) and b) are performed simultaneously.

[0085] In one particular embodiment, a bacterial strain comprising a group of rol genes and capable of infecting plants as defined above further comprises in its genome one or more expression cassettes, one or more of which contain genes encoding protein components necessary for the production of recombinant viral vectors.

[0086] Therefore, in this embodiment, the method for producing recombinant mammalian virus vectors from the hairy roots of plants includes the step of transforming the plants with bacterial strains such as Rhizobium rhisogenes and Agrobacterium tumefaciens. The bacterial strain contains a group of rol genes and one or more expression cassettes in its genome. One or more expression cassettes contain genes encoding protein components necessary for the production of recombinant viral vectors. The aforementioned plant belongs to the Brassicaceae family.

[0087] In another specific embodiment, two bacterial strains are used simultaneously, one of which contains a set of rol genes, and the other containing one or more expression cassettes encoding proteins necessary for the production of recombinant viral vectors.

[0088] In another specific embodiment, two or more bacterial strains are used simultaneously, each containing different expression cassettes encoding protein components necessary for the production of recombinant viral vectors, and at least one of the bacterial strains contains a group of rol genes.

[0089] In one particular embodiment, three or more bacterial strains are used simultaneously, one of which contains a group of rol genes, and at least two or more bacterial strains contain different expression cassettes encoding protein components necessary for the production of recombinant viral vectors.

[0090] step b) Transforming a plant with at least one vector containing one or more expression cassettes As described above, the method of the present invention comprises step b) transforming a plant with at least one vector comprising one or more expression cassettes, wherein one or more expression cassettes comprise genes encoding protein components necessary for the production of recombinant viral vectors.

[0091] In one particular embodiment, the plant is further transformed with a vector that encodes a viral helper function necessary for the efficient replication of the virus, for example, when the recombinant viral vector is an rAAV vector, a vector that encodes an adenovirus helper function.

[0092] In one particular embodiment, the plant is further transformed with a vector containing a viral genome that includes a gene encoding the product of interest. In another particular embodiment, the vector contains a gene encoding the product of interest, adjacent to two AAV-ITR sequences.

[0093] Any technique that enables the transformation of plants, particularly plants belonging to the Brassicaceae family, may be used. In particular, any physical method such as gene gun or microparticle gun system, electroporation, microinjection, or ultrasound-mediated transformation may be used. Chemical methods such as liposome-mediated transformation, silicon carbide fiber-mediated transformation, PEG-mediated transformation, calcium phosphate coprecipitation, polycationic DMSO technology, and DEAE dextran precipitation may also be used. PEI-mediated transformation may also be used.

[0094] In a preferred embodiment, transformation is performed using bacterial strains such as Rhizobium rhisogenes or Agrobacterium tumefaciens, which spontaneously transfer DNA located on a tumor induction (Ti) plasmid (T-DNA) to the nucleus of plant cells and stably integrate the DNA into the plant genome. In a particular embodiment, Rhizobium rhisogenes or Agrobacterium tumefaciens comprises a two-component vector containing one or more expression cassettes as defined below in its T-DNA region. The Rhizobium rhisogenes or Agrobacterium tumefaciens further comprises a helper plasmid containing vir genes originating from an Agrobacterium genus Ti plasmid. These genes encode a series of proteins that cleave the two-component plasmid at the left and right boundary sequences, thereby facilitating the transduction of T-DNA into the host plant cell.

[0095] In a preferred embodiment, a bacterial strain such as Rhizobium rhisogenes or Agrobacterium tumefaciens, which includes one or more expression cassettes encoding protein components necessary for the production of recombinant viral vectors, further includes a group of rol genes necessary for the formation of hairy roots.

[0096] In one particular embodiment, two or more bacterial strains are used, and these two or more bacterial strains include different expression cassettes encoding protein components necessary for the production of recombinant viral vectors.

[0097] As used herein, the term “expression cassette” has its common meaning in the art. This term refers to a combination of elements necessary for the expression of one or more genes.

[0098] In one embodiment, the present invention also relates to such an expression cassette.

[0099] The expression cassette can be contained in any suitable expression vector. Typically, the expression vector may be a two-component vector suitable for expression in plant cells, such as pRD400, pBIN19, pBINPlus, or pCAMBIA two-component vector, which has been modified to contain one or more expression cassettes of the present invention. Other examples of two-component vectors are listed in Table 2 of Bahramnejad et al., 2019. In one particular embodiment, the two-component vector is pRD400.

[0100] In one embodiment, the expression cassette comprises a promoter, one or more genes encoding protein components necessary for the production of a recombinant viral vector, and a polyadenylated sequence. In a particular embodiment, the expression cassette further comprises an enhancer.

[0101] Any promoter suitable for expression in plant cells may be used. In a particular embodiment, the promoter is suitable for expression in eukaryotic cells such as insect or mammalian cells, and is also suitable for expression in plant cells. Those skilled in the art can, based on their general knowledge of molecular biology, determine whether a eukaryotic cell promoter can motivate gene expression in plant cells. For example, the ability of a promoter to which a reporter gene is functionally linked in a construct can be tested by transfecting plant cells with a construct containing the reporter gene and the promoter using a reporter gene. In a particular embodiment, the promoter may be a p19 AAV reporter.

[0102] In a particular embodiment, the gene encoding the protein component necessary for the production of the recombinant viral vector is under the control of a constitutive promoter suitable for expression in plant cells. A non-extensive list of constitutive promoters suitable for expression in plant cells includes: cauliflower mosaic virus 35S (CaMV35S) (Odell et al., 1985), cassava leaf vein mosaic virus (CVMV) (Verdaguer et al., 1996), cotton leaf curl Multan virus (CLCuMV) C1 (Xie et al., 2003), lotus dwarf virus component 8 (Shirasawa-Seo et al., 2005), Australian banana streak virus (BSV) (Schenk et al., 2001), four o'clock mosaic virus (MMV) (Dey and Maiti, 1999), scrophularia mosaic virus (FMV) (Sanger et al., 1990), and maize polyubiquitin-1. Examples include the promoter of comeactin (Christensen et al., 1992), actin from Arabidopsis thalania (An et al., 1996), nopalin synthase (nos) from the Agrobacterium genus (An et al., 1988), the promoter of rolD from the Agrobacterium genus (Fei et al., 2003), or any functional variant thereof.

[0103] In another specific embodiment, the gene encoding the protein component required for the production of the recombinant viral vector is under the control of an inducible promoter suitable for expression in plant cells. Non-exclusive examples of promoters suitable for expression in plant cells include pristinamycin-responsive (Frey et al., 2001), maize-derived In2-2 (De Veylder et al., 1997), potato-derived wun1 (Siebertz et al., 1989), agrobacteria-derived mannopin synthase (Langridge et al., 1989), soybean-derived heat shock promoter Gmshp17.3 (Schoffl et al., 1989), alcohol dehydrogenase (Felenbok et al., 1988), or any functional variant thereof.

[0104] In one particular embodiment, the inducible promoter is an alcohol dehydrogenase promoter (Felenbok et al., 1988) or a functional variant thereof.

[0105] In one particular embodiment, the gene encoding the protein component required for the production of the recombinant viral vector is under the control of a promoter derived from a virus that infects cruciferous plants, such as the cauliflower mosaic virus 35S (CaMV35S) promoter or a functional variant thereof.

[0106] In a particular embodiment, the promoter is a functional variant of the CaMV35S promoter, exhibiting enhanced transcriptional activity compared to the wild-type CaMV35S promoter. Specifically, the functional variant of CaMV35S includes a duplication of a -343 to -90 bp fragment, as described by Kay et al., 1987. In a particular embodiment, the functional variant of CaMV35S has at least 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 13.

[0107] A "functional variant" refers to any variant that retains the function of the original promoter, i.e., is capable of initiating the transcription of a particular gene. More specifically, a functional variant may have at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least 100% of the transcriptional induction activity compared to the wild-type promoter. The transcriptional induction activity of a functional variant may exceed 100% of the activity of the wild-type promoter, for example, exceeding 110%, 120%, 130%, 140%, or even 150%.

[0108] In a particular embodiment, the promoter is the CaMV35S promoter, for example, the CaMV35S sequence of SEQ ID NO: 9 or a functional variant thereof. The CAMV35S functional variant may have at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least 100% of the transcriptional induction activity of the wild-type CAMV25S promoter of SEQ ID NO: 9. The transcriptional induction activity of the functional variant may exceed 100% of the activity of the wild-type CAMV25S promoter of SEQ ID NO: 9, for example, exceeding 110%, 120%, 130%, 140%, or even exceeding 150%.

[0109] In a particular embodiment, the promoter is a nopalin synthase (nos) promoter, for example, the nos promoter of the sequence of SEQ ID NO: 11 or a functional variant thereof. The nos functional variant may have at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least 100% of the transcriptional induction activity of the wild-type nos promoter of SEQ ID NO: 11. The transcriptional induction activity of the functional variant may exceed 100% of the activity of the wild-type nos promoter of SEQ ID NO: 11, for example, exceeding 110%, 120%, 130%, 140%, or even exceeding 150%.

[0110] In a particular embodiment, the promoter is an alcohol dehydrogenase promoter, for example, the alcohol dehydrogenase promoter of the sequence of SEQ ID NO: 14, or a functional variant thereof. The alcohol dehydrogenase functional variant may have at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least 100% of the transcriptional induction activity compared to the wild-type alcohol dehydrogenase promoter of SEQ ID NO: 14. The transcriptional induction activity of the functional variant may exceed 100% of the activity of the wild-type alcohol dehydrogenase promoter of SEQ ID NO: 14, for example, exceeding 110%, 120%, 130%, 140%, or even exceeding 150%.

[0111] In a particular embodiment, the expression cassette includes a terminator sequence downstream of a gene encoding a protein component necessary for the production of a recombinant viral vector. “Terminator sequence” means a sequence containing a 3'UTR sequence and a polyadenylation signal for terminating transcription. Any terminator sequence capable of causing transcriptional termination in plant cells may be used.

[0112] In one particular embodiment, the terminator sequence is a nopalin synthase (nos) terminator or a CAMV35S terminator.

[0113] The genes encoding the protein components necessary for the production of recombinant viral vectors are well known to those skilled in the art. Those skilled in the art can adapt the expression cassette used in the description of the experiment to the specific viral vector they wish to produce.

[0114] The genes encoding the protein components necessary for the production of recombinant viral vectors can be codon-optimized to improve their expression in plant cells.

[0115] The gene encoding the protein component necessary for the production of recombinant viral vectors contains a start codon. For example, the start codon may be the ATG trinucleotide, or an alternative start codon such as the ACG, CTG, GTG, and TTG start codons.

[0116] In a particular embodiment, one or more expression cassettes as described above include genes encoding protein components necessary for the production of a recombinant AAV viral vector.

[0117] In one particular embodiment, one or more expression cassettes of the present invention include AAV Cap and Rep genes.

[0118] In a particular embodiment, one or more expression cassettes of the present invention include genes encoding AAV VP1, VP2, and / or VP3 proteins, which correspond to the capsid (Cap) structural proteins of the rAAV vector. In a particular embodiment, one or more expression cassettes of the present invention further include genes encoding AAP proteins required for capsid assembly.

[0119] In a particular embodiment, one or more expression cassettes of the present invention include genes encoding the Rep52 protein and / or Rep78, which are proteins necessary for viral replication.

[0120] In a particular embodiment, the AAV Cap and Rep genes are contained in the same expression cassette. In a particular embodiment, the first expression cassette contains the AAV Cap gene, and the second expression cassette contains the AAV Rep gene. The first expression cassette containing the AAV Cap gene and the second expression cassette containing the AAV Rep gene may contain any of the promoter sequences suitable for expression in plant cells as described above. In a particular embodiment, the AAV Cap gene and / or the AAV Rep gene are under the control of a constitutive promoter suitable for expression in plant cells. In another particular embodiment, the AAV Cap gene and / or the AAV Rep gene are under the control of an inductive promoter suitable for expression in plant cells.

[0121] In one particular embodiment, the AAV Cap and Rep genes are each under the control of a promoter derived from a virus that infects cruciferous plants, such as the cauliflower mosaic virus 35S (CaMV35S) promoter. In this embodiment, the first expression cassette includes the AAV Cap gene under the control of the CaMV35S promoter, and the second expression cassette includes the AAV Rep gene under the control of the CaMV35S promoter.

[0122] In a particular embodiment, the first and second expression cassettes, each containing the AAV Cap gene and the AAV Rep gene, further comprise the terminator sequences described above. Any terminator sequence capable of causing transcriptional termination in plant cells may be used. In a particular embodiment, the first expression cassette containing the AAV Cap gene comprises the CaMV35S terminator sequence. In a particular embodiment, the second expression cassette containing the AAV Rep gene comprises the nopalin synthase (nos) terminator sequence.

[0123] In one particular embodiment, the first expression cassette includes a CaMV35S promoter, an AAV Cap gene, and a CaMV35S terminator sequence.

[0124] In one particular embodiment, the second expression cassette includes the CaMV35S promoter, the AAV Rep gene, and the nos terminator sequence.

[0125] In one particular embodiment, the first expression cassette and the second expression cassette further include an enhancer derived from tobacco mosaic virus, such as a tobacco mosaic virus omega (TMVΩ) enhancer.

[0126] In a particular embodiment, the first and second expression cassettes are cloned into one or two expression vectors. In a preferred embodiment, the first and second expression cassettes are cloned into a single expression vector. In a particular embodiment, the first and second expression cassettes are cloned into a two-component vector, for example, the pRD400 two-component vector.

[0127] Cap and Rep genes of any AAV serotype may be used. The Cap and Rep genes may be natural or artificial sequences.

[0128] In the present invention, the Cap gene, or the gene encoding the VP1, VP2, or VP3 capsid protein of the AAV vector, may be derived from naturally occurring or non-naturally occurring serotypes. In a particular embodiment, the serotype of the AAV vector capsid is selected from the natural AAV serotypes. Instead of using the natural AAV serotype, artificial AAV serotypes, including AAVs having non-naturally occurring capsid proteins, may be used in the context of the present invention, but are not limited to such artificial serotypes. Such artificial capsids can be generated by any appropriate technique, which may involve using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterologous sequence that may be obtained from a selected different AAV serotype, a non-proximate portion of the same AAV serotype, a non-AAV virus source, or a non-viral source. Capsids derived from artificial AAV serotypes may be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids.

[0129] According to one particular embodiment, the Cap gene, or the gene encoding VP1, VP2, or VP3, is AAV-1, -2, AAV-2 variant (e.g., AAV-2 optimized with a quadruple mutant capsid, including an engineered capsid having the Y44+500+730F+T491V modification, as disclosed by Ling et al., 2016), -3, and AAV-3 variant (e.g., AAV3-ST variant, including an engineered AAV3 capsid having two amino acid modifications S663V+T492V, as disclosed by Vercauteren et al., 2016), -3B, and AAV-3B variant. -4, -5, -6, and AAV-6 variants (e.g., AAV6 variants including the triple mutant AAV6 capsid Y731F / Y705F / T492V, disclosed in Rosario et al., 2016), -7, -8, -9, and AAV-9 variants (e.g., AAVhu68), -10 such as -2G9, -cy10 and -rh10, -rh39, -rh43, -rh74, -dj, Anc80, LK03, AAV.PHP, AAV2i8, porcine AAV such as AAVpo4 and AAVpo6, and the capsids of tyrosine, lysine, and serine capsid variants of AAV serotypes. In addition, the Cap gene may encode other manipulated non-natural variants (e.g., AAV-spark100), chimeric AAVs, or AAV serotype capsids obtained by truncation, rational design, error-prone PCR, and machine learning techniques. In a particular embodiment, the Cap gene encodes VP capsid proteins derived from at least two different AAV serotypes, or at least one chimeric VP protein having a combination of VP protein regions or domains derived from at least two AAV serotypes. For example, a chimeric AAV capsid can be obtained by combining an AAV8 capsid sequence with a sequence from an AAV serotype different from the AAV8 serotype, such as any of those described in detail above.In another embodiment, the AAV vector capsid comprises one or more variant VP capsid proteins, such as those described in WO2015013313, specifically RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6 capsid variants. In a particular embodiment, the AAV vector capsid is a hybrid of AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins. For example, the AAV serotype may be the -rh74-9 serotype as disclosed in WO2019 / 193119 (e.g., the Hybrid Cap rh74-9 serotype described in the examples of WO2019 / 193119; also referred herein as the rh74-9 serotype "-rh74-9", "AAVrh74-9", or "AAV-rh74-9"), or the -9-rh74 serotype as disclosed in WO2019 / 193119 (e.g., the Hybrid Cap 9-rh74 serotype described in the examples of WO2019 / 193119; also referred herein as the -9-rh74 serotype "-9-rh74", "AAV9-rh74", "AAV-9-rh74", or "rh74-AAV9"). In a particular embodiment, the AAV vector capsid is a peptide-modified hybrid of AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins, such as an AAV9-rh74 hybrid capsid or an AAVrh74-9 hybrid capsid modified with a P1 peptide, as described in PCT / EP2019 / 076958.

[0130] In a particular embodiment, the Cap gene, or the gene encoding VP1, VP2, or VP3, encodes a naturally occurring capsid such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-cy10, or AAVrh10 capsid. In a particular embodiment, the Cap gene, or the gene encoding VP1, VP2, or VP3 used in the present invention, encodes the AAV8 capsid.

[0131] In a particular embodiment, the Rep gene of any natural AAV vector may be used. Specifically, the Rep gene of the AAV2 vector may be used. In a particular embodiment, the genes encoding Rep52 and Rep78 of AAV2 may be used.

[0132] In one particular embodiment, the Cap gene contained in the expression cassette encodes a functional capsid protein, wherein the gene has identity with respect to the nucleotide sequence of SEQ ID NO: 1 of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0133] In one particular embodiment, the Rep gene contained in the expression cassette encodes a functional Rep protein, wherein the gene has identity with respect to the nucleotide sequence of SEQ ID NO: 2 of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0134] In one particular embodiment, a gene encoding the VP1 protein contained in the expression cassette encodes a functional VP1 protein, and the gene has identity with respect to the nucleotide sequence of SEQ ID NO: 3 of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0135] In a particular embodiment, the gene encoding the VP1 protein contained in the expression cassette encodes a functional VP1 protein whose identity to the amino acid sequence of SEQ ID NO: 17 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0136] In one particular embodiment, a gene encoding the VP2 protein contained in the expression cassette encodes a functional VP2 protein, and the gene has identity with respect to the nucleotide sequence of SEQ ID NO: 4 of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0137] In one particular embodiment, the gene encoding the VP2 protein contained in the expression cassette encodes a functional VP2 protein whose identity to the amino acid sequence of SEQ ID NO: 18 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0138] In one particular embodiment, a gene encoding a VP3 protein contained in an expression cassette encodes a functional VP3 protein, and the gene has identity with respect to the nucleotide sequence of SEQ ID NO: 5 of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0139] In a particular embodiment, the gene encoding the VP3 protein contained in the expression cassette encodes a functional VP3 protein whose identity to the amino acid sequence of SEQ ID NO: 19 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0140] In a particular embodiment, a gene encoding an AAP protein contained in an expression cassette encodes a functional AAP protein, and the gene has identity with respect to the nucleotide sequence of SEQ ID NO: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0141] In a particular embodiment, the gene encoding the AAP protein contained in the expression cassette encodes a functional AAP protein whose identity to the amino acid sequence of SEQ ID NO: 20 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0142] In one particular embodiment, a gene encoding the Rep52 protein contained in the expression cassette encodes a functional Rep52 protein, and the gene has identity with respect to the nucleotide sequence of SEQ ID NO: 7 of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0143] In one particular embodiment, the gene encoding the Rep52 protein contained in the expression cassette encodes a functional Rep52 protein whose identity to the amino acid sequence of SEQ ID NO: 21 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0144] In one particular embodiment, a gene encoding the Rep78 protein contained in the expression cassette encodes a functional Rep78 protein, wherein the gene has identity with respect to the nucleotide sequence of SEQ ID NO: 8 of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0145] In one particular embodiment, the gene encoding the Rep78 protein contained in the expression cassette encodes a functional Rep78 protein whose identity to the amino acid sequence of SEQ ID NO: 22 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0146] In a particular embodiment, one or more expression cassettes of the present invention include genes encoding VP1 protein, VP2 protein, VP3 protein, AAP (assembly activation protein), Rep52 protein, and / or Rep78 protein.

[0147] The genes encoding the VP1, VP2, VP3, AAP (Assembly Activation Protein), Rep52, and / or Rep78 proteins can be codon-optimized to improve their expression in plant cells.

[0148] The genes encoding the VP1, VP2, VP3, AAP (assembly activation protein), Rep52, and / or Rep78 proteins may be under the control of any promoter that enables their expression in plant cells.

[0149] In one particular embodiment, at least two of the genes encoding VP1, VP2, VP3, AAP, Rep52, and Rep78 are under the control of the same promoter.

[0150] In yet another embodiment, the gene encoding VP1 is included in an expression cassette including a promoter operably linked to the gene encoding VP1, the gene encoding VP2 is included in an expression cassette including a promoter operably linked to the gene encoding VP2, the gene encoding VP3 is included in an expression cassette including a promoter operably linked to the gene encoding VP3, the gene encoding AAP is included in an expression cassette including a promoter operably linked to the gene encoding AAP, the gene encoding Rep52 is included in an expression cassette including a promoter operably linked to the gene encoding Rep52, and the gene encoding Rep78 is included in an expression cassette including a promoter operably linked to the gene encoding Rep78.

[0151] In a particular embodiment, each cassette containing one gene selected from the genes encoding VP1, VP2, VP3, AAP, Rep52, or Rep78 proteins is cloned into one or more vectors. Preferably, all cassettes containing one gene each selected from the genes encoding VP1, VP2, VP3, AAP, Rep52, or Rep78 proteins are cloned into the same vector. In a particular embodiment, all cassettes are cloned into a two-component vector such as the pRD400 two-component vector.

[0152] In one particular embodiment, the genes encoding the VP1, VP2, VP3, AAP, Rep52, or Rep78 proteins are under the control of a constitutive promoter suitable for expression in plant cells. A non-extensive list of constitutive promoters suitable for expression in plant cells includes: cauliflower mosaic virus 35S (CaMV35S) (Odell et al., 1985), cassava leaf vein mosaic virus (CVMV) (Verdaguer et al., 1996), cotton leaf curl Multan virus (CLCuMV) C1 (Xie et al., 2003), lotus dwarf virus component 8 (Shirasawa-Seo et al., 2005), Australian banana streak virus (BSV) (Schenk et al., 2001), four o'clock mosaic virus (MMV) (Dey and Maiti, 1999), scrophularia mosaic virus (FMV) (Sanger et al., 1990), and maize polyubiquitin-1. Examples include the promoter of comeactin (Christensen et al., 1992), actin from Arabidopsis thalania (An et al., 1996), nopalin synthase (nos) from the Agrobacterium genus (An et al., 1988), the promoter of rolD from the Agrobacterium genus (Fei et al., 2003), or any functional variant thereof.

[0153] In another specific embodiment, the genes encoding the VP1, VP2, VP3, AAP, Rep52, or Rep78 proteins are under the control of an inducible promoter suitable for expression in plant cells. Non-exclusive examples of promoters suitable for expression in plant cells include pristinamycin-responsive (Frey et al., 2001), maize-derived In2-2 (De Veylder et al., 1997), potato-derived wun1 (Siebertz et al., 1989), agrobacteria-derived mannopin synthase (Langridge et al., 1989), soybean-derived heat shock promoter Gmshp17.3 (Schoffl et al., 1989), alcohol dehydrogenase (Felenbok et al., 1988), or any functional variant thereof.

[0154] "The functional variant" refers to any variant that retains the function of the original promoter, that is, one that can initiate the transcription of a specific gene.

[0155] In one particular embodiment, the genes encoding the VP1, VP2, VP3, AAP, Rep52, or Rep78 proteins are under the control of a promoter derived from a virus that infects cruciferous plants, such as the cauliflower mosaic virus 35S (CaMV35S) promoter or a functional variant thereof. In one particular embodiment, the promoter is a functional variant of the CaMV35S promoter, which has enhanced transcriptional activity compared to the wild-type CaMV35S promoter. Specifically, the functional variant of CaMV35S includes a duplication of a -343 to -90 bp fragment, as described by Kay et al., 1987. In one particular embodiment, the functional variant of CaMV35S has at least 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 13.

[0156] In one particular embodiment, - The gene encoding VP1 is under the control of a weak promoter, such as the nopalin synthase (nos) promoter or its functional variant. - The gene encoding VP2 is under the control of a weak promoter, such as the nopalin synthase (nos) promoter or its functional variant. - The gene encoding VP3 is under the control of a strong promoter, such as the CaMV35S promoter or a functional variant thereof, preferably a functional variant with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 13, and / or - The gene encoding AAP is under the control of a strong promoter, such as the CaMV35S promoter or a functional variant thereof, preferably a functional variant having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 13.

[0157] In a particular embodiment, at least one vector according to the present invention is - An expression cassette comprising a nos promoter or a functional variant thereof, a gene encoding VP1, and a terminator sequence such as a nos terminator sequence. - An expression cassette comprising a nos promoter or a functional variant thereof, a gene encoding VP2, and a terminator sequence such as a nos terminator sequence. - An expression cassette comprising the CaMV35S promoter or a functional variant thereof, preferably a functional variant having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 13, a gene encoding VP3, and a terminator sequence such as the CamV35S terminator sequence, and / or - An expression cassette comprising the CaMV35S promoter or a functional variant thereof, preferably a functional variant having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 13, the gene encoding AAP, and a terminator sequence such as the CamV35S terminator sequence. Includes.

[0158] In another specific embodiment, - The gene encoding VP1 is under the control of an inductive promoter, such as the alcohol dehydrogenase promoter or its functional variant. - The gene encoding VP2 is under the control of an inductive promoter, such as the alcohol dehydrogenase promoter or its functional variant. - The gene encoding VP3 is under the control of an inductive promoter such as the alcohol dehydrogenase promoter or a functional variant thereof, and / or - The gene encoding AAP is under the control of an inductive promoter, such as the alcohol dehydrogenase promoter or a functional variant thereof.

[0159] In a particular embodiment, at least one vector according to the present invention is - An expression cassette comprising an alcohol dehydrogenase promoter or a functional variant thereof, a gene encoding VP1, and a terminator sequence such as a nos terminator sequence. - An expression cassette comprising an alcohol dehydrogenase promoter or a functional variant thereof, a gene encoding VP2, and a terminator sequence such as a nos terminator sequence. - An expression cassette comprising an alcohol dehydrogenase promoter, a gene encoding VP3, a terminator sequence such as the CamV35S terminator sequence, and optionally an enhancer such as the TMVΩ enhancer, and / or - Expression cassette containing an alcohol dehydrogenase promoter, a gene encoding AAP, and a terminator sequence such as the CamV35S terminator sequence. Includes.

[0160] In a particular embodiment, the TMVΩ enhancer has a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 16.

[0161] In one particular embodiment, the genes encoding VP1, VP2, VP3, and / or AAP are further codon-optimized to improve their expression in plant cells.

[0162] When an inducible promoter for alcohol dehydrogenase is used, at least one vector according to the present invention further comprises an expression cassette encoding the ALCR protein required for activation of the alcohol dehydrogenase promoter. In a particular embodiment, the gene encoding the ALCR protein encodes a functional ALCR protein, wherein the gene has identity to the nucleotide sequence of SEQ ID NO: 15 of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In a particular embodiment, the gene encoding the ALCR protein contained in the expression cassette encodes a functional ALCR protein having identity to the amino acid sequence of SEQ ID NO: 23 of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100%.

[0163] In one particular embodiment, - The gene encoding Rep52 is under the control of an inductive promoter such as the alcohol dehydrogenase promoter or a functional variant thereof, and / or - The gene encoding Rep78 is under the control of an inductive promoter, such as the alcohol dehydrogenase promoter or a functional variant thereof.

[0164] In a particular embodiment, at least one vector according to the present invention is - An expression cassette comprising an alcohol dehydrogenase promoter or a functional variant thereof, a gene encoding Rep52, a terminator sequence such as the CamV35S terminator sequence, and optionally an enhancer such as the TMVΩ enhancer, and / or - An expression cassette comprising an alcohol dehydrogenase promoter or a functional variant thereof, a gene encoding Rep78, and a terminator sequence such as a nos terminator sequence. Includes.

[0165] In one particular embodiment, the genes encoding Rep52 and / or Rep78 are further codon-optimized to improve their expression in plant cells.

[0166] When an inducible promoter of alcohol dehydrogenase is used, at least one vector according to the present invention further comprises an expression cassette encoding an ALCR protein required for activation of the alcohol dehydrogenase promoter.

[0167] Recombinant viral vector recovery In one embodiment, the recombinant viral vector is recovered by harvesting the culture medium in which the hairy roots are cultured.

[0168] In one particular embodiment, the culture medium containing the recombinant is collected and used directly for subsequent applications.

[0169] Advantageously, the culture medium containing the recombinant viral vector according to this embodiment is suitable for oral use in humans and / or animals without the need for purification.

[0170] Therefore, another aspect of the present invention relates to a culture medium containing a recombinant viral vector obtainable by the method described above.

[0171] In one alternative embodiment, the recombinant viral vector is obtained after one or more purification steps. Appropriate protocols suitable for each recombinant viral vector are standard techniques in the industry and can be easily selected by those skilled in the art if there are purification steps suitable for the desired application.

[0172] In a second embodiment, the recombinant viral vector is extracted from the hairy root. More specifically, the recovery of the viral vector may be carried out by chemical extraction or by crushing the hairy root.

[0173] In one further specific embodiment, the method for producing a recombinant mammalian virus vector further includes the step of inducing the emergence of lateral roots in the hairy roots of a transformed plant, as described in WO16185122.

[0174] More specifically, the process of inducing the emergence of lateral roots in the hairy roots of transformed plants may be carried out by culturing the transformed hairy roots in the presence of at least one auxin.

[0175] In one particular embodiment, the hairy roots are cultured in a liquid medium containing at least one auxin.

[0176] Auxin can be selected from 2,4-dichlorophenoxyacetic acid (2,4-D), 3-indoleacetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,3,5-triiodoacetic acid, 4-chlorophenoxyacetic acid, 2-naphthoxyacetic acid, 1-naphthylacetic acid, 4-amino-3,5,6-trichloropicolinic acid, 3,6-dichloro-2-methoxybenzoic acid (Dicamba), and derivatives thereof.

[0177] Another aspect of the present invention is, a) A step of inducing the formation of hairy roots from a plant belonging to the Brassicaceae family according to any of the embodiments described above, and b) A process in which the plant is transformed with at least one vector comprising one or more expression cassettes, according to any embodiment described above, wherein the one or more expression cassettes comprises genes encoding protein components necessary for the production of a recombinant viral vector. This relates to hairy root cultures that can be obtained or have been obtained by [method / method].

[0178] Another aspect of the present invention relates to a transgenic plant transformed with at least one vector comprising one or more expression cassettes containing genes encoding protein components necessary for the production of a recombinant viral vector, the plant belonging to the Brassicaceae family.

[0179] Another aspect of the present invention relates to recombinant viral vectors that can be obtained or have been obtained by the method described above. [Examples]

[0180] Materials and methods A. Recombinant two-component plasmid for AAV protein expression The sequences used were extracted from plasmid p5e18_VD2_8_kanR (RepCap_cellules_293, provided by GENETHON). The coding sequences were manually optimized based on codon usage frequencies in human (initiator organism) and Brassica rapa (expression host organism) species.

[0181] Five constructs containing different expression cassettes were designed. Schematic diagrams of the five constructs are shown in Figure 1.

[0182] In construct 1, the expression of the CAP (AAV8-derived) and REP (AAV2-derived) genes is driven by the CaMV35S promoter.

[0183] In construct 2, the VP1 and VP2 genes are under the control of the Nos promoter, respectively. The VP3 and AAP genes are under the control of the "2*CaMV35S" promoter, respectively. The "2*CaMV35S" promoter is a variant of CaMV35S containing a -343 to -90 bp duplication fragment, as described by Kay et al., 1987. Since there is no specific anti-AAP antibody, the AAP protein was tagged with HA to enable its detection.

[0184] In construct 3, Rep52 and Rep78 are under the control of an ethanol-inducible system.

[0185] Architectural construct 4 is similar to construct 3, but with the addition of the tobacco mosaic virus omega (TMVΩ) enhancer to strengthen Rep52 expression.

[0186] In construct 5, an ethanol-inducible system was used to mobilize the expression of VP1, VP2, VP3, and AAP. Tobacco mosaic virus omega (TMVΩ) enhancer was added to increase VP3 expression. Since there is no specific anti-AAP antibody, the AAP protein was tagged with HA to enable its detection.

[0187] Next, each of these five constructs was cloned separately into the pRD400 plant expression vector (Datla et al., 1992).

[0188] The resulting plasmid pRD400-AAV was first cloned into electrocompetent Escherichia coli strain JM101, and then into Rhizobium rhisogenes strain ATCC 15834.

[0189] The R. rhizogenes strain ATCC 15834 was transformed by electroporation with an AAV encoding the pRD400 plasmid (pRD400-AAV).

[0190] B. Generation, selection, and culture of transgenic hairy roots 1. Plant species and in vitro culture Seeds from Brassica rapa cv 'Navet des vertus Marteau' were surface-sterilized for 10 minutes in a 5% bleach solution supplemented with a few drops of Triton X100, washed at least five times with sterile water, and then placed in a petri dish containing B5 agar. Germination and seedling growth occurred at 20°C with a photoperiod of 16h light / 8h dark.

[0191] 2. Plant infection by Rhizobium rhisogenes The inventors used Rhizobium rhizogenes strain 15834 (ATCC 15834) provided by the Pasteur Institute. Rhizobium rhizogenes was grown in MGL plates (2.5 g / l yeast extract, 5 g / l tryptone, 5 g / l mannitol, 5 g / l NaCl, 1.16 g / l sodium glutamate, 0.25 g / l KH2PO4, 0.1 g / l MgSO4, 1.0 mg / l biotin, 8 g / l agar, pH 7.0) supplemented with 50 mg / l kanamycin at the end to select a two-component plasmid. An inoculum was prepared from 20 ml of liquid bacterial culture grown overnight in MGL medium at 25°C. The suspension was centrifuged at 15,000 rpm for 5 minutes, and the collected cells were resuspended in fresh MGL medium, diluted, and an optical concentration of 1 ± 0.1 at 600 nm was obtained.

[0192] The plants were infected by puncturing the hypocotyl of 3-10 day old seedlings with a needle and applying a rhizobium inoculant to the injured area with a sterile cotton swab. Typically, hairy roots appeared from the wound site about two weeks after infection.

[0193] 3. Selection and culture of hairy root clones expressing AAV protein. Infected hypocotyls that produced hairy roots were excised from seedlings and placed on B5 (Duchefa) pH 5.8 supplemented with 3% saccharose and cefotaxime (300 mg / L) and 8 g / L agar. After 7 days, the tips of the isolated hairy roots were transferred to fresh B5-3% saccharose + cefotaxime (300 mg / L) and 8 g / L agar, where they were grown for 4-10 days.

[0194] To initiate culture in liquid medium, each hairy root clone was then transferred to 6 ml of B5 containing 3% saccharose for 10 days. After this, the culture medium was changed and further enriched with 2,4-dichlorophenoxyacetic acid (2,4D). The hairy root clones were cultured for another 10 days. After 20 days of culture, all samples were collected. 100 mg of fresh biomass was used for protein and RNA extraction.

[0195] C. SDS-PAGE and Western blot Protein extraction from hairy roots was performed using a commercially available kit (Macherey-Nagel, ref 740933) according to the supplier's protocol. A fixed volume of hairy root extract was sampled, mixed with 1 / 3 volume of 3X Laemmli buffer, and boiled for 5 minutes. 40 μl of each sample was placed on an AnykD mini protean TGX polyacrylamide gel (Bio-Rad). For Western blotting analysis, the proteins were transferred to a nitrocellulose membrane using the Bio-Rad Turbo Trans-Blot system (Bio-Rad, Hercules, California). The membranes were blocked in 5% nonfat milk in TBS buffer (Blotting grade blocker, Bio-Rad) and incubated with a 1:250 dilution of anti-AAV VP1 / VP2 / VP3 mouse monoclonal B1 (Progen 61058), or a 1:100 dilution of anti-AAV2 replicase mouse monoclonal 259,5 (Progen 61071), or a 1:1000 dilution of anti-HA tagged antibody mouse monoclonal (Abcam ab18181). Following incubation, the membranes were incubated with a 1:5000 dilution of m-IgGκBP-HRP antibody (Santa Cruz Biotechnology sc-516102). Staining was developed using a Western Clarity ECL development kit (170-5060, Bio-Rad).

[0196] Quantification of AAV particles in hairy roots of D. brassica rapa 1. rAAV2 / 8 vector produced in hairy roots of Brassica rapa Hairy root clones of Brassica rapa were generated as previously described. Aggregated AAV particles were detected in protein extracts from clones C1-38 (including construct number 1 as described above) and clones C2-8 and C2-53 (including construct number 2 as described above). Wild-type clones (i.e., without transgenes) were included as a negative control.

[0197] Clones were maintained for 20 days. Briefly, 1 g of fresh hairy roots were seeded in 100 mL of Gamborg B5 medium containing 30 g / L sucrose and incubated at 23°C and 100 rpm for 10 days. After 10 days of growth, the hairy roots were transferred to 100 mL of fresh Gamborg B5 medium containing 30 g / L sucrose, supplemented with 1 mg / L 2,4D, and incubated at 23°C and 100 rpm for another 10 days. At the end of the culture, 100 mg of fresh biomass was collected, frozen in liquid nitrogen, and stored at -80°C for subsequent protein extraction.

[0198] For each generated sample, three different extraction methods (Methods B, C, and D below) were tested and applied as shown in Table 1.

[0199] [Table 1]

[0200] Next, the concentration of aggregated virus particles (capsids / mL) was determined using the Progen AAV8 Capsid ELISA Kit described below.

[0201] 2. rAAV8-eGFP control vector preparation rAAV8-eGFP vectors produced from mammalian cells were used as positive controls in each experiment.

[0202] The control vector was prepared as follows: Briefly, HEK293T cells adapted for growth in suspension culture were seeded in 400 mL of F17 culture medium of known composition in a shaking flask (1 L). Three plasmids were transfused into the cells using a PTG1+ transfusion agent. Cells were collected 72 hours after transfusion and lysed by ultrasonic treatment. AAV8 particles in the culture supernatant were precipitated with polyethylene glycol (PEG), then purified by double CsCl density gradient ultracentrifugation, and finally formulated in 1× DPBS containing Ca2+ and Mg2+ by dialyzing in a Slide-A-Lyzer® 10K MWCO cassette (Thermo Scientific, Illkirch, France).

[0203] The viral genome concentration / mL (VG / mL) was determined from deoxyribonuclease-resistant particles using a TaqMan real-time PCR assay. The viral particle concentration (capsid / mL) was determined using the Progen AAV8 Capsid ELISA Kit described below. (5~10 7 5 μL of the control vector (corresponding to one capsid) was added to either 100 μL of extraction buffer (B, C, or D) or 100 μL of hairy root protein extract obtained from a wild-type clone (i.e., without the transgene) according to the extraction method described above (B, C, or D).

[0204] 3. rAAV8-eGFP denatured vector preparation The control vector was thermally denatured at 90°C for 15 minutes to serve as a negative control. This ensures that only the aggregated capsid yields a signal in the ELISA.

[0205] (5×10 7 5 μL of denatured vector (corresponding to one capsid) was added to 100 μL of extraction buffer (B, C, or D), or to 100 μL of hairy root protein extract from a wild-type clone (i.e., without the transgene).

[0206] 4. Titration of AAV8 capsid by ELISA The AAV8 aggregated capsid was titrated by ELISA using the Progen AAV8 Capsid ELISA Kit (PROGEN Biotechnik, Heidelberg, Germany) according to the manufacturer's instructions.

[0207] result The objective was to determine whether hairy roots from Brassica rapa can produce all the proteins necessary for AAV synthesis (REP, CAP, and AAP proteins).

[0208] To achieve this objective, the molecular constructs described above were designed, cloned, and introduced into Brassica rapa using Rhizobium rhizogenes. Transgenic hairy roots were induced in Brassica rapa and analyzed. The results for construct 1 and construct 2 are shown below.

[0209] 1. Analysis of AAV protein production from hairy roots containing construct 1. Western blot analysis was performed on protein extracts from hairy root clones containing construct 1, as detailed above.

[0210] Western blot analysis demonstrated the ability of hairy roots to produce VP1, VP2, and VP3 proteins.

[0211] Western blot analysis also demonstrated the Rep protein production ability of the hairy roots.

[0212] 2. Analysis of AAV protein production from hairy roots containing construct 2. Western blot analysis was performed on protein extracts from hairy root clones containing construct 2, as detailed above. Construct 2 was designed to test the expression capabilities of Brassica rapa VP1, VP2, VP3, and AAP (derived from AAV8) using different promoters, namely the "2*35S" promoter or the NOS promoter.

[0213] Western blot analysis demonstrated the ability of hairy roots to produce VP1, VP2, and VP3 proteins.

[0214] Western blot analysis also demonstrated the AAP-HA protein production capacity of the hairy roots.

[0215] 3. Quantification of AAV particles in hairy roots from Brassica rapa The inventors thus demonstrated the ability of hairy roots to produce AAV Rep, VP1, VP2, VP3, and AAP proteins. The next objective was to determine whether hairy roots from Brassica rapa can produce aggregate capsids.

[0216] The quantification of AAV particles present in hairy root protein extracts prepared by the three extraction methods (described in Materials and Methods) was determined by ELISA from clones (C1-38 / C2-8 / C2-53) that were shown by Western blotting to produce VP and / or AAP proteins.

[0217] Wild-type clones (i.e., without the transgene) were included as negative controls.

[0218] The results detailed in Table 2 below demonstrate that hairy roots from Brassica rapa can produce aggregate capsids.

[0219] [Table 2]

[0220] (1) The titers obtained with the control rAAV8-eGFP vector added to the extraction buffer or (2) the protein extract derived from wild-type hairy root clones were almost identical, and therefore it can be pointed out that the titration method is validated.

[0221] The titers (capsid / mL) obtained from protein extracts derived from clone C1-38 (referring to construct number 1) and clones C2-8 and C2-53 (referring to construct number 2) were high and comparable to those obtained with the control rAAV8-eGFP vector.

[0222] The titer obtained in the protein extract of hairy root clones is unexpectedly high, considering that no concentration step was performed and that very small amounts of starting material were used for extraction and quantification.

[0223] In addition, the results demonstrate the detection of AAV aggregate capsids according to three different extraction methods (B, C, and D), thus demonstrating the reproducibility of the methods.

[0224] [References] TIFF2026076163000003.tif181149TIFF2026076163000004.tif223167TIFF2026076163000005.tif224166TIFF2026076163000006.tif77166

Claims

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

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