Production of recombinant viral vectors from plant hairy roots

Abstract

The present invention relates to a method for producing a recombinant viral vector from the hairy roots of a plant, in particular from the hairy roots of a plant belonging to the Brassicaceae family. The present invention also relates to a transgenic plant, a hairy root culture, and a recombinant viral vector obtainable by the method of the present invention.

Description

[Technical Field]

[0001] The present invention relates to a method for producing recombinant viral vectors from hairy roots, in particular from hairy roots of plants belonging to the Brassicaceae family. [Background technology]

[0002] The increasing use of recombinant viral vectors for gene therapy and DNA vaccination applications has created a need for efficient production systems. Examples of recombinant viral vectors for therapeutic purposes include lentivirus-based vectors and adeno-associated virus-based vectors.

[0003] Among gene therapy products under development, recombinant adeno-associated virus (AAV)-based vectors are currently the most widely used and offer the greatest potential for in vivo delivery. The use of rAAV vector systems is favored, in part, because of the absence of disease associated with wild-type virus, the ability of AAV to transduce dividing as well as non-dividing cells, and the resulting long-term, robust transgene expression 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 marketing approval of recombinant adeno-associated virus gene therapy drugs in Europe and the United States (e.g., Luxturna® and Zolgensma®) represents a milestone achievement in the field of gene therapy.

[0004] AAVs are non-enveloped icosahedral particles containing a single-stranded DNA genome. They belong to the genus Dependoparvovirus, a genus that relies on a helper virus to provide essential genes in trans for productive infection (Weitzman and Linden, 2011). The 4.7-kb genome contains two major open reading frames, the regulatory (Rep) and structural capsid (Cap) genes, which encode several proteins required for viral replication, capsid structure, and viral genome packaging. Three proteins, VP1, VP2, and VP3, are naturally produced from the Cap gene by a combination of alternative splicing and leaky scanning of transcripts from the p40 promoter. All of these proteins share the same C-terminal sequence. The AAV capsid is composed of 60 units of VP1, VP2, and VP3 in an approximately 1:1:10 ratio. Proteins called assembly-activating proteins (AAPs) are translated from distinct open reading frames in the cap gene and are required 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, arise from transcription initiated using the p19 promoter. In addition to the Rep proteins, inverted terminal repeats (ITRs) are also required for AAV DNA replication and packaging (Balakrishnan and Jayandharan, 2014; Robert et al., 2017).

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

[0006] Transient transfection of mammalian cells with plasmid DNA to produce AAV viral vectors is the most commonly used strategy for the clinical-grade manufacture of these viral vectors. rAAV vectors are typically produced in human embryonic kidney 293 cells (HEK293) after transfection with typically three DNA plasmids carrying the Rep and Cap genes, the rAAV transgene, and specific genes providing helper adenovirus function.

[0007] Packaging or producer cell lines are generated by generating stable cell lines engineered with the introduction of both the Rep and Cap genes and / or the rAAV genome.

[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 BV was developed in which the number of BVs was reduced to two (Smith et al., 2009). In this method, the rep and cap sequences were inserted into a single baculovirus in a head-to-head configuration.

[0009] Additionally, 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 Summary of the Invention [Problem to be solved by the invention]

[0011] Despite improvements in these systems in recent years, there is a need to improve the productivity and / or quality of vectors to enable routine use in 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 viral vector from a plant hairy root, the method comprising: a) inducing the production of hairy roots from said plant; b) transforming said plant with at least one vector containing one or more expression cassettes, wherein the one or more expression cassettes contain genes encoding protein components required for the production of a recombinant viral vector; Including, The plant belongs to the Brassicaceae family. Regarding the method.

[0014] In particular, the plant 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, and the plant is in particular Brassica rapa.

[0015] In one particular embodiment, step a) is carried out by transforming a plant with a bacterial strain comprising the rol gene cluster, the bacterial strain being capable of infecting the plant.

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

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

[0018] In a specific embodiment, the one or more expression cassettes comprise the AAV rep and cap genes, each of which is under the control of a promoter derived from a virus that infects plants in the Brassicaceae family, such as the cauliflower mosaic virus 35S (CaMV35S) promoter.

[0019] In another specific embodiment, the one or more expression cassettes comprise genes encoding VP1, VP2, VP3, AAP (assembly activating protein), Rep52, and Rep78 proteins. In a specific embodiment, each gene encoding VP1, VP2, VP3, AAP, Rep52, or Rep78 protein is under the control of a constitutive promoter, such as the cauliflower mosaic virus 35S (CaMV35S) promoter or the nopaline synthase (nos) promoter, or under the control of an inducible promoter, such as the alcohol dehydrogenase (AlcA) promoter.

[0020] In one specific 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 one specific embodiment, the gene encoding VP3 is under the control of a functional variant of the CaMV35S promoter that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical 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 that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 13.

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

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

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

[0025] In one particular embodiment, the gene encoding Rep52 is further under the control of an enhancer, in particular the Tobacco Mosaic Virus Omega (TMVΩ) enhancer.

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

[0027] In one particular embodiment, the plant is further transformed with a vector encoding viral helper functions necessary for efficient viral replication, in particular a vector encoding adenoviral helper functions.

[0028] In a particular embodiment, the plant is further transformed with a vector comprising a viral genome comprising a gene encoding a product of interest, particularly a vector comprising a gene encoding a product of interest flanked by two AAV-ITR sequences.

[0029] Another aspect of the present invention is a) inducing the production of hairy roots from a plant belonging to the Brassicaceae family; and b) transforming said plant with at least one vector containing one or more expression cassettes, wherein the one or more expression cassettes contain genes encoding protein components required for the production of a recombinant viral vector. The present invention relates to hairy root cultures obtainable by

[0030] In one particular embodiment, the hairy root culture is obtainable by transforming a plant belonging to the Brassicaceae family, the plant being selected from the group consisting of Raphanus sativus, Raphanus sativus var. niger, Brassica oleracea L. var., Brassica napus, Arabidopsis thaliana, and Brassica rapa, and the plant is in particular Brassica rapa.

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

[0032] A further aspect of the present invention relates to a recombinant mammalian viral vector obtainable by the method described above. In a specific embodiment, the recombinant mammalian viral 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 var. niger, Brassica oleracea L. var. group, Brassica napus, Arabidopsis thaliana, and Brassica rapa, and the plant is particularly Brassica rapa. In a specific embodiment, the recombinant mammalian viral vector is a recombinant adeno-associated viral (AAV) viral vector. In another specific embodiment, the recombinant mammalian viral vector is produced from the hairy roots of a plant belonging to the Brassicaceae family, the plant being transformed with at least one vector containing one or more expression cassettes, the one or more expression cassettes comprising genes encoding protein components required for the production of the recombinant viral 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 containing one or more expression cassettes, the one or more expression cassettes including genes encoding protein components required for the production of a recombinant mammalian viral vector, wherein 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. var., Brassica napus, Arabidopsis thaliana, and Brassica rapa, and the plant is in particular Brassica rapa.

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

[0036] [Figure 1] FIG. 1 is a schematic diagram of a construct designed to produce AAV proteins in hairy roots.

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

[0038] Construct 1 contains (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 contains (i) a first expression cassette containing the nos promoter ("pNOS"), the VP1 gene ("VP1"), and the nos terminator ("tNOS"); (ii) a second expression cassette containing the nos promoter ("pNOS"), the VP2 gene ("VP2"), and the nos terminator ("tNOS"); (iii) a third expression cassette containing a variant of the CaMV35S promoter (referred to as "p2*35S"), the VP3 gene ("VP3"), and the CaMV35S terminator ("t35S"); and (iv) a fourth expression cassette containing a variant of the CaMV35S promoter (referred to as "p2*35S"), the AAP gene ("AAP"), and the CaMV35S terminator ("t35S"). The "p2*35S" promoter is a variant of the CaMV35S promoter containing a duplication of the -343 to -90 bp fragment as described by Kay et al., 1987.

[0040] Construct 3 contains (i) a first expression cassette containing an inducible promoter for alcohol dehydrogenase ("pAlcA"), 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 ("AlcR") required for activation of the alcohol dehydrogenase promoter, and a CaMV35S terminator ("t35S").

[0041] Construct 4 contains (i) a first expression cassette containing an inducible alcohol dehydrogenase promoter ("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 alcohol dehydrogenase promoter ("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 ("AlcR") required for activation of the alcohol dehydrogenase promoter, and a CaMV35S terminator ("t35S").

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

[0043] A first aspect of the present invention is a method for producing a recombinant mammalian viral vector from a plant hairy root, the method comprising: a) inducing the production of hairy roots from said plant; b) transforming said plant with at least one vector containing one or more expression cassettes, wherein the one or more expression cassettes contain genes encoding protein components required for the production of a recombinant viral vector; Including, The plant belongs to the Brassicaceae family. Regarding the method.

[0044] Recombinant Mammalian Viral Vectors The term "viral vector" relates, according to the present invention, to a carrier or "vector" derived from a virus. This term encompasses any viral particle, with or without a viral genome.

[0045] In the context of the present invention, viral vectors lacking the viral genome are also referred to as virus-like particles (VLPs). VLPs are highly organized structures that self-assemble from viral-derived structural proteins. These stable and versatile nanoparticles have excellent adjuvant properties that can induce innate and adaptive immune responses. Over the past few years, VLPs, taking advantage of their structural stability and resistance to manipulation, have been applied in other branches of biotechnology to carry and display heterologous molecules or serve as building blocks for novel nanomaterials. VLPs can be produced from members of a wide range of virus 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 transgene of interest. The term "transgene of interest" refers to a gene whose nucleic acid sequence is not naturally present in the viral genome. The transgene of interest may be a coding or non-coding sequence. In particular, recombinant viral vectors are used in gene therapy. As used herein, the term "gene therapy" refers to the transfer of genetic material of interest (e.g., DNA or RNA) into a host to treat or prevent a genetic or acquired disease or condition. The genetic material of interest encodes a product (e.g., a polypeptide or functional RNA) whose production in vivo is desired. For example, the genetic material of interest may encode a hormone, receptor, enzyme, or polypeptide of therapeutic value. Alternatively, the genetic material of interest may encode a functional RNA of therapeutic value, such as an antisense RNA of therapeutic value (e.g., an antisense RNA suitable for exon skipping) or shRNA.

[0047] In the context of the present invention, the viral vector is a mammalian viral vector, i.e., the viral vector is derived from a virus capable of infecting mammals, in particular humans, and therefore, in the context of the present invention, the viral vector is not derived from a virus capable of infecting plants.

[0048] Recombinant viral vectors may in particular 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 specific embodiment, the recombinant viral vector is a recombinant AAV (rAAV) vector.

[0050] In the present invention, the capsid of an AAV vector can be derived from a naturally occurring or non-naturally occurring serotype. In a specific embodiment, the serotype of the capsid of an AAV vector is selected from a natural AAV serotype. As an alternative to using a natural AAV serotype, artificial AAV capsids, including but not limited to AAVs with capsid proteins that do not occur in nature, may be used in the context of the present invention. Such artificial capsids can be generated by any suitable technique that uses a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterologous sequence that may be derived from a selected different AAV serotype, a non-contiguous portion of the same AAV serotype, a non-AAV viral 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 a specific embodiment, the capsid of the AAV vector is selected from the group consisting of AAV-1, -2, AAV-2 variants (e.g., AAV-2 optimized with a quadruple mutant capsid, including an engineered capsid with Y44+500+730F+T491V changes, as disclosed in Ling et al., 2016), -3 and AAV-3 variants (e.g., AAV3-ST variants, including an engineered AAV3 capsid with two amino acid changes S663V+T492V, as disclosed in Vercauteren et al., 2016), -3B and AAV-3B variants, -4, -5, -6 and AAV-6 variants (e.g., the AAV6 variant containing 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, AAVpo4 and AAVpo6, and tyrosine, lysine, and serine capsid mutants of AAV serotypes. Additionally, capsids of other engineered non-natural variants (e.g., AAV-spark100), chimeric AAVs, or AAV serotypes obtained by shuffling, rationale design, error-prone PCR, and machine learning techniques may also be useful.

[0052] In a specific embodiment, the AAV vector is a chimeric vector, i.e., its capsid comprises VP capsid proteins from at least two different AAV serotypes, or at least one chimeric VP protein that combines VP protein regions or domains 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 other than the AAV8 serotype, such as any of those specifically mentioned above. In another embodiment, the capsid of the AAV vector comprises one or more variant VP capsid proteins, such as those described in WO2015013313, specifically the RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6 capsid variants. In one specific embodiment, the capsid of the AAV vector is a hybrid of AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins. For example, the AAV serotype can 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; the rh74-9 serotype also referred to herein as "-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; the -9-rh74 serotype also referred to herein as "-9-rh74," "AAV9-rh74," "AAV-9-rh74," or "rh74-AAV9"). In a specific embodiment, the capsid of the AAV vector is a peptide-modified hybrid of AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins, such as the P1 peptide-modified AAV9-rh74 hybrid capsid or AAVrh74-9 hybrid capsid, as described in PCT / EP2019 / 076958.

[0053] In another embodiment, modified capsids can result from capsid modifications inserted by error-prone PCR and / or peptide insertion (e.g., as described in Bartel et al., 2011). Additionally, capsid variants can contain single amino acid alterations, such as tyrosine mutations (e.g., as described in Zhong et al., 2008).

[0054] In a specific embodiment, the AAV vector has a naturally occurring capsid, such as an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-cy10, or AAVrhlO capsid. In a specific embodiment, the recombinant AAV vector has an AAV8 capsid.

[0055] The genome of an AAV vector optionally comprises 5'- and 3'-AAV inverted terminal repeats (ITRs) flanking 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 AAVrhlO genome. In a specific embodiment, the genome of an AAV vector comprises 5'- and 3'-AAV2 ITRs.

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

[0057] Any combination of AAV serotype capsids and ITRs is feasible in the context of the present invention, in that an AAV vector may comprise a capsid and ITRs from the same AAV serotype, or may comprise a capsid from a first serotype and ITRs from a serotype different from the first serotype. Such vectors with capsid ITRs from different serotypes are also called "pseudotyped vectors."

[0058] Plants belonging to the Brassicaceae family As used herein, the phrase "a plant belonging to the Brassicaceae family" has its ordinary meaning in the art. The phrase includes any plant of the Brassicaceae family, also known as the Cruciferae, Mustardaceae, or Cabbage family.

[0059] According to the Royal Botanic Gardens, Kew, the Brassicaceae family includes over 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 a preferred embodiment, the plant belonging to the Brassicaceae family is selected from the group consisting of Brassica rapa, Raphanus sativus, Raphanus sativus var. niger, Brassica oleracea L. var. group, Brassica napus, and Arabidopsis thaliana.

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

[0063] Even more preferably, said plant belonging to the Brassicaceae family is Brassica rapa.

[0064] a) inducing the production of hairy roots from said plant Any technique that can induce the production of hairy roots in plants can be used in the present invention.

[0065] Hairy roots are a type of proliferative root that emerges at plant wound sites following 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 lack of gravitropism.

[0066] It should be noted that Agrobacterium rhizogenes is named Rhizobium rhizogenes following taxonomic changes to the genus Agrobacterium and the family Rhizobiaceae. Rhizobium rhizogenes can also be identified with the name Agrobacterium rhizogenes.

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

[0068] In one particular embodiment, hairy root production is induced by transforming a plant with a bacterial strain containing the rol gene cluster, which bacterial strain is capable of infecting the plant.

[0069] As used herein, the phrase "rol gene cluster" has its common meaning in the art. This phrase refers to a group of bacterial genes that can induce the production of hairy roots (Schmulling et al., 1988; Bulgakov et al., 2008). Typically, the rol gene cluster is carried by a plasmid, such as the pRi plasmid.

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

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

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

[0073] Several strains of Rhizobium rhizogenes can be used in the practice of the present invention. Suitable strains include, but are not limited to, Rhizobium rhizogenes strain TR7, also known as ATCC 25818, as well as strains 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.

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

[0075] Even more preferably, the Rhizobium rhizogenes strain is the ATCC 15834 strain.

[0076] In another embodiment, a strain of Agrobacterium tumefaciens is used. In a specific embodiment, the Agrobacterium tumefaciens strain used has been modified to include the rol gene cluster in its genome. In a specific embodiment, the Agrobacterium tumefaciens has been modified by transformation with a pRi plasmid containing the rol gene cluster.

[0077] In another embodiment, the Agrobacterium tumefaciens strain used does not contain the rol gene cluster. In this embodiment, transformation of a plant with Agrobacterium tumefaciens induces the formation of callus tissue. The callus tissue is then differentiated into hairy roots after addition of one or more hormonal substances. In a preferred embodiment, the hormonal substance is a hormone of the auxin family, 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 the present 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 rhizogenes and / or Agrobacterium tumefaciens is a technique known 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 can include, for example, but are not limited to, seeds, stems, leaves, stalks, cotyledonary nodes, hypocotyls, or other plant parts or cells.

[0080] Typically, infection with Rhizobium rhizogenes and / or Agrobacterium tumefaciens is carried out by applying a Rhizobium rhizogenes and / or Agrobacterium tumefaciens inoculum to pre-wounded plants.

[0081] Preferably, a semi-solid medium or liquid nutrient solution optimized for maintaining hairy roots is used, 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 medium amendments can be used that are optimized to meet the nutrient requirements of the host plants used in the production of sustainable hairy root cultures.

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

[0083] In another particular embodiment, step a) of inducing the production of hairy roots is carried out 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 a recombinant viral vector. In this embodiment, hairy roots can be obtained by transforming a transgenic plant expressing genes encoding protein components required for the production of a recombinant viral vector with a bacterial strain containing the rol gene cluster and capable of infecting plants as defined above.

[0084] In a preferred embodiment, steps a) and b) are carried out simultaneously.

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

[0086] Thus, in this embodiment, a method for producing a recombinant mammalian viral vector from a plant hairy root comprises transforming said plant with a bacterial strain such as Rhizobium rhizogenes or Agrobacterium tumefaciens; The bacterial strain comprises in its genome a rol gene cluster and one or more expression cassettes; the one or more expression cassettes contain genes encoding protein components required for the production of the recombinant viral vector; The plant belongs to the Brassicaceae family.

[0087] In another specific embodiment, two bacterial strains are used simultaneously, one containing the rol gene cluster and the other containing one or more expression cassettes encoding proteins required for the production of a recombinant viral vector.

[0088] In another specific embodiment, two or more bacterial strains are used simultaneously, the two or more bacterial strains containing different expression cassettes encoding protein components required for the production of a recombinant viral vector, and at least one bacterial strain containing the rol gene cluster.

[0089] In one specific embodiment, three or more bacterial strains are used simultaneously, one bacterial strain containing the rol gene cluster and at least two or more bacterial strains containing different expression cassettes encoding protein components required for the production of a recombinant viral vector.

[0090] b) transforming the plant with at least one vector containing one or more expression cassettes; As described above, the method of the present invention includes step b) of transforming a plant with at least one vector containing one or more expression cassettes, the one or more expression cassettes including genes encoding protein components required for the production of a recombinant viral vector.

[0091] In a particular embodiment, the plant is further transformed with a vector encoding viral helper functions necessary for efficient viral replication, e.g., a vector encoding adenoviral helper functions when the recombinant viral vector is an rAAV vector.

[0092] In a particular embodiment, the plant is further transformed with a vector comprising a viral genome comprising a gene encoding a product of interest, hi a further particular embodiment, the vector comprises a gene encoding a product of interest flanked by two AAV-ITR sequences.

[0093] Any technology that allows the transformation of plants, particularly plants belonging to the Brassicaceae family, may be used. In particular, any physical method may be used, such as gene gun or biolistic systems, electroporation, microinjection, or ultrasound-mediated transformation. Chemical methods may also be used, such as liposome-mediated transformation, silicon carbide fiber-mediated transformation, PEG-mediated transformation, calcium phosphate co-precipitation, polycation DMSO technology, DEAE-dextran precipitation, etc. PEI-mediated transformation may also be used.

[0094] In a preferred embodiment, transformation is performed using a bacterial strain, such as Rhizobium rhizogenes or Agrobacterium tumefaciens, which naturally transfers DNA (T-DNA) located on a tumor-inducing (Ti) plasmid into the nucleus of plant cells, stably integrating the DNA into the plant genome. In a specific embodiment, the Rhizobium rhizogenes or Agrobacterium tumefaciens contains a binary vector in its T-DNA region, comprising one or more expression cassettes, as defined below. The Rhizobium rhizogenes or Agrobacterium tumefaciens further contains a helper plasmid containing vir genes originating from the Ti plasmid of Agrobacterium. These genes encode a series of proteins that cleave the binary plasmid at the left and right border sequences and facilitate the transduction of the T-DNA into host plant cells.

[0095] In a preferred embodiment, a bacterial strain such as Rhizobium rhizogenes or Agrobacterium tumefaciens containing one or more expression cassettes encoding protein components required for the production of a recombinant viral vector further contains the rol gene cluster required for the production of hairy roots.

[0096] In one particular embodiment, two or more bacterial strains are used, the two or more bacterial strains containing different expression cassettes encoding the protein components required for the production of a recombinant viral vector.

[0097] As used herein, the term "expression cassette" has its general meaning in the art. The term refers to the combination of elements required for the expression of one or more genes.

[0098] In one aspect, 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 can be a binary vector suitable for expression in plant cells, such as pRD400, pBIN19, pBINPlus, or pCAMBIA binary vector, modified to include one or more expression cassettes of the invention. Other examples of binary vectors are listed in Table 2 of Bahramnejad et al., 2019. In one particular embodiment, the binary vector is pRD400.

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

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

[0102] In a specific embodiment, the genes encoding the protein components required for the production of the recombinant viral vector are under the control of constitutive promoters suitable for expression in plant cells, including, but not limited to, Cauliflower mosaic virus 35S (CaMV35S) (Odell et al., 1985), Cassava vein mosaic virus (CVMV) (Verdaguer et al., 1996), C1 of cotton leaf curl Multan virus (CLCuMV) (Xie et al., 2003), component 8 of milk vetch dwarf virus (Shirasawa-Seo et al., 2005), Australian banana streak virus (BSV) (Schenk et al., 2001), Mirabilis mosaic virus (MMV) (Dey and Maiti, 1999), Scrophulariaceae mosaic virus (FMV) (Sanger et al., 1990), and maize polyubiquitin-1. (Christensen et al., 1992), rice actin (McElroy et al., 1990), actin from Arabidopsis thalania (An et al., 1996), nopaline synthase (nos) from Agrobacterium (An et al., 1988), rolD from Agrobacterium (Fei et al., 2003), or functional variants of any of these.

[0103] In another specific embodiment, the genes encoding the protein components required for the production of the recombinant viral vector are under the control of inducible promoters suitable for expression in plant cells, including, but not limited to, pristinamycin-responsive promoters (Frey et al., 2001), In2-2 promoter from maize (De Veylder et al., 1997), wun1 promoter from potato (Siebertz et al., 1989), mannopine synthase promoter from Agrobacterium (Langridge et al., 1989), heat shock promoter Gmshp17.3 promoter from soybean (Schoffl et al., 1989), alcohol dehydrogenase promoter (Felenbok et al., 1988), or functional variants of any of these promoters.

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

[0105] In one particular embodiment, the genes encoding the protein components required for the production of the recombinant viral vector are under the control of a promoter derived from a virus that infects plants of the Brassicaceae family, such as the Cauliflower Mosaic Virus 35S (CaMV35S) promoter or a functional variant thereof.

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

[0107] "Functional variant thereof" refers to any variant that retains the function of the promoter from which it is derived, i.e., is capable of initiating transcription of a particular gene. In particular, a functional variant may have at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least 100% of the transcription-inducing activity of the wild-type promoter. The transcription-inducing activity of a functional variant may be more than 100%, e.g., more than 110%, 120%, 130%, 140%, or even more than 150% of the activity of the wild-type promoter.

[0108] In a particular embodiment, the promoter is the CaMV35S promoter, for example, CaMV35S of sequence 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 transcription-inducing activity compared to the wild-type CAMV25S promoter of SEQ ID NO: 9. The transcription-inducing activity of the functional variant may be more than 100%, for example more than 110%, 120%, 130%, 140%, or even more than 150% of the activity of the wild-type CAMV25S promoter of SEQ ID NO: 9.

[0109] In a particular embodiment, the promoter is a nopaline synthase (nos) promoter, such as the nos promoter of sequence 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 transcription-inducing activity of the wild-type nos promoter of SEQ ID NO: 11. The transcription-inducing activity of the functional variant may be greater than 100%, for example, greater than 110%, 120%, 130%, 140%, or even greater than 150% of the activity of the wild-type nos promoter of SEQ ID NO: 11.

[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 transcription-inducing activity compared to the wild-type alcohol dehydrogenase promoter of SEQ ID NO: 14. The transcription-inducing activity of the functional variant may be greater than 100%, for example, greater than 110%, 120%, 130%, 140%, or even greater than 150% of the activity of the wild-type alcohol dehydrogenase promoter of SEQ ID NO: 14.

[0111] In a specific embodiment, the expression cassette contains a terminator sequence downstream of the gene encoding a protein component required for producing a recombinant viral vector. The term "terminator sequence" refers to a sequence containing a 3'UTR sequence and a polyadenylation signal for terminating transcription. Any terminator sequence capable of terminating transcription in plant cells may be used.

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

[0113] The genes encoding the protein components required for the production of recombinant viral vectors are well known to those skilled in the art, who will be able to adapt the expression cassettes used in the experimental section to the particular viral vector they wish to produce.

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

[0115] Genes encoding protein components required for production of a recombinant viral vector include a start codon, for example, the start codon can be an ATG trinucleotide or an alternative start codon, such as ACG, CTG, GTG, and TTG start codons.

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

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

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

[0119] In a particular embodiment, the one or more expression cassettes of the invention comprise genes encoding the Rep52 protein and / or Rep78, which correspond to proteins required for viral replication.

[0120] In a specific embodiment, the AAV Cap and Rep genes are contained in the same expression cassette. In a specific embodiment, a first expression cassette contains the AAV Cap gene, and a 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 promoter sequence suitable for expression in plant cells, as described above. In a specific 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 specific embodiment, the AAV Cap gene and / or the AAV Rep gene are under the control of an inducible promoter suitable for expression in plant cells.

[0121] In one specific 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 which a first expression cassette comprises the AAV Cap gene under the control of the CaMV35S promoter, and a second expression cassette comprises the AAV Rep gene under the control of the CaMV35S promoter.

[0122] In a specific embodiment, the first and second expression cassettes containing the AAV Cap gene and AAV Rep gene, respectively, further comprise a terminator sequence as described above. Any terminator sequence capable of effecting transcription termination in plant cells may be used. In a specific embodiment, the first expression cassette containing the AAV Cap gene comprises a CaMV35S terminator sequence. In a specific embodiment, the second expression cassette containing the AAV Rep gene comprises a nopaline synthase (nos) terminator sequence.

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

[0124] In one specific embodiment, the second expression cassette comprises a CaMV35S promoter, an AAV Rep gene, and a nos terminator sequence.

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

[0126] In a specific embodiment, the first expression cassette and the second expression cassette are cloned into one or two expression vectors. In a preferred embodiment, the first expression cassette and the second expression cassette are cloned into a single expression vector. In a specific embodiment, the first expression cassette and the second expression cassette are cloned into a binary vector, for example, a pRD400 binary vector.

[0127] The 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 an AAV vector may be derived from a naturally occurring or non-naturally occurring serotype. In a specific embodiment, the serotype of the capsid of an AAV vector is selected from natural AAV serotypes. As an alternative to using natural AAV serotypes, artificial AAV serotypes, including but not limited to AAVs with non-naturally occurring capsid proteins, may be used in the context of the present invention. Such artificial capsids can be generated by any suitable technique using selected AAV sequences (e.g., fragments of the vp1 capsid protein) in combination with heterologous sequences, which may be from a selected different AAV serotype, a non-contiguous portion of the same AAV serotype, a non-AAV viral 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 a specific embodiment, the Cap gene, or the gene encoding VP1, VP2, or VP3, is selected from the group consisting of AAV-1, -2, AAV-2 variants (e.g., AAV-2 optimized with a quadruple mutant capsid, including an engineered capsid with Y44+500+730F+T491V changes, as disclosed in Ling et al., 2016), -3 and AAV-3 variants (e.g., the AAV3-ST variant, including an engineered AAV3 capsid with two amino acid changes S663V+T492V, as disclosed in Vercauteren et al., 2016), -3B and AAV-3B variants. , -4, -5, -6, and AAV-6 variants (e.g., AAV6 variants containing 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, -rh39, -rh43, -rh74, -dj, Anc80, LK03, AAV.PHP, AAV2i8, AAVpo4, and AAVpo6, such as AAV.PHP, AAV2i8, AAVpo4, and AAVpo6, and capsids of tyrosine, lysine, and serine capsid variants of porcine AAVs, as well as AAV serotypes. Additionally, the Cap gene may encode capsids of other engineered, non-natural variants (e.g., AAV-spark100), chimeric AAVs, or AAV serotypes obtained by shuffling, rational design, error-prone PCR, and machine learning techniques. In a specific embodiment, the Cap gene encodes VP capsid proteins from at least two different AAV serotypes, or encodes at least one chimeric VP protein that combines VP protein regions or domains 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 other than the AAV8 serotype, such as any of those mentioned in detail above.In another embodiment, the AAV vector capsid comprises one or more variant VP capsid proteins, such as those described in WO2015013313, particularly the 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 can 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; the rh74-9 serotype also referred to herein as "-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; the -9-rh74 serotype also referred to herein as "-9-rh74," "AAV9-rh74," "AAV-9-rh74," or "rh74-AAV9"). In a specific embodiment, the capsid of the AAV vector is a peptide-modified hybrid of AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins, such as the P1 peptide-modified AAV9-rh74 hybrid capsid or AAVrh74-9 hybrid capsid, as described in PCT / EP2019 / 076958.

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

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

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

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

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

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

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

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

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

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

[0140] In a specific embodiment, the gene encoding the AAP protein contained in the expression cassette encodes a functional AAP protein, and the gene has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100% identity to the nucleotide sequence of SEQ ID NO:6.

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

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

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

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

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

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

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

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

[0149] In a 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 contained in an expression cassette comprising a promoter operably linked to the gene encoding VP1, the gene encoding VP2 is contained in an expression cassette comprising a promoter operably linked to the gene encoding VP2, the gene encoding VP3 is contained in an expression cassette comprising a promoter operably linked to the gene encoding VP3, the gene encoding AAP is contained in an expression cassette comprising a promoter operably linked to the gene encoding AAP, the gene encoding Rep52 is contained in an expression cassette comprising a promoter operably linked to the gene encoding Rep52, and the gene encoding Rep78 is contained in an expression cassette comprising a promoter operably linked to the gene encoding Rep78.

[0151] In a specific 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 specific embodiment, all cassettes are cloned into a binary vector, such as the pRD400 binary vector.

[0152] In a particular embodiment, the gene encoding the VP1, VP2, VP3, AAP, Rep52, or Rep78 protein is under the control of a constitutive promoter suitable for expression in plant cells. A non-exhaustive list of constitutive promoters suitable for expression in plant cells includes Cauliflower mosaic virus 35S (CaMV35S) (Odell et al., 1985), Cassava vein mosaic virus (CVMV) (Verdaguer et al., 1996), C1 of cotton leaf curl Multan virus (CLCuMV) (Xie et al., 2003), component 8 of milk vetch dwarf virus (Shirasawa-Seo et al., 2005), Australian banana streak virus (BSV) (Schenk et al., 2001), Mirabilis mosaic virus (MMV) (Dey and Maiti, 1999), Scrophulariaceae mosaic virus (FMV) (Sanger et al., 1990), and maize polyubiquitin-1. (Christensen et al., 1992), rice actin (McElroy et al., 1990), actin from Arabidopsis thalania (An et al., 1996), nopaline synthase (nos) from Agrobacterium (An et al., 1988), rolD from Agrobacterium (Fei et al., 2003), or functional variants of any of these.

[0153] In another specific embodiment, the gene encoding the VP1, VP2, VP3, AAP, Rep52, or Rep78 protein is under the control of an inducible promoter suitable for expression in plant cells, including, but not limited to, pristinamycin-responsive promoters (Frey et al., 2001), In2-2 promoter from maize (De Veylder et al., 1997), wun1 promoter from potato (Siebertz et al., 1989), mannopine synthase promoter from Agrobacterium (Langridge et al., 1989), heat shock promoter Gmshp17.3 promoter from soybean (Schoffl et al., 1989), alcohol dehydrogenase promoter (Felenbok et al., 1988), or a functional variant of any of these promoters.

[0154] By "functional variant thereof" is meant any variant that retains the function of the promoter from which it was derived, i.e. is capable of initiating transcription of a particular gene.

[0155] In a specific embodiment, the gene encoding the VP1, VP2, VP3, AAP, Rep52, or Rep78 protein 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. In a specific embodiment, the promoter is a functional variant of the CaMV35S promoter that has enhanced transcriptional activity compared to the wild-type CaMV35S promoter. Specifically, the CaMV35S functional variant comprises a duplication of the -343 to -90 bp fragment as described by Kay et al., 1987. In a specific embodiment, the CaMV35S functional variant 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 nopaline synthase (nos) promoter or a functional variant thereof; - the gene encoding VP2 is under the control of a weak promoter, such as the nopaline synthase (nos) promoter or a functional variant thereof; - 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 having at least 80%, 85%, 90%, 95%, 99% or 100% identity to the nucleotide sequence of SEQ ID NO: 13, and / or - the gene encoding the 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 one particular embodiment, at least one vector according to the invention comprises: - an expression cassette comprising a nos promoter or a functional variant thereof, a gene encoding VP1, and a terminator sequence such as the 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 the 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, a gene encoding an AAP, and a terminator sequence, such as the CamV35S terminator sequence. Includes.

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

[0159] In one particular embodiment, at least one vector according to the invention comprises: - an expression cassette comprising an alcohol dehydrogenase promoter or a functional variant thereof, a gene encoding VP1, and a terminator sequence such as the 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 the 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 - an expression cassette comprising an alcohol dehydrogenase promoter, a gene encoding an AAP, and a terminator sequence, such as the CamV35S terminator sequence; Includes.

[0160] In one 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 a 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 alcohol dehydrogenase promoter 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. In a specific embodiment, the gene encoding the ALCR protein encodes a functional ALCR protein, which has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 15. In a specific embodiment, the gene encoding the ALCR protein contained in the expression cassette encodes a functional ALCR protein that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.8%, or 100% identity to the amino acid sequence of SEQ ID NO: 23.

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

[0164] In one particular embodiment, at least one vector according to the invention comprises: - 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 the 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 invention further comprises an expression cassette encoding an ALCR protein required for activation of the alcohol dehydrogenase promoter.

[0167] Recovery of recombinant viral vectors In one embodiment, the recombinant viral vector is recovered by harvesting the medium in which the hairy roots are cultured.

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

[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 any purification steps.

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

[0171] In an alternative embodiment, the recombinant viral vector is obtained after one or several purification steps. Appropriate protocols adapted to each recombinant viral vector are standard techniques in the art, and the person skilled in the art can easily select the appropriate purification step, if any, for the desired application.

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

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

[0174] In particular, the step of inducing the appearance of lateral roots in the hairy roots of the transformed plant 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] The auxin may 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) inducing the production of hairy roots from a plant belonging to the Brassicaceae family according to any of the embodiments described above; and b) transforming said plant with at least one vector comprising one or more expression cassettes according to any of the above embodiments, wherein the one or more expression cassettes comprise genes encoding protein components required for the production of a recombinant viral vector. The present invention relates to hairy root cultures obtainable or obtained by

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

[0179] Another aspect of the present invention relates to a recombinant viral vector obtainable or obtained by a method as described above. [Example]

[0180] Materials and Methods A. Recombinant binary plasmids for AAV protein expression The sequence used was extracted from the plasmid p5e18_VD2_8_kanR (RepCap_cellules_293, provided by GENETHON). The coding sequence was manually optimized based on codon usage in the human (source organism) and Brassica rapa (expression host organism) species.

[0181] Five constructs containing different expression cassettes were designed, and a schematic diagram of the five constructs is shown in Figure 1.

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

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

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

[0185] Construct 4 was similar to construct 3 but added the tobacco mosaic virus omega (TMVΩ) enhancer to enhance Rep52 expression.

[0186] In construct 5, an ethanol-inducible system was used to drive expression of VP1, VP2, VP3, and AAP. The tobacco mosaic virus omega (TMVΩ) enhancer was added to increase VP3 expression. Due to the lack of a specific anti-AAP antibody, the AAP protein was tagged with an HA tag to enable its detection.

[0187] Each of these five constructs was then separately cloned 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 rhizogenes strain ATCC 15834.

[0189] R. rhizogenes strain ATCC 15834 was transformed by electroporation with the AAV encoding 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 min in 5% bleach supplemented with a few drops of Triton X100, washed at least five times with sterile water, and then plated onto Petri dishes containing B5 agar. Germination and seedling growth occurred at 20°C under a 16-h light / 8-h dark photoperiod.

[0191] 2. Infection of Plants with Rhizobium rhizogenes We used Rhizobium rhizogenes strain 15834 (ATCC 15834), provided by the Institut Pasteur. Rhizobium rhizogenes was grown on MGL plates (2.5 g / L yeast extract, 5 g / L tryptone, 5 g / L mannitol, 5 g / L NaCl, 1.16 g / L Na-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 to select for binary plasmids. Inoculum was prepared from a 20 ml liquid bacterial culture grown overnight at 25°C in MGL medium. The suspension was centrifuged at 15,000 rpm for 5 min, and the collected cells were resuspended in fresh MGL medium and diluted to obtain an optical density of 1 ± 0.1 at 600 nm.

[0192] Plants were infected by pricking the hypocotyls of 3- to 10-day-old seedlings with a needle and applying Rhizobium inoculum to the wound area with a sterile cotton swab. Hairy roots usually emerged from the wound site approximately 2 weeks after infection.

[0193] 3. Selection and cultivation of hairy root clones expressing AAV proteins Infected hypocotyls developing hairy roots were excised from seedlings and placed on B5 (Duchefa) pH 5.8 supplemented with 3% sucrose and cefotaxime (300 mg / L), 8 g / L agar. After 7 days, independent hairy root tips were transferred onto fresh B5-3% sucrose + cefotaxime (300 mg / L), 8 g / L agar and grown there for 4–10 days.

[0194] To initiate liquid culture, each hairy root clone was then transferred to 6 ml of B5 containing 3% sucrose for 10 days. After this, the culture medium was changed to additionally contain 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. Aliquots of hairy root extract were sampled, mixed with 1 / 3 volume of 3X Laemmli buffer, and boiled for 5 minutes. 40 μl of each sample was loaded onto an AnykD mini protean TGX polyacrylamide gel (Bio-Rad). For Western blot analysis, proteins were transferred to nitrocellulose membranes using the Bio-Rad Turbo Trans-Blot system (Bio-Rad, Hercules, CA). The membrane was blocked in 5% nonfat milk (blotting-grade blocker, Bio-Rad) in TBS buffer and incubated with a 1:250 dilution of anti-AAV VP1 / VP2 / VP3 mouse monoclonal antibody B1 (Progen 61058), a 1:100 dilution of anti-AAV2 replicase mouse monoclonal antibody 259,5 (Progen 61071), or a 1:1000 dilution of anti-HA tag mouse monoclonal antibody (Abcam ab18181), followed by incubation with a 1:5000 dilution of m-IgGκBP-HRP antibody (Santa Cruz Biotechnology sc-516102). Post-development of the staining was performed using a Western Clarity ECL development kit (170-5060, Bio-Rad).

[0196] Quantification of AAV particles in hairy roots derived from D. brassicae rapa 1. rAAV2 / 8 Vector Produced in Hairy Roots from Brassica rapa Brassica rapa hairy root clones were generated as previously described. Assembled AAV particles were detected in protein extracts from clone C1-38 (containing construct #1 as described above) and clones C2-8 and C2-53 (containing construct #2 as described above). Wild-type clones (i.e., no transgene) were included as negative controls.

[0197] Clones were maintained for 20 days. Briefly, 1 g of fresh hairy roots was inoculated into 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 incubation, 100 mg of fresh biomass was harvested, frozen in liquid nitrogen, and stored at -80°C for subsequent protein extraction.

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

[0199] [Table 1]

[0200] The concentration of assembled viral particles (capsids / mL) was then determined using the Progen AAV8 Capsid ELISA Kit described below.

[0201] 2. rAAV8-eGFP Control Vector Preparation The rAAV8-eGFP vector produced from mammalian cells was used as a positive control in each experiment.

[0202] The control vector was prepared as follows. Briefly, HEK293T cells adapted to grow in suspension culture were seeded in 400 mL of F17 chemically defined culture medium in shaker flasks (1 L). Cells were transfected with three plasmids using PTG1+ transfection agent. 72 hours after transfection, cells were harvested and lysed by sonication. AAV8 particles in the culture supernatant were precipitated with polyethylene glycol (PEG), then purified by double CsCl density gradient ultracentrifugation, and finally formulated in 1x DPBS containing Ca2+ and Mg2+ by dialysis into Slide-A-Lyzer™ 10K MWCO cassettes (Thermo Scientific, Illkirch, France).

[0203] Viral genome concentration / mL (VG / mL) was determined from DNase-resistant particles by TaqMan real-time PCR assay. Viral particle concentration (capsids / mL) was determined using the Progen AAV8 Capsid ELISA Kit described below. (5~10 7 Five microliters of control vector (corresponding to capsids) 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 (B, C, or D) described above.

[0204] 3. rAAV8-eGFP Denatured Vector Preparation The control vector was heat denatured at 90°C for 15 minutes to serve as a negative control, ensuring that only assembled capsids would give a signal in the ELISA.

[0205] (5×10 7 Five microliters of denatured vector (corresponding to 10 capsids) 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., no transgene).

[0206] 4. AAV8 Capsid Titration by ELISA ELISA titration of AAV8 assembled capsids was performed 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 could produce all the proteins required for AAV production (REP, CAP, and AAP proteins).

[0208] To achieve this goal, molecular constructs as described above were designed, cloned, and introduced into Brassica rapa using Rhizobium rhizogenes. Transgenic Brassica rapa hairy roots were generated and analyzed. Results are shown below for Construct 1 and Construct 2.

[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 ability of hairy roots to produce Rep proteins.

[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 ability of Brassica rapa to express VP1, VP2, VP3, and AAP (derived from AAV8) using different promoters: 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 ability of hairy roots to produce AAP-HA protein.

[0215] 3. Quantification of AAV Particles in Hairy Roots from Brassica rapa Having thus demonstrated the ability of hairy roots to produce AAV Rep, VP1, VP2, VP3, and AAP proteins, we next aimed to determine whether hairy roots from Brassica rapa could produce assembled capsids.

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

[0217] A wild-type clone (i.e., no transgene) was included as a negative control.

[0218] The results detailed below in Table 2 demonstrate that hairy roots from Brassica rapa are capable of producing assembled capsids.

[0219] [Table 2]

[0220] It can be noted that the titers obtained with the control rAAV8-eGFP vector spiked into (1) extraction buffer or (2) protein extracts from wild-type hairy root clones were nearly identical, thus validating the titration method.

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

[0222] The titers obtained in the protein extracts of hairy root clones are 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 showed the detection of AAV assembled capsids according to three different extraction methods (B, C, D), thus demonstrating the reproducibility of the method.

[0224] [References] TIFF0007798767000003.tif193170TIFF0007798767000004.tif224170TIFF0007798767000005.tif224170TIFF0007798767000006.tif90170

Claims

1. 1. A method for producing a recombinant adeno-associated virus (AAV) vector from a plant hairy root, comprising: a) inducing the production of hairy roots from said plant; b) transforming the plant with at least one vector containing one or more expression cassettes, the one or more expression cassettes including genes encoding protein components required for the production of a recombinant AAV vector; Including, The plant belongs to the Brassicaceae family, the one or more expression cassettes comprise the AAV rep and cap genes; method.

2. 2. The method of claim 1, wherein the plant belonging to the Brassicaceae family is selected from the group consisting of Raphanus sativus, Raphanus sativus var. niger, Brassica oleracea L. convar, Brassica napus, Arabidopsis thaliana, and Brassica rapa.

3. 3. The method according to claim 1 or 2, wherein the plant belonging to the Brassicaceae family is Brassica rapa.

4. 4. The method according to claim 1, wherein step a) is carried out by transforming a plant with a bacterial strain containing the rol gene cluster, said bacterial strain being capable of infecting plants.

5. 5. The method of claim 4, wherein the bacterial strain is Rhizobium rhizogenes or Agrobacterium tumefaciens.

6. 6. The method of claim 1, wherein the AAV rep and cap genes are each under the control of a promoter derived from a virus that infects a Brassicaceae plant.

7. The method of claim 6, wherein the promoter is a cauliflower mosaic virus 35S (CaMV35S) promoter.

8. 8. The method of claim 1, wherein the one or more expression cassettes comprise genes encoding VP1, VP2, VP3, AAP (assembly activating protein), Rep52, and Rep78 proteins.

9. 9. The method of claim 8, wherein each gene encoding the VP1, VP2, VP3, AAP, Rep52, or Rep78 protein is under the control of a constitutive promoter or under the control of an inducible promoter.

10. 10. The method of claim 9, wherein the constitutive promoter is a cauliflower mosaic virus 35S (CaMV35S) promoter or a nopaline synthase (nos) promoter.

11. 10. The method of claim 9, wherein the inducible promoter is an alcohol dehydrogenase (AlcA) promoter.

12. 9. The method of claim 8, wherein 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 having at least 90% identity to the nucleotide sequence of SEQ ID NO: 13, and the gene encoding AAP is under the control of the CaMV35S promoter or a functional variant thereof having at least 90% identity to the nucleotide sequence of SEQ ID NO:

13.

13. The method of claim 12, wherein the gene encoding VP3 is under the control of a functional variant of the CaMV35S promoter having at least 90% 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 90% identity to the nucleotide sequence of SEQ ID NO:

13.

14. The method of claim 8, wherein each of the genes encoding VP1, VP2, VP3, and AAP is under the control of an AlcA promoter.

15. 15. The method of claim 8, wherein the gene encoding VP3 is further under the control of an enhancer.

16. 16. The method of claim 15, wherein the enhancer is a tobacco mosaic virus omega (TMVΩ) enhancer.

17. 17. The method according to any one of claims 8 to 16, wherein the genes encoding Rep52 and Rep78 are under the control of an AlcA promoter.

18. 18. The method of claim 8, wherein the gene encoding Rep52 is further under the control of an enhancer.

19. 19. The method of claim 18, wherein the enhancer is a tobacco mosaic virus omega (TMVΩ) enhancer.

20. 20. The method of any one of claims 8 to 19, wherein the genes encoding the VP1, VP2, VP3, AAP, Rep52, and / or Rep78 proteins are codon-optimized.

21. 21. The method of any one of claims 1 to 20, wherein the plant is further transformed with a vector encoding viral helper functions required for efficient replication of the recombinant AAV vector.

22. 22. The method of claim 21, wherein the plant is transformed with a vector encoding adenoviral helper functions.

23. 23. The method of any one of claims 1 to 22, wherein the plant is further transformed with a vector comprising an AAV genome containing a gene encoding a product of interest.

24. 24. The method of claim 23, wherein the plant is transformed with a vector containing a gene encoding a product of interest flanked by two AAV-ITR sequences.

25. a) inducing the production of hairy roots from a plant belonging to the Brassicaceae family; and b) transforming the plant with at least one vector containing one or more expression cassettes, the one or more expression cassettes including the AAV rep and cap genes encoding protein components required for the production of a recombinant adeno-associated virus (AAV) vector. Hairy root cultures obtainable by

26. 26. The hairy root culture of claim 25, wherein the plant belonging to the Brassicaceae family is selected from the group consisting of Raphanus sativus, Raphanus sativus var. niger, Brassica oleracea L. convar, Brassica napus, Arabidopsis thaliana, and Brassica rapa.

27. 27. The hairy root culture of claim 26, wherein the plant belonging to the Brassicaceae family is Brassica rapa.

28. 28. A hairy root culture according to any one of claims 25 to 27, wherein the one or more expression cassettes are as defined in any one of claims 1 to 20.

29. A transgenic plant transformed with at least one vector containing one or more expression cassettes, wherein the one or more expression cassettes include the AAV rep and cap genes encoding protein components required for the production of a recombinant adeno-associated virus (AAV) vector, and the plant belongs to the Brassicaceae family.

30. 30. The transgenic plant of claim 29, wherein the plant of the Brassicaceae family is selected from the group consisting of Raphanus sativus, Raphanus sativus var. niger, Brassica oleracea L. convar, Brassica napus, Arabidopsis thaliana, and Brassica rapa.

31. 31. The transgenic plant of claim 30, wherein the plant belonging to the Brassicaceae family is Brassica rapa.

32. 32. A transgenic plant according to any one of claims 29 to 31, wherein the one or more expression cassettes are as defined in any one of claims 1 to 20.

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