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WO2025151846A1 - Compositions et procédés d'expression de protéines recombinantes et d'arn dans des plastes de graines, de gousses et de racines - Google Patents

Compositions et procédés d'expression de protéines recombinantes et d'arn dans des plastes de graines, de gousses et de racines

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Publication number
WO2025151846A1
WO2025151846A1 PCT/US2025/011323 US2025011323W WO2025151846A1 WO 2025151846 A1 WO2025151846 A1 WO 2025151846A1 US 2025011323 W US2025011323 W US 2025011323W WO 2025151846 A1 WO2025151846 A1 WO 2025151846A1
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WO
WIPO (PCT)
Prior art keywords
promoter
plastid
plant
seq
seed
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Pending
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PCT/US2025/011323
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English (en)
Inventor
Pal Maliga
Malihe MIRZAEE
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Rutgers State University of New Jersey
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Rutgers State University of New Jersey
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Application filed by Rutgers State University of New Jersey filed Critical Rutgers State University of New Jersey
Publication of WO2025151846A1 publication Critical patent/WO2025151846A1/fr
Pending legal-status Critical Current
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8214—Plastid transformation

Definitions

  • Plastids are semi-autonomous plant organelles with thousands of copies of the ⁇ 155-kb genome localized in 10 to 100 plastids per cell.
  • the plastid genome of flowering plants encodes about one hundred genes, the products of which assemble with -3,000 nucleus-encoded proteins to form the plastid transcription and translation machinery and carry out complex metabolic functions, including photosynthesis, fatty acid and amino acid biosynthesis. Transformation of the plastid genome in flowering plants was first accomplished in tobacco (Nicotiana tabacum), the current model species of plastid engineering 1 2 .
  • the soybean is the most important source of vegetable protein for human and animal consumption as well as ingredients for many chemical products. 90% of US oilseed production is from soybeans (USDA).
  • the second component of the system includes: ii) a nuclear transformation vector comprising a nucleic acid construct encoding a plant codon optimized RNA polymerase operably linked to a seed specific promoter, and a plastid targeting sequence, expression of said construct transporting said RNA polymerase protein into seed plastids where it binds to the RNA polymerase promoter of step a), thereby activating transcription of sequence encoding said one or more heterologous proteins of interest.
  • the RNA polymerase promoter is selected from a T7, a T3 or a SP6 promoter and the recombinant protein or RNA of interest can be one or more of GFP, mScarlet, RFP, GUS, a vaccine, an antibody, a fusion protein, a protein conferring abiotic resistance, an enzyme, a fungicidal protein, an insecticidal protein and a RNA that confers insect resistance.
  • Suitable plastid transformation vectors for delivery of said heterologous proteins or RNAs of interest include TVV1 of SEQ ID NO: 3 and GPV1 of SEQ ID NO: 4, each comprising a multicloning site for insertion of a sequence encoding said heterologous protein of interest.
  • An exemplary method comprises introducing the two- component expression system described above into the target plant; selecting plant cells surviving selection pressure and expressing said at least one nucleic acid encoding said heterologous protein or RNA of interest; and c) regenerating a homoplastomic plant from the surviving plant cells.
  • the plant is selected from a soybean plant and said plastid transformation vector is GPV1 of SEQ ID NO: 4.
  • FIGS 1A - IB Two-component systems for seed-specific expression of recombinant proteins.
  • Fig. 1A In the leaf, the nuclear T7RNAP is not expressed because it’s seed specific promoter is inactive.
  • Fig. IB In the seed, the T7RNAP is transcribed from the seed specific promoter. The mRNA is exported to the cytoplasm where it is translated. The plastid-targeted T7RNAP enters the seed plastids where it transcribes the gfp coding sequence from the phage T7gl0 promoter and the mRNA is translated on plastid ribosomes.
  • the TVV vectors carry the spectinomycin resistance (aadA) marker gene fused with the c-Myc epitope.
  • VT7glO is the T7 phage gene 10 promoter; Histidine/Threonine attenuator and psbA 3' UTR His/Thre Atten and ⁇ psbA) are symbolized as stem-loops.
  • phrases "consisting essentially of' when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO.
  • the phrase when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the functional and novel characteristics of the sequence.
  • Homoplastomic refers to a pure population of plastid genomes, either within a plastid or within a population contained in plant cells and tissues. Homoplastomic plastids, cells or tissues are genetically stable because they contain only one type of plastid genome. Hence, they remain homoplastomic even after the selection pressure has been removed, and selfed progeny are also homoplastomic.
  • Transplastome refers to a transformed plastid genome.
  • the nopaline synthase (NOS) promoter the nopaline synthase (NOS) promoter
  • OCS octapine synthase
  • caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter, the CaMV 35S promoter, and the figwort mosaic virus 35S promoter
  • the light inducible promoter from the small subunit of rubisco the Adh promoter
  • the sucrose synthase promoter the R gene complex promoter
  • chlorophyll a/b binding protein gene promoter etc.
  • RNA transcript refers to a product resulting from RNA polymerase-catalyzed transcription of a DNA sequence.
  • a primary transcript When an RNA transcript is a perfect complementary copy of a DNA sequence, it is referred to as a primary transcript or it may be a RNA sequence derived from posttranscriptional processing of a primary transcript and is referred to as a mature RNA.
  • mRNA RNA that is without introns and that can be translated into protein by the cell.
  • cDNA refers to a DNA that is complementary to and synthesized from an mRNA template using the enzyme reverse transcriptase.
  • a reporter sequence encodes a detectable molecule.
  • Exemplary reporter sequences encoding reporters including GFP, RFP, mScarlet, GUS, etc, Applicability of the Technology
  • Seed protein expression in plastids would be useful in any crop where the seed, or plant part other than leaf, is consumed as food, serves as feed to livestock, or is the source of pharmaceutical proteins or industrial raw materials. Plastids are present in all plant cell types each of which have an identical genome. Different tissue types may include chloroplasts in leaves, proplastids in seed, chromoplasts in tomato and pumpkin fruit and carrot root, amyloplasts in potato tuber tissue.
  • Plants of interest include without limitation, Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, canola/rapeseed, etc.), Cucurbitaceae (melons and cucumber), Gramineae (wheat, barley, rice, maize, etc.), Solanaceae (potato, tobacco, tomato, peppers), various floral crops, such as sunflower, and nut-bearing trees, such as almonds, cashews, walnuts, and pecans.
  • Methods and compositions for transforming plants by introducing a transgenic DNA construct into a plant genome in the practice of this invention can include any of the well-known and demonstrated methods.
  • the expression cassette or vector is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of number of standard breeding techniques can be used, depending upon the species to be crossed.
  • Plant transformants containing a desired genetic modification as a result of any of the above-described methods resulting in decreased or increased expression of the seed protein of interest can be selected by various methods known in the art. These methods include, but are not limited to, methods such as SDS-PAGE analysis, immunoblotting using antibodies which bind to the seed protein of interest, single nucleotide polymorphism (SNP) analysis, or assaying for the products of a reporter or marker gene, and the like.
  • SNP single nucleotide polymorphism
  • Juncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye ⁇ Secale cereale), sorghum ⁇ Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet ⁇ Pennisetum glaucum), proso millet ⁇ Panicum miliaceum), foxtail millet ⁇ Setaria italica), finger millet ⁇ Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat ⁇ Triticum aestivum), tobacco ⁇ Nicotiana tabacum), potato ⁇ Solanum tuberosum), peanuts (Arachis hypogaea), cotton ⁇ Gossypium barbadense, Gossypium hirsutum), sweet potato ⁇ Ipomoea batatus), cassava ⁇ Mani
  • Vegetables include tomatoes ⁇ Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans ⁇ Phaseolus vulgaris), lima beans ⁇ Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • lettuce e.g., Lactuca sativa
  • green beans ⁇ Phaseolus vulgaris
  • peas Lathyrus spp.
  • members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • Ornamentals include azalea ⁇ Rhododendron spp ), hydrangea ⁇ Macrophylla hydrangea , hibiscus ⁇ Hibiscus rosasanensis), roses ⁇ Rosa spp.), tulips ⁇ Tulipa spp.), daffodils ⁇ Narcissus spp ), petunias ⁇ Petunia hybrida), carnation ⁇ Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.
  • Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine ⁇ Pinus taeda), slash pine ⁇ Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine ⁇ Pinus contorta), and Monterey pine ⁇ Pin us radiata),' Douglas-fir (Pseudotsuga menziesii),' Western hemlock ⁇ Tsuga canadensis),' Sitka spruce (Picea glaucdy, redwood ⁇ Sequoia sempervirensj, true firs such as silver fir ⁇ Abies amabilis) and balsam fir (Abies balsamed), and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).
  • pines such as loblolly pine ⁇ Pin
  • a "cell line” is a clone of a primary cell or cell population that is capable of stable growth in vitro for many generations.
  • Tobacco seeds (Nicotiana tabacum var. Petit Havana) were sown on Murashige and Skoog (MS) medium, 0.7% agar (7 g/L) and 3% sucrose (30 g/L). The plants were maintained by growing cuttings on the same medium. The plants were incubated in a growth chamber at 28 °C with 16 hour light/8 hour dark cycles. The regenerated shoots were rooted on the same medium and transplanted to soil to collect seed and for crossing.
  • Plastid transformation is carried out by the biolistic process. Bombardment of leaves, selection of transplastomic events on spectinomycin medium, and characterization of the tobacco plants is carried out as previously described 2, 16, 17
  • Transplastomic soybean plants are obtained by the bombardment of embryogenic cell cultures of cv Jack or Williams 82. Transplastomic soybean are selected by spectinomycin resistance according to the protocol published by Dubald et al. 18 . Plastid transformation vectors and sequences are listed in Table 1.
  • SEQ ID NO: 16 Cryl4Ab coding sequence in Ncol-Xbal fragment cccATGGATTGTAATTTACAATCACAACAAAATATTCCATATAATGTATTAGCAATAC
  • SEQ ID NO: 18 Plastid dual promoter (ptDP) dsRNA construct to target vATPase A in
  • Protein Extraction Total leaf and seed protein were extracted from plants grown in the soil using previously established protocols with minor modifications 39- 40 . Briefly, this protocol is described below. About 100 mg leaf or 50 mg seed were powdered in liquid nitrogen and resuspended in either: a) 400 pl leaf extraction buffer (100 mM HEPES (pH 7), 5 mM EDTA, 10 mM dithiothreitol (DTT), 2 mM phenylmethyl sulfonyl fluoride (PMSF), 10% glycerol, and protease inhibitor cocktail (15 pl/ml; Sigma-Aldrich)), or b) 800 pl seed extraction buffer (50 Mm Tris-HCl (pH 8), 500 mM NaCl, 5 mM EDTA, 5% glycerol, 10 mM dithiothreitol (DTT), 2 mM PMSF, and protease inhibitor cocktail (15 pl/ml; Sigma-Aldrich)).
  • the leaf and seed supernatants were collected after centrifugation at 14,000 g for 20 min at 4 °C.
  • concentration of leaf and seed TSP was determined using a BSA protein standard curve.
  • the membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween®' 20 detergent (TBS-T), for 90 min at room temperature.
  • GFP was detected by 90 min incubation of membranes at room temperature using 1: 1000 dilution of anti -GFP (Living color peptide, Clontech) antibody.
  • TBS-T Tris-buffered saline with 0.1% Tween®' 20 detergent
  • HRP horseradish peroxidase
  • IgG horseradish peroxidase
  • T7RNAP T7 phage RNA polymerase
  • T7gl0 T7 phage gene 10
  • the plastid genes have leader sequences with efficient ribosome binding sites, such as the T7gl0 51 or cry9Aa2 n ’ 52 leaders.
  • T7gl0 51 or cry9Aa2 n ’ 52 leaders A brief description of recombinant protein expression from plastid transgenes with two leader sequences, driven by a tissue specific T7RNAP, is provided below.
  • T7gl0 leader transcriptionally fused with the plastid rRNA operon PEP promoter, yielded high level of recombinant protein accumulation in tobacco and lettuce chloroplasts 60, 6 68 . Therefore, we used the T7 phage gene 10 leader for translation of recombinant proteins, using constructs like pMM7 ( Figure 2, SEQ ID NO: 1). The construct was introduced into the plastid genome by standard protocols.
  • the leader of Cry9Aa2 gene leader also promotes high level recombinant protein expression in chloroplasts and could be a suitable alternative to the T7gl0 leader 11, 52 . Therefore, we use the Cry9Aa2 gene leader for translation of recombinant proteins, using constructs like pMM33 ( Figure 2, SEQ ID NO: 2). The construct was introduced into the plastid genome by standard protocols.
  • T7 RNA polymerase promoter sequence is 5' TAATACGACTCACTATAG 3' and the transcription starts at the underlined G in the promoter sequence
  • a nucleotide SEQ ID NO: 2. The lOx lower protein levels in part are due to the suboptiraal T7 RNA polymerase promoter.
  • the marker gene is flanked by minimal attB (34 bp) lattP (39 bp) sequences, which are target sites for the PhiC31 phage-site-specific integrase for posttransformation excision of the marker gene. Between the multiple cloning site and attB site a tRNA (trnP) has been included to facilitate efficient processing in polycistronic mRNAs 54 .
  • trnP tRNA
  • marker genes may also be excised by alternative site-specific recombinases
  • Protein output in seed plastids driven by the T7 RNA polymerase depends on: (1) The strength of the plant promoter, driving transcription of the gene in the plant nucleus that produces mRNA from which the plastid targeted T7 RNA polymerase s translated in the cytoplasm. (2) The T7 promoter variant inside the plastid (chloroplast) driving the transcription of the plastid transgene and producing more or less of the mRNA which is translated on plastid ribosomes.
  • chloroplast chloroplast
  • Example 1 we have shown that in tobacco seed protein output could be obtained from variants of the T7 promoter in a 40-fold range, 4 mg/kg to 120 mg/kg fresh seed.
  • recombinant proteins may accumulate in seed plastids using a transcriptionally regulated, two-component expression system.
  • Seed-based bioreactors when the proteins are stable, enable long-term storage of the proteins of interest.
  • Good candidates for seedspecific expression in plastids would be bacterial vaccine antigens which do not require glycosylation, such as the anthrax protective antigen 58 , the porcine post-weaning diarrhea vaccine 59 and tuberculosis antigen 60, 61 .
  • the non-photosynthetic plastids in the seed are prospective hosts for new metabolic pathways 62 .
  • a ptDP dsRNA construct targeting vATPase that would be suitable to control P. guildinii adults is shown SEQ ID NO: 18.
  • the gene fragment could be cloned as EcoRI-Hindlll fragment in soybean plastid transformation vector GPV1 (SEQ ID NO: 4).
  • the publication by Schvartzman et al. 70 cites publications on several different sting bug species which are susceptible to control by RNAi. This includes the brown marmorated stink bug Halyomorpha halys 7 , the neotropical brown stink bug Euschitus heros 78 , southern green stink bug Nezara viridula 19 and the brown-winged green sting bug Plauytia stali 80 .
  • the armyworm complex is another group of major insect pests which can also be controlled by Bt insecticidal proteins. However, change in the climatic events favored the emergence of insect resistance 82 . Combination of different approaches is necessary to achieve efficient pest control. Tissue specific expression of plastid transgenes using the transcriptionally regulated plastid expression system with its adjustable output should make a major impact on advancing army worm pest control.

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Abstract

L'invention concerne des compositions et des procédés pour la production rapide et efficace de soja transgénique exprimant des protéines hétérologues ou des ARN d'intérêt dans des plastes de graines, de gousses et de racines.
PCT/US2025/011323 2024-01-11 2025-01-12 Compositions et procédés d'expression de protéines recombinantes et d'arn dans des plastes de graines, de gousses et de racines Pending WO2025151846A1 (fr)

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US63/620,037 2024-01-11

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WO2025151846A1 true WO2025151846A1 (fr) 2025-07-17

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108522290A (zh) * 2017-03-02 2018-09-14 云南纳博生物科技有限公司 一种自发光烟草及转基因方法
US20200017868A1 (en) * 2017-01-09 2020-01-16 Rutgers, The State University Of New Jersey Compositions and methods for improving plastid transformation efficiency in higher plants
US20210147862A1 (en) * 2019-10-23 2021-05-20 Pairwise Plants Services, Inc. Compositions and methods for rna-templated editing in plants
US20230203512A1 (en) * 2021-07-20 2023-06-29 Rutgers, The State University Of New Jersey Compositions and methods for agrobacterium mediated transformation of chloroplasts in seed plants

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200017868A1 (en) * 2017-01-09 2020-01-16 Rutgers, The State University Of New Jersey Compositions and methods for improving plastid transformation efficiency in higher plants
CN108522290A (zh) * 2017-03-02 2018-09-14 云南纳博生物科技有限公司 一种自发光烟草及转基因方法
US20210147862A1 (en) * 2019-10-23 2021-05-20 Pairwise Plants Services, Inc. Compositions and methods for rna-templated editing in plants
US20230203512A1 (en) * 2021-07-20 2023-06-29 Rutgers, The State University Of New Jersey Compositions and methods for agrobacterium mediated transformation of chloroplasts in seed plants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MCBRIDE K. E., ET AL.: "CONTROLLED EXPRESSION OF PLASTID TRANSGENES IN PLANTS BASED ON A NUCLEAR DNA-ENCODED AND PLASTID-TARGETTED T7 RNA POLYMERASE.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (PNAS), NATIONAL ACADEMY OF SCIENCES, vol. 91., 1 July 1994 (1994-07-01), pages 7301 - 7305., XP002058229, ISSN: 0027-8424, DOI: 10.1073/pnas.91.15.7301 *

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