WO2024018036A1 - Microalgues exprimant des produits biologiquement actifs - Google Patents

Microalgues exprimant des produits biologiquement actifs Download PDF

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WO2024018036A1
WO2024018036A1 PCT/EP2023/070231 EP2023070231W WO2024018036A1 WO 2024018036 A1 WO2024018036 A1 WO 2024018036A1 EP 2023070231 W EP2023070231 W EP 2023070231W WO 2024018036 A1 WO2024018036 A1 WO 2024018036A1
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seq
sequence
host cell
polynucleotide
nucleic acid
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PCT/EP2023/070231
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Raffaele Ingenito
Marco MATTU
Giuseppe Martelli
Rosa Paola RADICE
Maria Carmela PADULA
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Bioinnova S.R.L.S.
Naturamla Srl
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Publication of WO2024018036A1 publication Critical patent/WO2024018036A1/fr

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    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/11—Preparation or pretreatment of starting material involving culturing conditions, e.g. cultivation in the dark or under defined water stress
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a platform based on unicellular algae, grown in a controlled environment, which serve as a biofactory for the production of biologically active metabolites.
  • an editing methodology has been developed in which the genomic structure is modified through a natural process attributable to a targeted genetic improvement methodology.
  • Biofactories Plant organisms are commonly used as biofactories for the production of molecules of biological or pharmaceutical interest.
  • biofactory refers to unicellular organisms capable of producing bioactive molecules, recombinant or otherwise, for application in pharmaceutical, nutraceutical, cosmeceutical, and more generally in the industrial field.
  • Such molecules can be of various kinds, particularly peptides and biofunctional proteins for human, animal and/or plant use such as enzymes, antigens or therapeutic proteins.
  • Microalgae are unicellular plant organisms belonging to both the prokaryotic and eukaryotic kingdoms and are from a biophysiological point of view full-fledged plants. Microalgae produce energy through the photosynthetic process, removing carbon dioxide from the atmosphere and releasing oxygen at the end of the process. Their special position in phylogenetic evolution makes them model organisms, as they share characteristics common to both bacteria and higher plants. The unicellular nature of microalgae, means that they must respond to environmental stresses quickly and efficiently (to ensure their survival), maintaining the genome in a highly reactive euchromatic state.
  • Microalgae comprise a wide range of mainly aquatic, unicellular, and eukaryotic organisms (including green algae, diatoms, and brown algae) that engage in photosynthetic activity, and contain cytoplasmic organelles such as mitochondria and chloroplasts.
  • microalgae The chromatinstructure of microalgae is distinct from other eukaryotic organisms; in fact, this is heavily coloredhighlighting a more compact nucleosomal structure and a close association of DNA with histone protein components. These differences, which are more present, in green microalgae, highlight a differential mechanism in the regulation of gene expression at the histone chromatin level.
  • structural chromatin differences in microalgae may be explanatory regarding stable nuclear transgene expression that is, in fact, difficult and transient due to chromatin-mediated genesilencing itself (H. Cerutti, A.M. J., N.W. Gillham, J.E. Boynton, Epigenetic silencing of a foreigngene in nuclear transformants of Chlamydomonas, The Plant Cell 9:925-945 (1997)).
  • GMOs genetically modified organisms
  • Microalgae are an important source of healthy nutrients for human needs and are also further used for biomass and biofuel production. Genetic engineering and stable expression (maintained over multiple generations) of different transgenes would open up new horizons and greatly improve the value and opportunity of cultivating microalgae for multiple applications. However, as described earlier, it has been difficult to achieve a stable and sufficiently high level of gene expression, so a new methodology to help overcome this obstacle is extremely useful. Such an approach must take into account gene silencing related to the unique and resistant histone presence of microalgae, including green microalgae.
  • the present invention reports a totally innovative approach regarding the use of microalgae and specific growth and reproduction techniques to express and produce biologically active metabolites for use, in the form of freeze-dried biomass, as functional food for animal and humanuse.
  • the invention is based on the development of a novel strategy for the biosynthesis of active compounds by unicellular microalgae by implementation of the targeted horizontal gene transfer (THGT) conditions allowing introduction of specific oligos coding for peptides/proteins without using the state of the art transfection methodologies such as viral vectors, microinjection, Laserfection/Optical Transfection, Lipofection, Biolistic Methods or other kind of transfection methodologies know in the art.
  • THGT targeted horizontal gene transfer
  • Chlorophyceae are one of the classes of green microalgae, distinguished mainly on the basis of ultrastructural morphology. Usually, their coloration is caused by the predominance of photosynthetic pigments such as chlorophyll a and chlorophyll b.
  • the chloroplast can have a discoidal, plate-shaped, reticulate, cup-shaped, spiral or ribbon-like conformation depending on the species to which it belongs. Most species belonging to this class, have one or more storage compartments called pyrenoids, located within the chloroplast. Pyrenoids, in turn, contend for various proteins including starch.
  • Some species of green microalgae can store nutrient sources in the form of oily droplets. They usually have a cell wall consisting of an inner layer of cellulose and an outer layer of pectose.
  • the present invention is directed toward the production of biofunctional peptides and proteins for human and animal use, preferably biologically active proteins or therapeutic proteins, within a host cell, represented by a microalgal cell such as microalgae belonging to the division Chlorophyta with particular attention to the class: Chlorophyceae and Trebouxiophyceae.
  • the host cell is used as a biofactory for protein production.
  • the recombinant Chlorophyceae host cell is Haematococcus pluvialis.
  • an host cell comprising an isolated nucleic acid molecule comprising or consisting of: a) a polynucleotide sequence encoding at least one peptide; and b) at least one polynucleotide linker sequence with at least 90% of sequence homology to any of SEQ ID No 1-21; wherein the polynucleotide linker sequence is linked to the 5’ end and/or the 3' end of the encoding polynucleotide sequence of a) and in which the encoding polynucleotide sequence is not SEQ ID No 116; and wherein said host cell is an algal cell, preferably said host cell belongs to the Chlorophyceae class.
  • the isolated nucleic acid comprises or consists of: a) the polynucleotide sequence encoding at least one peptide; and b) at least one polynucleotide linker sequence with at least 90% of sequence homology to any of SEQ ID No 1-21, linked at the 5’ end of the polynucleotide sequence of a); and c) at least one polynucleotide linker sequence with at least 90% of sequence homology to any of SEQ ID No 1-21, linked at the 3’ end of the polynucleotide sequence of a).
  • the isolated nucleic acid molecule comprises a coding polynucleotide sequence has at least 90% homology to any of SEQ ID No 22-47.
  • the isolated nucleic acid molecule comprises a coding polynucleotide sequence has at least 90% homology to any of SEQ ID No 221-222.
  • the host cell belongs to the species Hematococcus pluvialis and/or wherein said host cell belongs to the class of Trebouxiophyceae; preferably said host 1) cell belongs to the species Haematococcus spp and/or Chlorella spp.; more preferably said host cell belongs to Haematococcus pluvialis and/or Chlorella vulgaris.
  • algal biomass comprising at least one host cell as defined above and/or a lysate and/or an extract of said host cell; preferably said algal biomass comprises at least one peptide encoded by the nucleic acid molecule as defined in any of claims 1 to 4.
  • a. induce thermal stress in a culture of microalgae, preferably belonging to the class of Chlorophyceae by heating at a temperature between 35 and 50 ° C for a time between 300 and 600 seconds
  • a mixture of lytic enzymes comprising at least one of the following enzymes: cellulase, cellulase CP, hemicellulose, chitinase, 0-D- glucanase, macerozyme, helicase, driselasi, lytic enzyme L, pectinase, protease, xylanase, cutinase, P-D-glucuronidanase, cellobiohydrolase, mixtures of them; e.
  • step d incubate the microalgae culture treated in step d) at a temperature between 30 - 40° C for 4-8 h; f. combine the culture incubated from step e) in 10 ml of culture medium, with 0.1 - 0.5% of a solution comprising the isolated nucleic acid previously defined, in the presence of 10-35 % polyethylene glycol (PEG-X).
  • PEG-X polyethylene glycol
  • the above algal biomass according or an algal biomass obtainable from the above method for medical use, preferably for use in the treatment and/or prevention of infection caused by airborn viruses, more preferably for use in the treatment and/or prevention of a SARS COV2 infection.
  • Airborne viruses are most commonly transmitted through small respiratory droplets. These droplets are expelled when someone with the airborne disease sneezes, coughs, laughs, or otherwise exhales in some way. These infectious vehicles can travel along air currents, linger in the air, or cling to surfaces, where they are eventually inhaled by someone else.
  • the list of airborn virsuses includes and not limited to SARS, MERS, Chickenpox, Respiratory Syncytial Virus (RSV), Influenza, Measles, swine-origin influenza. It is a further object of the invention a pharmaceutical composition comprising the above host cell or the above biomass or the algal biomass obtainable from the above method and at least one pharmacologically acceptable excipient.
  • nucleic acid molecule comprising or consisting of: a) a polynucleotide sequence encoding peptides; and b) at least one polynucleotide linker sequence with at least 90% sequence homology to any of SEQ ID No 1-21; wherein the polynucleotide linker sequence is linked to the 5’ end and/or the 3' end of the coding polynucleotide sequence of a) and in which the coding polynucleotide sequence is not SEQ ID No 116.
  • said isolated nucleic acid molecule comprises or consists of: a) a polynucleotide sequence encoding peptides and biofunctional proteins; and b) at least one polynucleotide linker sequence with at least 90% sequence homology to any of SEQ ID No 1-21, linked at the 5’ end of the polynucleotide coding sequence of a); and c) at least one polynucleotide linker sequence with at least 90% sequence homology to any of SEQ ID No 1-21, linked at the 3’ end of the polynucleotide coding sequence of a).
  • the coding polynucleotide sequence a) has at least 90% sequence homology to any of SEQ ID No 22-47, or its functional derivatives.
  • FIG. 1 Schematic representation of the expression method according to an embodiment of the invention.
  • FIG. 4 Monocytes after treatment with microalgae.
  • 1 st bar corresponds to day 0 (DAYO)
  • 2 nd bar corresponds to control - 48 h
  • 3 rd bar Haematoccus pluvialis P, 4 th bar Chlorella
  • a - intMo have a higher capacity to secrete cytokines in the blood and are reflective of an inflammatory environment
  • B - High CD 163 expression is associated to proinflammatory response
  • the present invention is directed to a platform (biofactory) for the production of biofunctional peptides and proteins for human, animal and/or plant use, particularly peptides and therapeutic proteins of interest through targeted genome editing of unicellular algae.
  • a methodology has been developed that enables targeted genome editing in microalgae.
  • the developed methodology allows for the introgression of exogenous genetic material, in a stable form, within the genome of the algae thus realizing an efficient and specializedbioreactor.
  • the specialization lies in the fact that the methodology allows the creation of special algal lines that are characterized in relation to the gene and metabolite that they are able to biosynthesize in a specific and efficient form.
  • Microalgae suitable for the present invention include microalgae chosen from the group consisting Chlorophyta (green algae), Rhodophyta (red algae), Stramenopiles (heterokonts), Xanthophyceae (yellow-green algae), Glaucocystophyceae (glaucocystophytes), Chlorarachniophyceae (chlorarachniophytes), Euglenida (euglenids), Haptophyceae (coccolithophorids), Chrysophyceae (golden algae), Cryptophyta (cryptomonads), Dinophyceae (dinoflagellates), Haptophyceae (coccolithophorids), Bacillariophyta (diatoms), Eustigmatophyceae (eustigmatophytes), Raphidophyceae (raphidophytes), Scenedesmaceae, Phaeophyceae (brown algae).
  • said microalgae is chosen from the group consisting of Chlamydomonas, Chlorella, Dunaliella, Haematococcus, diatoms, Scenedesmaceae, Tetraselmis, Ostreococcus, Porphyridium, and Nannochloropsis.
  • the algal lines belonging to the Chlorophiceae class are used, initially pretreated through the application of a genetic improvement scheme aimed at improving both biophy siological parameters and the ability to incorporate exogenous DNA fragments.
  • a construct which is nothing more than an artificially structured polynucleotide sequence capableof first being transcribed and consequently translated realizing a specific gene product
  • biological vectors that are capable, by different mechanisms, of infecting microalgal cells.
  • the biological vectors insert the construct within the microalgal cell, which is first incorporated within the genomic DNA and then transcribed and consequently generating the corresponding gene product by exploiting the transcription apparatus of the plant cell.
  • the technology described here constitutes an innovative and revolutionary methodology in that it involves and exploits biophysiological mechanisms inherent to the selected algal lines that enable them to incorporate gene constructs in an autonomous form. In essence, it is the selected algal lines themselves, through specific biomolecular mechanisms inherent in them, that incorporate the exogenous gene fragment. The same mechanism, in nature, would allow them to implement an evolutionary process resulting in improved fitness and survival.
  • microalgal species are subjected to particular changes in environmental parameters and consequent conditioning of specific biophysiological activities that make them suitable for incorporating the construct by biomolecular mechanisms innate in them. Then, in a targeted manner and with a specific procedure, normal biophysiological conditions are restored so that the algal cells resume their normal life cycle. At this point the algae having incorporated the exogenous gene fragment through their natural life functions transcribe and translate the information contained therein becoming in fact specialized biofactories (specialized in that each algal line after incorporation of a specific construct becomes a specific biofactory).
  • biotechnological process that is the subject of the invention reproduces in a controlled manner what would happen in nature in a spontaneous manner, effectively allowing a modified cell to be obtained.
  • the same biomolecular mechanism exploited in the biotechnological process that is the subject of the invention allows microalgae to create new variability and consequently adapt to changes in their habitat.
  • this mechanism allows the microalgae to incorporate exogenous gene fragments into their genome that could bring them a possible selective advantage from a survival perspective in the short term, consequently determining an evolutionaryprocess in the medium to long term.
  • Further advantage of this methodology lies in the fact that it allows obtaining genomic structures that are structurally and functionally stable over time since the microalga, once it incorporates the gene construct, is able to pass it on to future generations, making it an integral part of its genome.
  • the elaborated protocol is simple to execute, inexpensive, and applicable to all microalgae belonging to the class Chlorophiceae and especially to some genera such as, by way of example only, Chlorella, Haematococcus, and Chlamidomonas for which supercompetent genotypes particularly responsive to treatment are available.
  • the gene construct designed for use in the expression platform of the invention requires the use of specific linker sequences at the ends of the coding sequence for the exogenous protein of interest and said specific linker sequences are derived from the algal genome of interest, and have been identified and selected within said algal genome for the development of the platform of the invention.
  • Efficient growth medium is defined as any culture medium in which a microalgal cell according to the invention, preferably a Chlorophyceae cell, is usually grown.
  • Such medium typically includes an aqueous phase containing assimilable sources of carbon, nitrogen andphosphate, as well as mineral salts, metals and other appropriate nutrients such as, for example, vitamins.
  • suitable media and growth conditions are described in the "Examples" section.
  • Cells of the present invention can be cultured in conventional fermentationphotobioreactors, shaking flasks, test tubes, microtiter plates, and Petri dishes.
  • the culture can becarried out at temperature, pH and oxygen content appropriate for the recombinant cell.
  • the term "transformation” identifies any methodology bywhich an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can beinserted within microbial cells.
  • transformation is used primarilyto describe a genetic, heritable change caused by the acquisition of exogenous nucleic acids by the microorganism, and is essentially synonymous with the term “transfection.”
  • a methodology for transforming a competent algal host cell involves two main steps: a) pre-stratifying the cell with physical stressors, b) treating the host cell with an enzyme or mixture of enzymes, and c) introducing an exogenous nucleic acid molecule into the host cell.
  • the enzyme can havecellulase, protease, P-glucoronase and various combinations of these activities.
  • the expression system of the invention which allows a protein of interest to be expressed within the algal cell, includes regulatory control elements that are active in microalgal cells.
  • novel regulatory or linker sequences described as aspects of the invention, can be used not only in the algal cells described here, but also in cells belonging to different species.
  • the design and construction of the expression systems covered by the invention use standard biomolecular technologies known to persons skilled in the art. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd edition.
  • the expression system or expression vector comprises a polynucleotide sequence that codes for a protein of interest, for example, a SARS-COV2 virus protein, such as the spike protein or a peptide derived from it, or the GLP-2 protein hormone, or PYY protein hormone, or Xenin-25 peptide associated with any promoter sequence, or 5' linker, and possibly a terminator sequence, or 3' linker.
  • the 5' linker, coding sequence and 3' linker are operatively linked in such a way as to be functional within the host cell.
  • the expression system may also include additional regulatory sequences that are functional within the genome of the hostcell. Inducible or constitutively active sequences can be used. Suitable control elements, also include any regulatory element associated with the expression of the nucleic acid molecules described here.
  • the present invention is also directed to the algal host cell comprising the expression system described above and/or to a biomass obtained from or comprising said algal cell.
  • a biomass according to the present invention is a composition comprising transformed algal cells and/or their secretions and/or extracts and/or lysates or other derivatives.
  • the expression system of the invention preferably comprises at least one of the nucleic acid molecules isolated in the present invention and described herein.
  • all genetic elements of the expression system are sequences associated with previously isolated nucleic acid molecules.
  • the nucleic acid sequence encoding for the protein of interest, or coding sequence is stably integrated into the genome of the host cell, while in others, said coding sequence is operatively linked to a promoter linker sequence and/or a teminator linker sequence, both of which are functional in the host cell.
  • the linker sequences to which the coding sequence is operatively linked include, but are not limited to, the novel nucleic acid sequences described inthe present invention.
  • the coding sequence is optimized for the codon belonging specifically to the host cell of Haematococcus pluvialis so as to maximize translation efficiency.
  • the proteins of interest produced by a recombinant host cell that are the subject of the invention include, but are not limited to, peptides and biofunctional proteins for human, animal, and/or plant use, also referred to herein as therapeutic proteins.
  • a biofunctional peptide or protein for human, animal, and/or plant use, as used herein, includes proteins useful for the treatment or prevention of diseases, pathological conditions, and various disorders in both animals and humans and the plant kingdom.
  • cure and “treatment” refer to both therapeutic treatment and prophylactic or preventive measures in which the objective is to preventer slow down (reduce) an unwanted pathophysiological condition, disease, or disorder, or to achieve beneficial or desired clinical results.
  • beneficialor desired clinical results include, but are not limited to, the alleviation of symptoms or signs associated with a pathological condition or disorder of normal physiology; diminution in the magnitude of a condition, disease, or disorder; stabilization of a condition, disease, or disorder (orbetter, situations in which the condition, disease, or disorder is stable and does not worsen over time) delay in the onset or progression of the condition, disease, or disorder; improvement of the condition, disease, or disorder; remission (total or partial and detectable or undetectable) of the condition, disease, or disorder; or enhancement or improvement of a condition, disease, or disorder.
  • Treatment includes eliciting a clinically meaningful response without excessive side effects and also prolonging survival over expected survival if treatment is not received.
  • therapeutic proteins include, but are not limited to, biologically active proteins, such as enzymes, antibodies, or antigenic proteins, while in other forms of realization, therapeutic proteins include, but are not limited to: a viral protein such as a coronavirus Spike protein, PYY, Xenin-25, a human vaccine, an animal vaccine, and a veterinary drug.
  • the GLP-2 peptide is a 33 -amino acid peptide, having the following sequence: HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID No. 192).
  • Peptides derived from the SARS C0V2 Spike protein (or functional fragments of the Spike protein) used for the purposes of the present invention are as follows:
  • C0V 1.1 ATRFASVYAWNRKRISNCVADYSVLYNSASF (31 aminoacids) (SEQ ID No. 194)
  • COV-1.2 ASVYAWNRKRISNCVADYSVLYNSASFSTFK (31 aminoacids) (SEQ ID No. 195)
  • COV 2.1 ADYSVLYNSASFSTFKCYGVSPTKLNDLCFT (31 aminoacids) (SEQ ID No. 197)
  • COV 3.1 PYRVWLSFELLHAPATVCGPKKSTNLVKNK (31 aminoacids) (SEQ ID No. 203)
  • COV 3.2 WLSFELLHAPATVCGPKKSTNLVKNKCVNF (31 aminoacids) (SEQ ID No. 204)
  • COV 4.1 TSGWTFGAGAALQIPFAMQMAYRFNG (26 aminoacids) (SEQ ID No. 209)
  • COV 4.2 TSGWTFGAGAALQIPFAMQMAYRFNGIGVTQ (31 aminoacids) (SEQ ID No. 210) COV 4.3: FGAGAALQIPFAMQMAYRFNGIGVTQNVL (29 aminoacids) (SEQ ID No. 211) COV 5: NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI (35 aminoacids) (SEQ ID No. 212)
  • COV 5.2 LDKYFKNHTSPDVDLGDISGI (21 aminoacids) (SEQ ID No. 214)
  • COV 6.1 ESLIDLQELGKYEQYIKWPWYIWLGFIAG (29 aminoacids) (SEQ ID No. 216)
  • COV 6.2 EQYIKWPWYIWLGFIAGLIAIVMVTIML (28 aminoacids) (SEQ ID No. 217)
  • C0V_7 GSCCKFDEDDSEPVLKGVKLHYT (22 aminoacids) (SEQ ID No. 218)
  • Peptide YY also known as peptide tyrosine tyrosine is a peptide that in humans is encoded by the PYY gene.
  • PYY is a short (36-amino acid) peptide released from cells in the ileum and colon in response to feeding. In the blood, gut, and other elements of periphery, PYY acts to reduce appetite; similarly, when injected directly into the central nervous system, PYY is also anorexigenic, i.e., it reduces appetite.
  • the PYY(peptide tyrosine tyrosine) peptide is a 33 -amino acid peptide, having the following sequence:
  • Xenin-25 is a 25-amino acid peptide hormone co-secreted from the same enteroendocrine K-cell as the incretin peptide glucose-dependent insulinotropic polypeptide. Xenin-25 has been demonstrated in pancreatic islets and recently shown to possess actions in relation to the regulation of insulin and glucagon secretion, as well as promoting beta-cell survival.
  • the Xenin-25 peptide is a 25-amino acid peptide, having the following sequence: MLTKFETKSARVKGLSFHPKRPWIL (25 aminoacids) (SEQ ID No. 220)
  • proteins produced by a recombinant host cell included in the invention include, but are not limited to, industrial enzymes.
  • Industrial enzymes include, but are not limited to, enzymes used in the production, preparation, storage, nutrient mobilization or processing of products, including food, medical, chemical, mechanical and other industrial products.
  • Industrial enzymes include, but are not limited to: alpha-amylase, alpha-galactosidase, betaamylase, cellulase, beta-glucanase, dextrin dextranase, glucoamylase, hemicellulase/pentosanase, xylanase, invertase, lactase, naringinase, pectinase, pullulase, acid proteinase, alkaline protease, bromelain, papain, pepsin, aminopeptidase, endo-peptidase (trypsin, chemotrypsin, pepsin, elastase), renin/reninachemosin, sibtilism, thermolysin, aminoacylase, glutaminase, lysozyme, penicillin acylase, triglyceridase, phospholipase, pre
  • the proteins produced by a recombinant host cell included in the invention include an auxotrophic marker, a dominant selection marker (such as, for example, an enzyme that degrades antibiotic resistance) or another protein involved in transformation selection, a reporter protein, an enzyme involved in protein glycosylation, and an enzyme involved in cellular metabolism.
  • Protein produced or expressed by the algae cell according to the invention can be produced on a commercial scale.
  • Commercial scale includes protein production from a microorganism grown in an aerated biofermentor of size > 100 L, > 1,000 L, > 10,000 L, or > 100,000 L. In some forms of implementation, commercial-scale production is performed in an aerated biofermentor with agitation.
  • the protein produced by the algae cell can also accumulate within the cell or can be secreted by the cell, for example, into the culture medium as a soluble protein.
  • the protein produced can be recovered from the cell, the culture medium, or the fermentation medium in which the cell itself is grown.
  • the same biomass expressing the GLP-2 protein or PYY protein or Xenin-25 or the SARS COV2 spike protein, or peptides derived from it, can be used directly for the purposes of the invention, which include the therapeutic and preventive uses of a pharmaceutical product, composition or kit, and non therapeutic uses, such as in the preparation of a nutraceutical product.
  • the present invention is directed to a method for producing a recombinant protein; the methodology also includes culture conditions for the microalgal cells of the invention such that a polynucleotide sequence coding for a protein can be expressed.
  • Proteins produced by the method elaborated in this invention can also be purified using a variety of standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reversed phase chromatography, chromatography using concanavallin A, chromatofocusing, and differential solubilization.
  • proteins produced by the method described in the present invention are isolated in "substantially pure” form. In this context, “substantially pure” refers to a purity that allows effective use of the protein as a commercial product.
  • the recombinant protein accumulates within the cell and is recovered from the cell; in some embodiments, the host cell of the method belongs to the Chlorophyceae class, while in other embodiments, the host cell is from Haematococcus pluvialis.
  • the recombinant protein is a therapeutic protein, a food enzyme or an industrial enzyme.
  • the recombinant protein is the spike protein of SARS- COV2 or a peptide derived from it.
  • the recombinant protein is the PYY protein or a peptide derived from it (a functional derivative).
  • the recombinant protein is the Xenin-25 peptide or a peptide derived from it (a functional derivative).
  • the recombinant protein is a therapeutic protein that includes a secretion signal sequence.
  • a “peptide derived from” or a “functional derivative” is an elongated version of the referred peptide comprising one or more aminoacids at the C and/or N- terminal and maintaining the same biological properties.
  • Nucleic acids Isolated nucleic acid molecules or polynucleotide sequences that constitute the expression system in the algal cell form the object of the invention.
  • the nucleic acid sequences described here include the 5' and 3' linker sequences and coding sequences.
  • the GLP-2 coding sequence and sequences encoding functional fragments of the SARS-COV2 spike protein are shown as examples. Other examples are the coding sequences of the PYY peptide and of the Xenin-25 peptide.
  • nucleic acid molecule can be a DNA molecule, RNA molecule (e.g., mRNA) or derivatives of them (e.g., cDNA).
  • RNA molecule e.g., mRNA
  • cDNA e.g., cDNA
  • nucleic acid molecule refers primarily to the physical nucleic acid molecule
  • nucleic acid sequence or “polynucleotide sequence” refer primarily to the sequence of nucleotides present on the nucleic acid molecule, the phrases are used interchangeably, especially in reference to a nucleic acid molecule, polynucleotide sequence, or nucleic acid sequence encoding a protein.
  • a nucleic acid molecule isolated by the present invention is produced using recombinant DNA technology (such as, for example, cloning and amplification by polymerase in reaction (PCR)) or by chemical synthesis.
  • Isolated nucleic acid molecules include naturally occurring nucleic acid molecules and their homologs, including, but not limited to, naturally occurring allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or reversed in such a way that these modifications provide the desired effect on the sequence, function, and/or biological activity of the encoded peptide or protein.
  • a double-stranded DNA present in this invention includes a single-stranded DNA and its complementary strand, the sequence of which mirrors the sequence of the single-stranded DNA.
  • nucleic acid molecules of the present invention may be double-stranded or single- stranded and also include those nucleic acid molecules that form stable hybrids under high "stringency" conditions with a sequence of the invention and/or with a sequence complementary to a sequence of the invention. Methods for tracing a complementary sequence are known to experts in the field.
  • the term "protein” includes single-chain polypeptide molecules as well as multiple polypeptide complexes in which the individual constituent polypeptides are bound through covalent and noncovalent means.
  • polypeptide includes peptides of two or more amino acids in length, typically having more than 5, 10 or 20 amino acids.
  • the new nucleic acid molecules of the present invention can be used in any genus of microalgae in which they are found to be functional.
  • the nucleic acid molecules of the invention are used in algae including Chlorophyta (green algae), Rhodophyta (red algae), Stramenopiles (heterokonts), Xanthophyceae (yellow-green algae), Glaucocystophyceae (glaucocystophytes), Chlorarachniophyceae (chlorarachniophytes), Euglenida (euglenids), Haptophyceae (coccolithophorids), Chrysophyceae (golden algae), Cryptophyta (cryptomonads), Dinophyceae (dinoflagellates), Haptophyceae (coccolithophorids), Bacillariophyta (diatoms), Eustigmatophyceae (eustigmatophytes), Raphidophy
  • the nucleic acid molecules are used in algae belonging to the class Chlorophyceae.
  • recombinant nucleic acid molecules are used in the species Haematococccus pluvialis.
  • recombinant microorganism has a genome that has been modified (i.e., mutated or changed) from its natural (i.e., naturally occurring or wild-type) form using recombinant technology.
  • a recombinant microorganism according to the present invention may include a microorganism in which nucleic acid molecules have been inserted, deleted, or modified (i.e., mutated, e.g., by nucleotide insertion, deletion, substitution, and/or inversion) such that the modifications provide the desired effect within the microorganism.
  • the present invention is directed to the 5' and 3' linker sequences.
  • the 5' or promoter linker is a DNA sequence that directs transcription of a coding region associated with it.
  • the 3' or terminal linker is the gene sequence that marks the end of transcription of genomic DNA.
  • the linker of the invention is any of the following sequences: CGGGGCAACTCAAGAAATTC (SEQ ID No 1) GTCTGGCCGAGGTCTGGTTCCTGTGCC (SEQ ID No 2) ACTGCACATCGCTGCAGTCT (SEQ ID No 3) CGCGTCGGGGCCTGCCTAAG (SEQ ID No 4) TTACCTGCCACACAAGCCTG (SEQ ID No 5) CGTGCTACTGGGGTCTGGCAG (SEQ ID No 6) CACATGCCATCCGAGTCGTC (SEQ ID No 7) CACAACCATACTGGCGAAGT (SEQ ID No 8) ATGGCCACGC (SEQ ID No 9) CTCTACCCAC (SEQ ID No 10)
  • CCGGACTGCCATAGCACAGCTAGACGA (SEQ ID No 11) GTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 12) ACTGACTGCCATAGCACAGCTAGACGA (SEQ ID No 13) ATTTGCTGCATGACTGGATCAATGCGACGA (SEQ ID No 14) GTCTGGCCTGACGTATGATCGATGCCATAAATGC (SEQ ID No 15) ATGCCCTGATCCCAATGATGGACGA (SEQ ID No 16) GTCTGGCCGAAACTGATTTGGCCATGAC (SEQ ID No 17) GAGCGTGCTGAAATGCATGCGACGA (SEQ ID No 18) GTCTGGCCCCCGGGTATAGTAGCTGAC (SEQ ID No 19) CCCGGGTATAGTAGCTGACTGCGACGA (SEQ ID No 20) GTCTGGGAGCGTGCTGAAATGCATG (SEQ ID No 21)
  • nucleic acid molecule comprising a polynucleotide sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto any one of SEQ ID NO: 1 - SEQ ID NO:21, in which the polynucleotide sequence functions as a promoter and/or terminal linker at least in algae belonging to the class Chlorophyceae.
  • the invention also relates to an isolated nucleic acid molecule comprising a polynucleotide sequence that hybridizes with any of the SEQ ID NO: 1 - SEQ ID NO:21 sequences or that hybridizes with a polynucleotide sequence that is at least 95 percent identical to any of the SEQ ID NO:1 - SEQ ID NO:21 sequences.
  • the isolated nucleic acid molecule may include a polynucleotide sequence that is completely complementary to any of the SEQ ID NO: 1 - SEQ ID NO:21 sequences or to a polynucleotide sequence that is at least 95% identical to any of the SEQ ID NO:1 - SEQ ID NO:21 sequences.
  • Microalgae belonging to the genus Chlorophyceae, were grown in the culture medium shown in Table 1, under illumination intensity of 120 mmol photons m s -2-1 in an alternating cycle of 16 h of light and 8 h of dark, at a temperature of 25 °C.
  • the cultures were agitated by mechanical shaker(g24 environmental incubator shaker, American Laboratory Trading) at 70 rpm throughout the growth time.
  • the percentages shown in the table may vary depending on the species, genotype and initial concentration of microalgae cultures.
  • microalgae belonging to the class Chlorophyceae were subjected to stress pretreatment with the aim of making the algae optimized and responsive to the treatment.
  • the selection process applicable to different microalgae species, involves: thermal stress induction in a temperature range of 35-50° C for a time interval of 600 to 300 seconds;
  • UV-A, UV-B and UV-C Exposure of cultures to physical mutagenic (UV) agents.
  • UV-A, UV-B and UV-C Exposure of cultures to physical mutagenic (UV) agents.
  • UV-A, UV-B and UV-C Exposure of cultures to physical mutagenic (UV) agents.
  • UV-A, UV-B and UV-C Exposure of cultures to physical mutagenic (UV) agents.
  • UV-A, UV-B and UV-C UV-A, UV-B and UV-C
  • Cultures were characterized according to the following parameters: growth capacity, cell size, adaptive capacity, and ability to incorporate exogenous fragments.
  • microalgae-specific genes related to the photosynthesis pathway were analyzed.
  • the following sequences were derived from specific algal sequences:
  • CACATGCCATCCGAGTCGTC (SEQ ID No 7)
  • CTCTACCCAC (SEQ ID No 10)
  • the coding sequences for the Spike fragments of SARS-CoV- 2 are as follows:
  • AATGTGTCAATTTCAACTTCAATGGT (SEQ. ID No 35) COV 3.5:
  • AAATTA (SEQ ID No 59)
  • oligonucleotides comprising the gene sequence of GLP2 : CATGCTGATGGTTCTTTCTCTGATGAGATGAACACCATTCTTGATAATCTTGCCGCCAGGGAC TTTATAAA CTGGTTGATTCAGACCAAAATCACTGAC (SEQ ID No. 116) and at least one promoter linker, preferably a promoter linker and a terminal linker (underlined in the list below), derived from the specific algal sequences and previously described, were synthesized. Relative sequences are reported below:
  • TCTACCCAC (SEQ ID No 123) CCGGACTGCCATAGCACAGCTAGACGACATGCTGATGGTTCTTTCTCTGATGAGA TGAACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCA GACCAAAATCACTGACGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 124)
  • the coding sequence for the PYY peptide is as follows:
  • the coding sequence for Xenin-25 peptide is as follows:
  • oligonucleotides comprising the above coding gene sequence of PYY and Xenin-25 and at least one promoter linker, preferably a promoter linker and a terminal linker (underlined in the list below), derived from the specific algal sequences and previously described, were synthesized.
  • the microalgae culture was resuspended at a ratio of 1 : 5 to 1 : 25, depending on cell concentrations, in a macerating solution which constitutes a lytic mixture.
  • the macerating solution consisted of 16- 50% (v/v) 0.35 M mannitol and 0.2-0.6% lytic enzyme mixture (Table 2).
  • the lytic mixtures were then incubated at 30-37°C for 4-8 h.
  • the composition of the lytic mixture, temperature and incubation time changed according to the microalgae species.
  • microalgae solutions after treatment with the lytic mixture were combined, in a final volumeof 10 ml of culture medium, with 0.1 to 0.5 % v/v of polynucleotide diluted 2, 5, 10, 20, 25, or 50times (initial concentration 100 pM), depending on the nucleotide length and the resulting molecular weight of each polynucleotide (Table 3)
  • Steps i, ii and iii are repeated 2 to 5 times.
  • the pellet is resuspended in 1 to 50 ml of standard culture medium. Centrifugation speed and duration vary according to the concentrations of PEG used (Table 4)
  • the amplification protocol is as suggested in the Phire Plant Direct PCR Master Mix brochure. (Thermo ScientificTM - Cat. Number: Fl 60S - https : //www. thermofisher, com/ order/ catalo g/ product/F 160S )
  • the table below shows the polynucleotide sequences related to the Spike fragments inserted in the algae, the product resulting from the sequencing analysis, and the percentage of identity that this product has, compared to the sequences deposited in the databases, indicated by their respective unique ID codes.
  • GLP2 human glucagon-like peptide 2
  • a human glucagon-like peptide 2 (GLP2) ELISA kit (0.156-10 ng/mL) was used. 100 mg of dry algal biomass, obtained by freeze- drying the crude product, was dissolved in 10 mL of water containing 50% acetonitrile and 0.1% trifluoroacetic acid. The extraction step performed is reported in the materials and methods, in fact, the solution was sonicated for 15 min and then, by centrifugation, the algal biomass was separated from the supernatant. The supernatant was lyophilized and 100 pl was resuspended in diluent buffer (provided by the kit).
  • diluent buffer provided by the kit.
  • the analysis protocol used includes the following steps:
  • the amount of peptide present in the sample is in the range of 150 to 2000 pg/ml.
  • IEC-6 cells 5x10 3 IEC-6 cells were seeded and allowed to grow overnight.
  • CTRL and GLP-2 cells of H. pluvialis were sonicated and filtered to separate the precipitated phase from the liquid phase.
  • Different concentrations of algal extracts were tested (starting from 25% v/v. Serial 1:2 dilutions were made until the final concentration of 0.39 % v/v was reached).
  • MTT assay was conducted after
  • the protocol used involves the following steps:
  • PBMCs were purified using standard Ficoll-Paque gradient centrifugation according to the instructions of the manufacturer (Biochrom, Germany). Briefly, 20 ml of Ficoll-Paque gradient was pipetted into 50-ml centrifuge tubes. The heparinized blood was diluted 1 :3 in IX phosphate-buffered saline (PBS) (Euroclone s.p.a, Italy) and carefully layered over the Ficoll-Paque gradient. The tubes were centrifuged for 20 min at 2000 rpm.
  • PBS IX phosphate-buffered saline
  • Mononuclear cells stratified in the Ficoll-plasma interface were extracted at the end of centrifugation, and the cells were washed twice in PBS (for 5 min at 1500 rpm), resuspended in PBS and counted in a Neubauer chamber. Dead cells were excluded using 1% trypan blue.
  • PBMCs were frozen in freezing medium (FBS/10% DMSO) and stored in liquid nitrogen.
  • PBMCs obtained from healthy donors were counted and plated at the concentration of 4xl06/ml in RPMI medium (Cell Genix, USA) with 10% FBS.
  • PBMCs were stimulated with SARS CoV-2 antigenic peptide containing biomass The plate was incubated at 37 °C with 5% CO2 for 2 days and then analysed by FACS.
  • Immune cells collected from Spike-stimulated PBMC cultures were stained with a viability fluorescent dye that irreversibly labels dead cells prior to fixation and/or permeabilization procedures (AQUA ThermoFisher L34966 for T, B and NK) and LIVE/DEAD ( Fixable Blue Dead Cell Thermofisher L34961 for Monocyte-MAcrophages), allowing dead cells to be excluded from analysis.
  • LIVE/DEAD Fixable Blue Dead Cell Thermofisher L34961 for Monocyte-MAcrophages
  • Monocyte-Macrophages identification LIVE/DEAD (Fixable Blue Dead Cell Thermofisher L34961), CD16 (FITC eBioscience), CD80 (PE BD), CD163 (PE-CF594 BD), CD14 (BV510 Biolegend), PD-L1 (BV421 BD), CDl lc (BUV395 BD)
  • T, B and NK lymphocyte identification LIVE/DEAD AQUA (ThermoFisher L34966), CDl lc (BUV395 BD), CD4 (BUV496, Biolegend), CD3 (BUV661, BD Pharmingen), IgG (BV650 BD), CD24 (BV785 Biolegend), IgD (BB515, BD Pharmingen), CD21 (PE/Dazzle 594 Biolegend), CD56 (PE BD), CD38 (PE Cy7, Biolegend), CD19 (PercPCy5.5, Biolegend), CD27 (APC, Biolegend), CD8 (PeCy5.5 Invitrogen), HLA-DR (AlexaFluor 780, Biolegend)
  • the immune system has two distinct components: mucosal and circulatory.
  • the mucosal immune system provides protection at the mucosal surfaces of the body. These include the mouth, eyes, middle ear, the mammary and other glands, and the gastrointestinal, respiratory and urogenital tracts. Antibodies and a variety of other anti-microbial proteins in the sticky secretions that cover these surfaces, as well as immune cells located in the lining of these surfaces, directly attack invading pathogens. Almost all infectious diseases in people and other animals are acquired through mucosal surfaces. As evident from the virus that causes COVID-19, SARS-CoV-2, enters the body via droplets or aerosols that get into nose, mouth or eyes. It can cause severe disease if it descends deep into the lungs and causes an overactive, inflammatory immune response.
  • Sars CoV-2 is classified as airborn viurses and all the Airborn viruses have the same mechanism of infection through the mucosal compartments.
  • IgA antibody responses form as a result of vaccination or prior infection, or occur quickly enough in response to a new infection, they could prevent serious disease by confining the virus to the upper respiratory tract until it is eliminated.
  • Nasal vaccination can be effective in boositn or preventing viral infection for airborn viruses.
  • Microalgae expressing Sars CoV-2 peptide antigens can be in appropriate formulation spray used as boosting or preventing vaccine for airborn viruses.
  • the sars cov-2 antigen containing biomass produced according to the present invention methodology was tested against PMBC (Periferal Mononucler Blood cells) of subject received vaccination against Sars Cov-2 to evaluate toxicity on blood cells, pro- or anti-inflammatory activity , ability for modulating specific macrophage families involved in the cascade events involved in immune response.
  • PMBC Periferal Mononucler Blood cells
  • the SARS COV-2 antigenic peptide containing biomass was incubated with PBMC ad time course analysis perfomed at 24h and 48h to analyze the variation in macrophages populations.

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Abstract

La présente invention concerne une plate-forme basée sur des algues unicellulaires, cultivées dans un environnement contrôlé, qui servent de centre de bioproduction pour la production de métabolites biologiquement actifs.
PCT/EP2023/070231 2022-07-20 2023-07-20 Microalgues exprimant des produits biologiquement actifs WO2024018036A1 (fr)

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