Classifications

• • 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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
• • 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
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)

Definitions

• the present invention relates to the field of genetic engineering applicable to various industries including the pharmaceuticals, chemicals, agri-foods, cosmetics and biotechnologies industries.
• the invention relates to a vector useful for the production of a substance of interest by a bacterium, particularly a bacterium belonging to the order Actinomycetales, more particularly to the genus Propionibacterium, and even more particularly to the species Propionibacterium freudenreichii.
• Said vector can be particularly useful in the pharmaceutical field, for the large-scale production of substances whose activity is of interest in industries as diverse as pharmaceuticals, chemicals, agri-foods, cosmetics but also as a biotechnology tool for the production and secretion of substance of interest in bacteria particularly belonging to the order Actinomycetales.
• baculovirus baculovirus, Sf , Sf21, etc.
• plant cells e.g., CHO, HEK, COS, etc.
• mammalian cells e.g., CHO, HEK, COS, etc.
• propionibacteria are very robust and adaptable to particular media (e.g. media containing milk, milk derivatives or molasses) that are possibly hostile to the growth and development of other living systems (e.g. due to the presence in the medium of lactic acid, salt, etc.).
• media e.g. media containing milk, milk derivatives or molasses
• These bacteria have also good tolerance with respect to variations, changes or disturbances of the environmental conditions likely to occur during large-scale culture operations.
• propionibacteria can be described as "natural antifungals” since they naturally produce metabolites (for example, propionate) that inhibit the development of contaminating fungi. Furthermore, they are able to produce recombinant proteins of significant size (for example, proteins of more than 500 amino acids) and can even produce several different proteins simultaneously (for example, more than a dozen different proteins).
• Propionibacteria are also particularly useful for applications in the pharmaceutical and dietary fields.
• propionibacteria can be used as a tool for addressing or targeting substances to the intestine. Indeed, in mammals, including humans, propionibacteria are naturally able to target the intestine where their survival time can reach two weeks, compared to only two or three days for lactic bacteria, even though they are natural hosts of this environment.
• propionibacteria over various other bacteria proposed so far for use in anti-tumoral therapy (e.g., in WO 01/25397 in the name of Vion Pharmaceuticals, Inc. and in WO 2009/11 1177 in the name of Mount Sinai School of Medicine of New York University) is that propionibacteria per se are efficient and specific anti-tumoral agents that can safely be used in mammals, in particular in humans. This inherent property of propionibacteria can thus be further enhanced upon using propionibacteria to deliver therapeutic agents, such as drugs, to eradicate tumor cells while at the same time preventing damage to normal cells.
• therapeutic agents such as drugs
• Propionibacteria do not need to be genetically attenuated or enhanced by genomic mutations as it is the case for other bacteria such as Clostridium, Salmonella, Listeria, and the like.
• propionibacteria can be particularly useful anti-tumoral agents thanks to both their intrinsic anti-tumoral properties and their ability to efficiently, specifically and safely deliver other anti- tumoral drugs in order to kill cancer cells.
• the Inventors have previously demonstrated that Propionibacterium freudenreichii (Falentin et al., 2010, Plos One; Genbank accession No. FN806773) has particular cytotoxic properties with respect to colon cancer cells (Jan et al., 2002; Lan et al., 2007).
• Propionibacterium freudenreichii adheres to colonic epithelial cells and has no toxic effect on healthy cells (Lan et al., 2008).
• the Inventors have recently developed new vectors allowing the production and secretion of various substances at a high level by a bacterium, particularly belonging to the order Actinomycetales, including the genus Propionibacterium.
• the Inventors have shown that said signal peptides particularly in combination with promoters of genes encoding a surface layer protein of a Propionibacterium freudenreichii, allow the secretion into the extracellular medium of a substance of interest by a bacterium, in particular a propionibacterium, at a level up to 100 times greater than that obtained with other signal peptides in the same strain.
• the invention relates to a vector comprising:
• nucleic acid sequence encoding a signal peptide is selected from the group consisting of: - the sequence of the signal peptide of a gene encoding a surface layer protein of a Propionibacterium freudenreichii; and
• sequences having at least 80%, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with said sequence over the entire length of said sequence;
• sequence of said promoter is selected from the group consisting of:
• sequences having at least 80%>, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with said sequence over the entire length of said sequence.
• a vector can be a cloning or an expression vector.
• the vectors according to the invention can be viral vectors such as bacteriophages, or non-viral, such as plasmid vectors.
• a plasmid vector is a non-viral DNA molecule hosted by a cell, distinct from the natural chromosomal DNA of said host cell and capable of autonomous replication. The choice of plasmid vector and, more particularly, the origin of replication it carries thus depend on the host cell. According to the type of host cell, several copies of a plasmid vector and/or several different plasmid vectors can be hosted simultaneously.
• a plasmid vector according to the invention can possibly be carried by (or "integrated in” or "inserted in") the chromosome of the host cell.
• nucleic acid sequence refers to the sequence of a nucleic acid molecule, DNA and RNA, wherein the former can be genomic DNA, plasmid DNA, recombinant DNA or complementary DNA (cDNA), for example, and the latter can be messenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA).
• mRNA messenger RNA
• rRNA ribosomal RNA
• tRNA transfer RNA
• nucleic acid sequences of the invention refer to sequences of DNA molecules.
• the promoter comprised in the vector according to the invention can be a promoter suitable for the expression of said substance of interest in a bacterium, particularly a priopionibacterium.
• said promoter is a promoter for bacterial RNA polymerase, in particular for propionibacterial RNA polymerase.
• Said promoter can be a constitutive and/or inducible promoter well known by one skilled in the art.
• said promoter is inducible.
• the promoter can be developmentally regulated, inducible or tissue specific, preferably inducible in the digestive tract.
• said promoter is a promoter of propionic origin, in particular of P. freudenheimii origin, such as the promoter of protein PF963 of P. freudenreichii. More particularly, said promoter is a strong propionic promoter.
• the sequence of said promoter is selected from the group consisting of:
• sequences having at least 80%, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with said sequence over the entire length of said sequence.
• promoters of genes encoding a surface layer protein of a Propionibacterium freudenreichii in combination with signal peptides of genes encoding a surface layer protein of a Propionibacterium freudenreichii allow the secretion into the extracellular medium of a substance of interest by a propionibacterium at a level up to 100 times greater than that obtained with other promoters and peptide signals in the same propionibacterium strain.
• sequence of said promoter is selected from the group consisting of:
• said surface layer protein is selected from the group consisting of the surface layer protein A, the surface layer protein B, the surface layer protein C, the surface layer protein D, the surface layer protein E and the surface layer protein F, in particular of the surface layer protein A.
• sequence of said promoter is selected from the group consisting of:
• sequences having at least 80%>, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with said sequence over the entire length of said sequence;
• CIRM BIA 118 strain refers to the strain numbered 118 available at the International Centre for Microbial Resources dedicated to food bacteria interest ("Centre international de lives microbiennes dedie au. bacteries d'interets") located in Renncs (France).
• the vector according to the invention can comprise at least two promoters such as defined above which, even more advantageously, will be situated preferably in a series (but not necessarily coupled to each other), in order to increase the expression level of said substance of interest ("expression booster" effect).
• the recombinant vector according to the present invention comprises at least one nucleic acid sequence encoding a signal peptide translationally fused to at least one nucleic acid sequence encoding a substance of interest.
• said signal peptide is a secretory signal peptide.
• secretory signal peptide refers to any peptide allowing the secretion, in particular into the extracellular medium, of said substance of interest, by a host cell comprising the vector according to the invention, particularly by a propionibacterium.
• said signal peptide is a propionibacterial secretory signal peptide, allowing the secretion, in particular into the extracellular medium, of said substance of interest, by a host cell comprising the vector according to the invention, particularly by a propionibacterium.
• a signal peptide of a propionibacterium selected from dairy propionibacterium and so-called "cutaneous" propionibacterium (CPB) can be used.
• dairy propionibacterium can be cited: Propionibacterium freudenreichii, P. jensenii, P. thoenii P. acidipropionici and P. microaerophilum.
• the following cutaneous propionibacterium can also be cited: P.
• a signal peptide of a propionibacterium selected from the following species can be used: Propionibacterium freudenreichii, more particularly the subspecies P. freudenreichii subsp. freudenreichii and P. freudenheimii subsp. shermanii, and Propionibacterium acnes, wherein said propionibacterium is preferentially P. freudenreichii subsp. shermanii.
• said nucleic acid sequence encoding a signal peptide can be selected from the group consisting of:
• sequences having at least 80%, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with one of said sequences over the entire length of said sequence.
• Table I presents the nucleotide (NT) sequences, as well as the corresponding amino acid (AA) sequences of signal peptides, identified by the Inventors using proteomic analysis of P. freudenreichii CIRM-BIA 1 exoproteome.
• Table II below presents amino acid sequences of signal peptide, identified by the Inventors from the genomic sequence of P. acnes, accessible from databases (strain P. acnes KPA171202; accession number: NCBI: NC 006085; 5 GenBank: AEO 17283). Table II
• nucleic acid sequence encoding a signal peptide can be selected from the group consisting of:
• sequences having at least 80% more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%o and even more particularly at least 99% of identity with said sequence over the entire length of said sequence.
• Said surface layer protein can be selected from the group consisting of the surface layer protein A, the surface layer protein B, the surface layer protein C, the surface layer protein D, the surface layer protein E and the surface layer protein F of a Propionibacterium freudenreichii, in particular the surface layer protein A of a Propionibacterium freudenreichii.
• the Inventors have shown that said signal peptides, in particular in combination with promoters of genes encoding a surface layer protein of a Propionibacterium freudenreichii, allow the secretion into the extracellular medium of a substance of interest by a propionibacterium at a level up to 100 times greater than that obtained with other signal peptides in the same propionibacterium strain.
• said promoter and said signal peptide are the promoter and the signal peptide of the same gene encoding a surface layer protein of a Propionibacterium freudenreichii.
• nucleic acid sequence encoding a signal peptide is selected from the group consisting of:
• sequences having at least 80%>, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with one of said sequences over the entire length of said sequence and particularly corresponding to nucleic acid sequences encoding a signal peptide allowing the secretion, in particular into the extracellular medium, of said substance of interest by a propionibacterium.
• the inventors have demonstrated that the signal peptide having the sequence set forth in SEQ ID NO: 59 in combination with the promoter of the gene encoding the Surface layer protein A of a Propionibacterium freudenreichii allows the secretion into the extracellular medium of a substance of interest by different strains of Propionibacterium freudenreichii at a level up to 100 times greater than that obtained with other signal peptides and promoters in the same Propionibacterium freudenreichii strain.
• the vector according to the invention comprises the nucleic acid sequence SEQ ID NO: 69 or a sequence having at least 80%, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%o, at least 97%, at least 98%> and even more particularly at least 99% of identity with SEQ ID NO: 69 over the entire length of said sequence, trans lationally fused to said nucleic acid sequence encoding a substance of interest.
• sequences having at least 80% of identity with the nucleic acid sequence SEQ ID NO: 69 over the entire length of said sequence comprise a promoter and a sequence encoding a signal peptide, particularly a secretory signal peptide.
• the vector according to the invention comprises the nucleic acid sequence SEQ ID NO: 69 translationally fused to said nucleic acid sequence encoding a substance of interest.
• the nucleic acid sequence SEQ ID NO: 69 comprises the promoter and the signal peptide of the Surface layer protein A of the Propionibacterium freudenheimii CIRM BIA 118.
• the percentage of identity to which reference is made in the presentation of the invention are determined on the basis of global alignment of sequences to be compared, that is to say, on an alignment of sequences over their entire length, using for example the algorithm of Needlman and Wunsch 1970.
• This sequence comparison can be done for example using the needle software by using the parameter "Gap open” equal to 10.0, the parameter “Gap Extend” equal to 0.5, and a matrix "BLOSUM 62".
• Software such as needle is available on the website ebi.ac.uk worldwide, under the name "needle”.
• Said nucleic acid sequence encoding a signal peptide can be translationally fused with several nucleic acid sequences encoding a substance of interest. Said nucleic acid sequence encoding a signal peptide can be operatively linked to several promoters.
• said substance of interest can be any molecule of interest.
• said substance of interest is of eukaryotic origin, more particularly of animal origin and even more particularly of mammalian origin.
• said substance of interest can originate from mammals selected from the group consisting of rodents (e.g. mice, rats, rabbits, Chinese pigs, hamsters), canidae (e.g., dogs),felidae (e.g., cats), domestic livestock (e.g. cows, pigs, goats, sheeps, horses), and humans.
• rodents e.g. mice, rats, rabbits, Chinese pigs, hamsters
• canidae e.g., dogs
• felidae e.g., cats
• domestic livestock e.g. cows, pigs, goats, sheeps, horses
• said substance of interest is a peptide or a protein chosen in the group consisting of antibodies, receptor ligands, hormones, cytokines, growth factors, cell adhesion molecules, blood clotting factors, enzymes, fragments thereof and combinations thereof, more particularly in the group consisting of cytokines, antibodies and hormones, and even more particularly from the group consisting of proapoptotic TRAIL protein and the C- terminal extracellular domain of the TRAIL protein.
• a “fragment” of a protein or peptide is a protein or a peptide of amino acid sequence of smaller length but included in the one of said initial peptide or protein.
• a protein "fragment” could be a peptide, for example.
• the vector according to the present invention makes it possible to express and secrete the proapoptotic TRAIL protein (TNF- related apoptosis-inducing ligand, also called TNSF10, TL2, CD253 and Apo- 2L), a cytokine of the TNF family.
• the sequence of the Homo sapiens TRAIL protein can be the sequence set forth in SEQ ID NO: 58 (accession number AAC50332.1).
• said substance of interest can be the proapoptotic TRAIL protein or the C-terminal extracellular domain of the TRAIL protein, in particular the Homo sapiens proapoptotic TRAIL protein or the C- terminal extracellular domain of the Homo sapiens TRAIL protein.
• said substance of interest is the C-terminal extracellular domain of the TRAIL protein consisting of the sequence from amino acids 1 14 to 281 of the sequence set forth in SEQ ID NO: 58.
• TRAIL is an antineoplastic agent with strong potential because it induces the death of many tumor cells, independently of p53 and Pgpl80 (MDR, multidrug resistance). TRAIL also inhibits the growth of xenografted colon tumors in nude mice (Ashkenazi et al., 1999). Quite interestingly, TRAIL has little cytotoxic effect on most normal tissues (Ashkenazi et al., 1999), including human colon epithelium (Strater et al., 2002).
• propionibacteria have major advantages compared to other bacteria such as salmonellas, bifidobacteria and coliform bacteria.
• dairy propionibacteria enable local delivery because they target colon epithelial cells and have an active fermentative metabolism in the human colon (Herve et al., 2007), which enables site-specific delivery of TRAIL.
• GRAS food-quality bacteria
• these food-quality (GRAS: generally regarded as safe) bacteria adapt and survive in the digestive tract of animals and humans with an efficiency that, although strain-dependent, exceeds that of other probiotics (Herve et al., 2007).
• GRAS food-quality bacteria
• they express in the intestine enzymatic activities characteristic of their fermentative metabolism by producing an increase in SCFA concentrations (Lan et al, 2007b) and induce an increase in apoptosis in the mucosa of the colon of rats treated with 1 ,2-dimethylhydrazine (Lan et al., 2008).
• the Inventors have further shown a synergistic action with TRAIL in vitro as illustrated in the examples below.
• the vector according to the invention comprises:
• nucleic acid sequence encoding a signal peptide is selected from the group consisting of:
• sequence of said promoter is selected from the group consisting of:
• sequences having at least 80%>, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with said sequence over the entire length of said sequence.
• a particularly preferred vector according to the invention is the vector pCHH04: TRAIL comprising:
• nucleic acid sequence encoding the signal peptide of the Surface layer protein A of the Propionibacterium freudenreichii CIRM BIA 118, translationally fused to
• nucleic acid sequence encoding the sequence from amino acids 114 to 281 of the TRAIL protein sequence set forth in SEQ ID NO: 58.
• a P. freudenreichii CIRM-BIA-118 comprising said vector pCHH04: TRAIL has been deposited on November 13, 2012 under number CNCM 1-4692 with the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Dondel Roux, 75724 Paris Cedex 15, France).
• a particularly preferred vector according to the invention is the vector comprised in the propionibacterium deposited on November 13, 2012 under number CNCM 1-4692 with the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Dondel Roux, 75724 Paris Cedex 15, France).
• the present invention further relates to a bacterium comprising at least one vector according to the invention as previously defined.
• Said bacterium comprising a vector according to the invention can belong to the order Actinomycetales, in particular to the genus Propionibacterium, and more particularly to a species selected from the group consisting of Propionibacterium freudenreichii, Propionibacterium jensenii, Propionibacterium thoenii, Propionibacterium acidipropionicii, Propionibacterium acnes, Propionibacterium granulosum, Propionibacterium avidum, Propionibacterium propionicum and Propionibacterium microaerophilum and even more particularly the species Propionibacterium freudenreichii.
• said bacterium comprising a vector according to the invention can be selected from the group consisting of Propionibacterium freudenreichii freudenreichii and Propionibacterium freudenreichii shermanii.
• the propionibacterium comprising at least one vector according to the invention can be selected from the group consisting of CIRM BIA 512 strain, CIRM BIA 125 strain, CIRM BIA 118 strain, CIRM BIA 129 strain, CIRM BIA 138 and CIRM BIA 122 strain, particularly CIRM BIA 512 strain, CIRM BIA 125 strain, CIRM BIA 118 strain, CIRM BIA 129 strain and CIRM BIA 122 strain, more particularly CIRM BIA 512 strain, CIRM BIA 125 strain, CIRM BIA 118 strain and CIRM BIA 129 strain, even more particularly CIRM BIA 512 strain, CIRM BIA 125 strain, CIRM BIA 118 strain and even more particularly CIRM BIA 512 strain and CIRM BIA 1 18 strain and even more particularly CIRM BIA 118 strain.
• the propionibacterium comprising at least one vector according to the invention is a CIRM BIA 138 strain.
• This strain also named TL 133 strain in the article of Lan et al. (2007b) has the advantage to be particularly tolerant towards digestive stresses.
• the vector can be carried by the chromosome of the bacterium according to the invention.
• the vector can be integrated, for example, in the chromosome of the host cell by homologous recombination as described by Deutsch e* al. (2012).
• One particularly preferred bacterium according to the invention is the propionibacterium deposited on November 13, 2012 under number CNCM 1-4692 with the Collection Nationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 rue du Dondel Roux, 75724 Paris Cedex 15, France). If the substance of interest to be secreted lends itself to such a use (an anorexiant peptide, for example), the bacterium according to the invention could be used as a probiotic food or dietary supplement for mammals, in particular humans.
• the bacterium can be integrated in the mammal's food in the form of a fermented dairy product (e.g., fermented milk, fermented whey, cheese).
• the present invention further relates to a composition comprising at least one vector according to the invention and/or at least one bacterium according to the invention.
• composition according to the invention can be a dietary or a pharmaceutical composition.
• dietary compositions comprises any type of food/feed and beverages, also including clinical, nutrition and dietary supplements.
• the dietary compositions according to the present invention may further contain protecting components (such as carbohydrates, betaines, fibers, polyois), protective hydrocolloids (such as gums, proteins, modified starches), binders, film-forming agents, encapsulating agents/materials, wail/shell materials, matrix compounds, coatings, emu isi tiers, surface active agents, solubil ising agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste-masking agents, weighting agents, jei!yfying agents, gel-forming agents, antioxidants and ant imicrobials.
• protecting components such as carbohydrates, betaines, fibers, polyois
• protective hydrocolloids such as gums, proteins, modified starches
• binders film
• Said pharmaceutical composition according to the invention can comprise an effective amount of at least one vector according to the invention and/or at least one bacterium according to the invention and at least one pharmaceutically acceptable carrier (or excipient).
• Such therapeutically effective amount can be determined by one skilled in the art by routine tests including assessment of the effect of administration of said components (vector according to the invention and/or bacterium according to the invention) on the pathologies and/or disorders which are sought to be prevent and/or to be treated by the administration of said drug (or pharmaceutical composition).
• such tests can be implemented by analyzing both quantitative and qualitative effect of the administration of different amounts of said aforementioned components (vector according to the invention and/or bacterium according to the invention) on a set of markers (biological and/or clinical) characteristics of said pathologies and/or of said disorders, in particular from a biological sample of a subject.
• the present invention further relates to a vector according to the invention or a bacterium according to the invention for use as a medicament.
• the invention further relates to a vector according to the invention or a bacterium according to the invention for use as a medicament in the prevention and/or treatment of at least one disease selected from the group consisting of allergies, hypertension (e.g., the substance of interest has a hypotensive activity), obesity (e.g., the substance of interest has an anorexiant activity), cancers, in particular colorectal cancers (e.g., the substance of interest has a proapoptotic activity) and inflammatory colon diseases, in particular Crohn's disease (wherein the substance of interest is advantageously an anti- inflammatory cytokine, for example IL-10).
• a disease selected from the group consisting of allergies, hypertension (e.g., the substance of interest has a hypotensive activity), obesity (e.g., the substance of interest has an anorexiant activity), cancers, in particular colorectal cancers (e.g., the substance of interest has a proapoptotic activity) and inflammatory colon diseases, in
• the invention further relates to a vector according to the invention or a bacterium according to the invention for use as a medicament in the prevention of at least one microbial infection, including a viral, bacterial, fungal or parasitic infection.
• the substance of interest can be an antigen or an epitope.
• said medicament can be a vaccine, which can further comprise an immune adjuvant.
• the invention further relates to a vector according to the invention or a bacterium according to the invention, in particular a bacterium belonging to the genus Propionibacterium and more particularly to the species Propionibacterium freudenreichii for use as a medicament in the prevention and/or treatment of cancer; in particular wherein said substance of interest is an anti-tumoral agent like the proapoptotic TRAIL protein or the C-terminal extracellular domain of the TRAIL protein (in particular of sequence from amino acids 114 to 281 of the protein TRAIL sequence set forth in SEQ ID NO: 58).
• anti-tumoral agent relates to any compound having an anti-tumoral effect.
• the anti-tumoral agent can have a preventing or inhibiting effect on the formation or growth of tumors or tumor cells. This can be a pro-apoptotic, an anti-proliferative, a cytostatic or a cytotoxic effect.
• the invention further relates to the use of a vector or a bacterium according to the invention for the preparation of a medicament for the prevention and/or treatment of at least one disease, in particular selected from the group consisting of allergies, hypertension (e.g., the substance of interest has a hypotensive activity), obesity (e.g., the substance of interest has an anorexiant activity), cancers, in particular colorectal cancers (e.g., the substance of interest has a proapoptotic activity) and inflammatory colon diseases, in particular Crohn's disease (wherein the substance of interest is advantageously an antiinflammatory cytokine, for example IL-10).
• a vector or a bacterium according to the invention for the preparation of a medicament for the prevention and/or treatment of at least one disease, in particular selected from the group consisting of allergies, hypertension (e.g., the substance of interest has a hypotensive activity), obesity (e.g., the substance of interest has an anorexiant activity), cancers, in particular
• the invention further relates to the use of a vector or a bacterium according to the invention for the preparation of a medicament in the prevention of at least one microbial infection, including a viral, bacterial, fungal or parasitic infection.
• the substance of interest can be an antigen or an epitope.
• said medicament can be a vaccine, which can further comprise an immune adjuvant.
• the medicament or the composition according to the invention can be manufactured in a conventional way.
• the medicament (pharmaceutical composition) in accordance with the invention can moreover include one or more pharmaceutically acceptable excipients or additives such as diluents, adjuvants, anti-foaming agents, stabilizers, dispersants, colorants, preservatives, etc.
• Inert excipients or adjuvants can be used in such a way that, in the drugs according to the present invention, the only therapeutic agents will be the vector and/or the bacterium.
• the medicament (pharmaceutical composition) according to the present invention can include one or more other therapeutically or prophylactically active agents, in addition to the vector and/or the bacterium.
• the combination of several therapeutic agents including at least the vector and/or the bacterium, will have a better therapeutic or prophylactic action than when the vector and/or the bacterium are the only therapeutic agents present in the medicament (pharmaceutical composition).
• This better action can be, among others:
• the various means of the present invention are preferably administered to a mammal for the secretion of the substance of interest in the small intestine and/or the colon, preferably the colon, of said mammal.
• mammal is defined in its usual sense. Examples of mammals include bovines; pigs; goats; sheep; horses; rodents such as mice, rabbits, rats and hamsters; felines and canines, including domestic animals such as cats and dogs. A preferred mammal in the context of the invention is a human.
• the means of the invention can be administered by any suitable conventional route, in particular selected from the oral, subcutaneous, intramuscular, intravenous, intrarectal, enema and intratracheal routes.
• Oral administration is preferred, wherein the drug is in the form of tablets, hard gelatin capsules (e.g., gastroprotective gelatin capsules), soft capsules, powders for direct use or for dilution (e.g., lyophilisates), syrups, gels, etc.
• Said means can be administered in a single or repeated dose one or more times spaced over a certain interval of time.
• the suitable administration route and dosing schedule can vary according to various parameters, such as the subject to be treated and/or the substance of interest.
• the invention also relates to a method for therapeutic or prophylactic treatment of a subject in need thereof, comprising the step of administering to said subject a therapeutically effective amount of at least one compound selected from the group consisting of:
• the invention also relates to a method for therapeutic or prophylactic treatment of a disease, in particular a cancer, more particularly a colorectal cancer, comprising the step of administering to a subject in need thereof a therapeutically effective amount of at least one compound selected from the group consisting of:
• a bacterium according to the invention in particular a bacterium belonging to the genus Propionibacterium and more particularly to the species Propionibacterium freudenreichii; and a medicament (pharmaceutical composition) according to the invention;
• said substance of interest is the proapoptotic TRAIL protein or the C-terminal extracellular domain of the TRAIL protein (in particular of sequence from amino acids 114 to 281 of the protein TRAIL sequence set forth in SEQ ID NO: 58).
• the present invention further relates to the use of a vector and/or a bacterium according to the invention to produce and secrete, preferably into the extracellular medium, one or more substance of interest.
• the present invention further relates to a method for producing and secreting into the extracellular medium, at least one substance of interest, by at least one bacterium according to the invention, said method comprising the steps of:
• the method according to the invention makes it possible to produce and secrete substances of interest on a large scale, that is, on an industrial scale.
• suitable conditions in terms of the composition of the culture medium, temperature, time, ventilation, stirring, etc.
• suitable conditions in terms of the composition of the culture medium, temperature, time, ventilation, stirring, etc.
• propionibacteria are known to those persons skilled in the art (see in particular documents US 20090312425 in the name of Meiji Dairies Corp. and CN 101045910 in the name of Nanjing University of Technology).
• propionibacteria are robust, are able to grow on particular substrates such as whey or molasses and are able to adapt to non-standard culture conditions, characteristics which a large number of other bacteria do not share.
• the invention further relates to a product comprising:
• At least one vector according to the invention and/or at least one bacterium according to the invention and/or another active agent, in particular chosen in the group consisting of an anti-tumoral agent, an anti- inflammatory agent and an immunomodulatory agent, more particularly an anti-tumoral agent;
• the substance of interest has a hypotensive activity
• obesity e.g., the substance of interest has an anorexiant activity
• cancers in particular colorectal cancers (e.g., the substance of interest has a proapoptotic activity) and inflammatory colon diseases, in particular Crohn's disease (wherein the substance of interest is advantageously an antiinflammatory cytokine, for example IL-10).
• Said active agent can be a short-chain fatty acid (SCFA), preferably propionate or acetate.
• SCFA short-chain fatty acid
• the invention also relates to an isolated nucleic acid molecule of sequence selected from the group consisting of:
• sequences having at least 80%, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with one of said sequences over the entire length of said sequence.
• sequences having at least 80% of identity with one of the nucleic acid sequences encoding the peptides of sequences SEQ ID NO: 59 to 68 over the entire length of said sequence encode a signal peptide, particularly a secretory signal peptide.
• sequences having at least 80% of identity with the nucleic acid sequence SEQ ID NO: 69 over the entire length of said sequence comprise a promoter and a sequence encoding a signal peptide, particularly a secretory signal peptide.
• the invention further relates to a vector comprising at least one nucleic acid sequence selected from the group consisting of: - the nucleic acid sequences encoding the peptides of sequences SEQ ID NO: 59 to 68;
• sequences having at least 80%, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with one of said sequences over the entire length of said sequence.
• the invention also relates to a bacterium comprising a vector comprising at least one nucleic acid sequence selected from the group consisting of:
• sequences having at least 80%>, more particularly at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and even more particularly at least 99% of identity with one of said sequences over the entire length of said sequence.
• FIG. 1 Graphs illustrating the synergy observed in vitro between TRAIL and P. freudenreichii metabolites.
• HT29 colon cancer cells were treated with sublethal doses of TRAIL (25 ng/ml, 50 ng/ml and 100 ng/ml).
• SCFA propionate/acetate SCFA, figure 1 A
• P. freudenreichii supernatant FIG. IB
• Figure 2 Identification of the protein secreted in the majority by P. freudenreichii, PF963.
• A growth of two strains of P . freudenreichii, one auto lytic ( ⁇ ) and the other nonlytic (o).
• B and C electrophoretic analysis (SDS-PAGE) of proteins secreted by a strain of lytic (B) and nonlytic (C) P. freudenheimii.
• Protein PF963 was identified by mass spectrometry.
• Figure 3 Diagram detailing the cloning strategy to obtain the pFB4:TRAIL plasmid (deposited with the CNCM on July 23, 2009 under number 1-4213).
• A the promoter region and signal peptide of the P.
• freudenreichii protein PF963 were amplified by PCR with introduction (PCR mutagenesis) of restriction sites Nde ⁇ and ⁇ .
• the active extracellular portion of TRAIL (residues 1 14 to 281) was amplified by PCR with introduction of restriction sites HmdIII and Pstl .
• B and C the two PCR products were purified and linked in order to obtain the ligation product SP-TRAIL.
• D plasmid pK705 was opened by digestion using two enzymes Ndel and Pstl . The ligation product SP-TRAIL was introduced into the open plasmid.
• the new plasmid pFB4 includes a promoter and a signal peptide enabling the secretion, in particular into the extracellular medium, by P. freudenreichii of a heterologous protein.
• the arrows and the scissors represent PCR primers and restriction sites, respectively.
• Figure 4 Map of the pFB4:TRAIL plasmid (deposited with the CNCM, Institut Pasteur, 28 rue du Dondel Roux, F-75724 Paris Cedex 15, on July 23, 2009 under number 1-4213).
• Figure 5 Sequence of the fusion protein coded for by the pFB4:TRAIL plasmid.
• the underlined region corresponds to the PF963 protein signal sequence and the region in bold corresponds to the TRAIL extracellular domain sequence (residues 1 14 to 281 ).
• Figure 6 Detection by Western blot of the fusion protein coded for by the pFB4:TRAIL plasmid.
• the samples deposited were culture supernatants of wild P. freudenreichii CIP103027 (1) or of P. freudenheimii CIP103027 carrying the pFB4:TRAIL plasmid (2 and 3).
• a solution of Superjft/ZerTRAILTM (Alexis Biochemicals, Coger, France) was deposited as positive control (4).
• the Western blot was developed using a commercial "PAb to TRAIL" antibody (Alexis Biochemicals).
• Figure 7 Map of the pCHH04: TRAIL plasmid (deposited with the CNCM on November 13, 2012 under number CNCM 1-4692).
• Figure 8 Proteomic comparative analysis of Propionibacterium freudenreichii exoproteomes.
• Strains CIRM BIA 1 , 129, 1 18, 456, 127, 508, 516 and 512 were grown in YEL medium and the culture supernatants analyzed by SDS PAGE followed by Coomassie Blue staining.
• the reference strain CIRM BIA 1 the first sequenced in 2010, secretes 4 proteins with equivalent efficiency, including PF963.
• Figure 9 Western Blot detection.
• the plasmids pFB4:TRAIL and pCHH04:TRAIL are compared within the same CIRM BIA 118 strain with respect to TRAIL secretion into the extracellular medium. Secretion of TRAIL into the extracellular medium is then compared in different strains harboring the pCHH04:TRAIL plasmid :The pCHH04:TRAIL plasmid was transformed into different strains of Propionibacterium freudenreichii and the resulting supernatants were analyzed with respect to TRAIL secretion by western blotting.
• this bacterium induced the apoptosis of human colon adenocarcinoma cells in vitro via these SCFA which act on cancer cell mitochondria (Jan et al., 2002).
• the mitochondrial pathway of apoptosis induction has been clearly identified in the triggering of programmed cell death of HT29 cells by dairy propionibacteria (Jan et al., 2002; Lan et al., 2007a).
• Said SCFA cause the opening of mitochondrial permeability transition pores (PTP), the depolarization of mitochondria, the leaking of proapoptotic mitochondrial proteins and the activation of effector caspases.
• PTP mitochondrial permeability transition pores
• TRAIL is a cytokine capable of inducing the apoptosis of human colon cancer cells by binding to death receptors. TRAIL thus induces a different apoptotic pathway on the cellular and molecular levels and potentiates the action of other proapoptotic molecules used in cancer chemotherapy (Lacour et al., 2001; Lacour et al, 2003; Meurette et al, 2005; Meurette et al, 2006). Cell death induced by TRAIL or by SCFA is promoted by an acidic environment (Meurette et al, 2007; Lan et al, 2007a).
• Figure 1 shows that sublethal doses of TRAIL (25 ng/ml, 50 ng/ml and lOO ng/ml) do not significantly induce cell death during the treatment period. Moreover, the smallest doses of SCFA alone induce little or no cell death, but induce massive death in the presence of TRAIL (figures 1A and IB). These results show a synergy of proapoptotic action on human colon cancer cells between SCFA metabolites produced by P. freudenreichii and TRAIL. Ill-Development of a first recombinant propionibacterium with the goal of inducing both the intrinsic and extrinsic apoptotic pathways.
• the Inventors sought to make a bacterium, harmless to healthy cells, produce inducers of the two apoptotic pathways. These inducers are the SCFA produced by P. freudenreichii for the intrinsic pathway and TRAIL for the extrinsic pathway. Since propionibacteria have a positive tropism for the mucosa of the colon, said recombinant bacterium will not only be likely to produce TRAIL in situ in the colon, but also to carry SCFA and TRAIL toward colon epithelial cells.
• PF963 the major protein secreted by P. freudenreichii during its growth and in the absence of lysis.
• the experimental procedure e.g., electrophoresis, trypsino lysis, nano-LC and MS/MS
• PF963 the major protein secreted by P. freudenreichii during its growth and in the absence of lysis.
• the experimental procedure e.g., electrophoresis, trypsino lysis, nano-LC and MS/MS
• the supernatant of the nonlytic strain of P. freudenreichii was analyzed by electrophoresis.
• the gel fragment containing the major protein secreted was removed, rinsed and then subjected to "in gel" trypsin proteolysis.
• the resulting peptides were separated by nano-LC and then analyzed with tandem mass spectrometry (MS/MS).
• PF963 is an enzyme secreted via the machinery of the "Sec” pathway which recognizes and cleaves a signal peptide (SP).
• SP signal peptide
• This construction was carried out in E. coli on a cloning plasmid.
• the fusion thus obtained was introduced into an expression vector (pK705) previously developed for the cloning and expression of propionibacterial genes in dairy propionibacteria and efficient in P . freudenreichii (Kiatpapan et al., 2000) in order to express the fusion protein.
• the expression and the extracellular addressing of the fusion protein were then analyzed by Western blot.
• strains were screened on the basis of aptitude for autolysis. Indeed, it is known that certain strains of said bacterium make use of a programmed cell suicide, autolysis. In this case, cytoplasmic proteins are released in the surrounding medium.
• other strains including strain CIRM BIA 1 , do not undergo autolysis and on the contrary make use of a tolerance reaction with respect to various stresses, called the starvation- induced multi-tolerance response. In principle, these nonlytic strains thus only release actively secreted proteins and do not release proteins by accident.
• Figure 2A shows the evolution of the bacterial population for an autolytic strain ( ⁇ ) and for a nonlytic strain (o) of Propionibacterium freudenreichii subsp. shermanii.
• Figure 2C shows the electrophoretic analysis (SDS-PAGE) of proteins secreted by a nonlytic strain, CIRM BIA 1. This analysis reveals several secreted proteins, including protein PF963, identified in the culture supernatant of all the nonlytic strains tested. This protein was cut out of a preparative SDS-PAGE gel and subjected to digestion by trypsin.
• SDS-PAGE electrophoretic analysis
• ESI-MS/MS electrospray ionization tandem mass spectrometry
• QSTAR®XL hybrid triple quadrupole time-of- flight apparatus
• protein PF963 a secreted bacterial wall peptidase belonging to the NlpC/P60 family.
• the complete 5 sequence of protein PF963 (SEQ ID NO 36; table I) can be deduced after determination of the complete sequence of the genome of strain CIP 103027 by the Inventors.
• a signal peptide at the N-terminal end of PF963 indicates that this enzyme is secreted via the Sec secretion pathway.
• the sequence of said signal peptide is SEQ ID NO 36.
• the sequence of the pFB4:TRAIL plasmid was verified (SEQ ID NO 42).
• the portion corresponding to the fusion protein ranges from nucleotides 8451 to 9070. This portion is translated in figure 5 (SEQ ID NO 43): the sequence corresponding to the PF963 peptide signal protein is underlined and the Val 114 - Gly 281 sequence of TRAIL appears in bold.
• the fusion protein has a sequence of 205 amino acid residues corresponding to a mass of 23,190 Da and an isoelectric point of 9.08.
• the elimination of the signal peptide leads to a sequence of 171 amino acid residues corresponding to a mass of 19,822 Da and an isoelectric point of 8.60.
• the inventors have optimized the plasmid pFB4: TRAIL by changing both the promoter region and the signal peptide to obtain the pCHH04: TRAIL plasmid, allowing secretion into the extracellular medium, of a higher amount of TRAIL into the supernatant.
• pCHH04:TRAIL The construction of pCHH04:TRAIL is described in Figure 7.
• the pFB4:TRAIL plasmid which is a shuttle plasmid, was transformed into Escherichia coli.
• the propionibacterial insert containing the promoter PF963 and the signal peptide PS963 were excised using the appropriate restriction endonucleases.
• a new propionibacterial DNA fragment (set forth in SEQ ID NO: 69), containing the promoter region and the signal peptide of protein slpA of the Propionibacterium freudenreichii CIRM BIA 118 strain, was introduced in order to put the signal peptide in frame with the TRAIL coding sequence.
• the corresponding sequence was amplified by PCR (PSlpA fwd BamHl, which has the sequence set forth in SEQ ID NO: 70 and PS SlpA-EcoRV-Rev bis, which has the sequence set forth in SEQ ID NO: 71) from Propionibacterium freudenreichii CIRM BIA 1 genomic sequence, which was previously sequenced and annotated (Falentin et al., 2010a).
• This new plasmid, pCHH04:TRAIL was then transformed into different strains of Propionibacterium freudenreichii. The corresponding supernatants were then analyzed by western blot with respect to TRAIL secretion as above described.
• TRAIL concentration was further quantified by Enzyme- Linked Immunosorbent Assay (ELISA) in supernatant of Propionibacterium freudenreichii transformed with pFB4:TRAIL or pCHH04:TRAIL plasmid according to the manufacturer's instructions (R&D System Europe, Lille, France). Human TRAIL standard concentration-response curves were used to quantify TRAIL levels in supernatants. TRAIL concentrations were determined using an automatic plate reader associated with genesis software (LabSystems Spectrophotometer, Cambridge, UK) and data were expressed in ng/ml.
• TRAIL allows higher amounts of TRAIL secretion into the extracellular medium than the pFB4: TRAIL construction.
• the sip A promoter isolated from strain CIRM BIA 118, is most probably stronger than PF963 promoter.
• the slpA signal peptide allows efficient secretion of the TRAIL human cytokine in different Propionibacterium strains.
• Other sip proteins were then identified from the genomic sequence of several Propionibacterium freudenreichii strains.
• the corresponding signal peptides are presented in Table III and may also be used to allow heterologous protein expression in Bacteria, in particular belonging to the order Actinomycetales, in particular to the genus Propionibacterium, and more particularly to the species Propionibacterium freudenreichii.

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