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WO2023175124A1 - Bactéries d'administration de médicament - Google Patents

Bactéries d'administration de médicament Download PDF

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Publication number
WO2023175124A1
WO2023175124A1 PCT/EP2023/056845 EP2023056845W WO2023175124A1 WO 2023175124 A1 WO2023175124 A1 WO 2023175124A1 EP 2023056845 W EP2023056845 W EP 2023056845W WO 2023175124 A1 WO2023175124 A1 WO 2023175124A1
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Prior art keywords
bacterium
gene
seq
sequence identity
functional
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Inventor
Morten Otto Alexander SOMMER
Kira SARUP-LYTZEN
Mareike BONGERS
Ruben Vazquez URIBE
Max VAN 'T HOF
Frederik Bartholdy Flensmark NEERGAARD
Felipe Gonzalo TUEROS FARFAN
Susanne KAMMLER
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Danmarks Tekniske Universitet
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Danmarks Tekniske Universitet
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    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • 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
    • A61K35/741—Probiotics
    • 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
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • 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
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12Y104/03005—Pyridoxal 5'-phosphate synthase (1.4.3.5), i.e. pyridoxamine 5-phosphate oxidase
    • C—CHEMISTRY; METALLURGY
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    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)
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    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01003—Thiamine-phosphate diphosphorylase (2.5.1.3)
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04016—Thiamine-phosphate kinase (2.7.4.16)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Definitions

  • the present invention relates to a bacterium deficient in at least two functional genes in the redox pathway or at least one functional gene in the vitamin B1 synthetic pathway and at least one functional gene in the vitamin B6 pathway.
  • the invention also relates to the bacterium comprising an expression cassette for producing a therapeutic molecule.
  • the invention also relates to a kit with the bacterium and a plasmid; as well as a pharmaceutical formulation comprising the bacterium.
  • the invention also relates to use of the bacterium as a medicament, for example for treating Parkinson’s disease.
  • Genetically engineered bacteria can be used to deliver therapeutic molecules in the mammalian gut. However, their ability to survive and reproduce inside and outside a patient must be controlled to ensure safety and minimize release into the environment.
  • the inventors have developed novel therapeutic bacteria to deliver therapeutic molecules (medicaments).
  • a bacterium comprising an expression cassette for producing a therapeutic molecule (or medicament), wherein the bacterium is deficient in: a) at least two functional genes in the redox detoxification pathway; or b) at least one functional gene in the vitamin B1 synthetic pathway and at least one functional gene in the vitamin B6 synthetic pathway.
  • a kit comprising the bacterium according to the first aspect and a plasmid.
  • a pharmaceutical formulation comprising the bacterium of the first aspect.
  • a bacterium of the first aspect or a pharmaceutical formulation thereof for use as a medicament, for example, for use in a method of treating Parkinson’s disease.
  • the bacteria may be a therapeutic bacteria. By therapeutic is meant these cells are non- pathogenic.
  • the bacteria may be commensal. That is, the bacterium may be non- pathogenic and commensal.
  • the bacteria may be capable of colonizing the gut.
  • the bacteria may be a gut microbe (a bacteria belonging to the human gut microbiome or microflora). Bacteria capable of colonizing the gut may be tested using in vitro artificial models (e.g. SHIME, Simulator of Human Intestinal Microbial Ecosystem®).
  • non-pathogenic bacteria that are not capable of causing disease or harmful responses in a host.
  • the non-pathogenic bacteria may be from the genus Bacteroides or Escherichia.
  • the non-pathogenic bacteria may be a probiotic bacteria.
  • probiotic live, non-pathogenic microorganisms which can confer health benefits to a host organism, e.g. a human.
  • the non-pathogenic bacteria may be a synthetic bacteria.
  • the test for haemolytic activity may be used. This test uses blood agar plates and tests for the ability of the bacteria to lyse red blood cells in the vicinity of the bacterial colony. Other types of testing are also available such as sequencing and screening the genome of the bacteria for known pathogenicity factors (de Nies et al (2021), Microbiome 9: article number 49).
  • Suitable bacteria include Escherichia coli, for example E. coli Nissle.
  • suitable therapeutic cells include lactic acid bacteria for example Lactobacillus and/or Lactococcus.
  • Other examples of therapeutic cells include Akkermansia, for example Akkermansia muciniphila, Bifidobacterium, Bacteroides, Salmonella or Listeria.
  • Clostridium or Anaerobutyricum soehngenii for example where the deficiency is in the vitamin B1 or vitamin B6 pathways.
  • the genes specified are rendered inoperable or are not present in the genome to begin with in terms of a synthetic bacteria.
  • the genes or gene functions are deleted.
  • the bacteria does not contain the functional gene.
  • the enzyme encoded by the gene is not produced (conversely, functional means that the enzyme encoded by the gene is produced). This may be as a result of complete deletion of the coding sequences of the genes or partial deletion.
  • removing the functional gene may be accomplished by mutation of the coding sequence, for example by way of insertion or substitution of other sequences.
  • one or more mutations (such as frameshift mutations) may be generated such that any resulting RNA transcript codes for a non-functional or truncated protein.
  • an insertion may be made into the chromosomal sequence to disrupt the amino acid code.
  • the gene may be completely deleted or partially deleted, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the coding sequence may be deleted. This percentage is with respect to the entire sequence.
  • Non-functionality may be tested by:
  • the bacteria would be grown on +/- PLP (Vitamin B6); to test thiL deficiency, the bacteria would be grown on +/- TPP (Vitamin B1); to test sodA/B deficiency, the bacteria would be grown on minimal media +/- oxygen.
  • the bacteria if deficient will not grow without the supplemented media (or cannot grow in oxygen in the case of sodA/B).
  • the resulting cells lacking the Vitamin B1 and Vitamin B6 genes are also known as auxotrophs meaning they are mutant organisms that require a particular additional nutrient which the normal unmutated strain does not.
  • the expression cassette includes the machinery needed to produce the therapeutic molecule (medicament). This may include a promoter and the gene sequence of the therapeutic molecule (medicament). Alternatively this may include a promoter and a gene encoding an enzyme which when expressed produces the therapeutic molecule.
  • the bacterium is genetically engineered to include the expression cassette. The bacterium is a genetically engineered bacterium.
  • the expression cassette may be cloned into one of the native plasmids of a therapeutic bacteria.
  • the expression cassette may be cloned into a plasmid which is not native to the bacteria. The plasmid is then transformed into the bacteria.
  • the expression cassette may be integrated into the genome of the bacteria (the bacterial chromosome). This can be done using the CRISPR technique. Alternatively this can be done by various other methods including clonetegration (Shearwin et al (2013), ACS Synthetic Biology, Vol 2, pp537-541).
  • a promoter is a nucleotide sequence capable of controlling the expression of a gene.
  • the promoter may be a o 70 promoter or a modified version of such a promoter where the nucleotide composition has been optimized for in vivo expression levels.
  • the promoters claimed have been tested for predictability and robustness in the mammalian Gl tract. They have been selected from a large library of promoters, causing the most stable gene expression under any conditions (e.g. +/- oxygen, in exponential or stationary growth phase, in the upper and lower part of the Gl tract, in the lumen vs. in the mucus layer), which are important for making robust therapeutic bacteria.
  • any conditions e.g. +/- oxygen, in exponential or stationary growth phase, in the upper and lower part of the Gl tract, in the lumen vs. in the mucus layer
  • the promoter may have at least 70, 75, 80, 85, 90, 95 or 100% sequence identity to SEQ ID NO. 74 or 75.
  • the promoter may comprise or consist of SEQ ID NO.s 74 or 75.
  • the promoter for the tyrosine hydroxylase may have a consensus sequence as follows:
  • the promoter may be SEQ ID NO. 59 or 60 or a sequence comprising 90, 95 or 98% sequence identity with either SEQ ID NO. 59 or 60.
  • the promoter for any or all of FolE, FolM and/or FoIX may be an Anderson promoter (http://parts.igem.org/Promoters/Catalog/Anderson).
  • the promoter for any or all of FolE, FolM and/or FoIX may have a consensus sequence as follows (again with reference to the IIIPAC code above):
  • the promoter may be SEQ ID NO. 55-73 or a sequence comprising 70, 75, 80, 85, 90, 95 or 98% sequence identity with either SEQ ID NO. 55-73.
  • the promoter may consist of consensus sequence SEQ ID NO. 74 or 75.
  • Functional variants with different degrees of sequence identity can be checked for retention of activity by comparing expression of a suitable reporter under the control of the variant promoter and compare this activity with the reporter under the control of the non-variant promoter. It is generally preferred that a promoter with less that 100% sequence identity retains at least 25, 50, 75, 80, 85, 90, 95 or 100% activity of the reference promoter.
  • the promoters may be shortened at 1 or both ends of the sequence. This shortening may be by 1 or 2 nucleotides at 1 or both ends. These shortened variants can be checked for retention of activity as explained above.
  • kit or kit of parts
  • kit two or more components packaged together.
  • the bacterium in the kit are as defined herein.
  • the plasmid may comprise any of the promoters described herein.
  • the therapeutic molecule may be any of benefit to a patient. That is, the therapeutic molecule is a medicament.
  • the therapeutic molecule or medicament may be a protein encoded in the expression cassette; or the expression cassette may encode an enzyme which enables production of the therapeutic molecule.
  • the enzyme may also be referred to as a medicament, for example, tyrosine hydroxylase as described herein.
  • the medicament may be a heterologous medicament, for example a heterologous protein. By heterologous is meant not from a bacterial species.
  • the medicament may be a mammalian protein.
  • the bacterium may comprise an expression cassette for expression of a gene which encodes a medicament, wherein the bacterium is deficient in: a) at least two functional genes in the redox detoxification pathway; or b) at least one functional gene in the vitamin B1 synthetic pathway and at least one functional gene in the vitamin B6 synthetic pathway.
  • the gene may be a heterologous gene.
  • the medicament may be tyrosine hydroxylase.
  • therapeutic molecules examples include L-DOPA, serotonin, dopamine, nor-adrenaline, adrenaline and other tryptophan or tyrosine derivatives.
  • L-DOPA is a prodrug of dopamine that is administered to patients with Parkinson's due to its ability to cross the blood-brain barrier.
  • Dopamine which is produced by decarboxylation of L-DOPA, modulates blood pressure, and also has a role in immune modulation, adipose tissue metabolism, nutrient absorption, and modulation of gut-brain axis functions.
  • the expression cassette may comprise tyrosine hydroxylase as described above and a L-DOPA decarboxylase.
  • tryptophan or tyrosine derivatives molecules resulting from the biosynthetic pathways of tryptophan or tyrosine.
  • tryptophan or tyrosine derivatives that have therapeutic potential include: tryptamine (TRM) and/or phenethylamine (PEA).
  • PKA phenethylamine
  • others include: adrenaline, 5-HTP (5-hydroxytryptophan) and 5-HT (5-hyroxytryptamine).
  • genetically engineered is meant the bacteria has been modified from its native state.
  • the bacteria is a recombinant bacteria.
  • the vitamin B6 synthetic pathway is shown in Figure 6.
  • the bacteria may be deficient in any gene from this pathway as shown.
  • the non-functional gene may be epd, pdxB, se/C, pdxA, dxs, pdxJ or pdxH.
  • the non-functional gene is pdxH.
  • the non-functional gene may be Pyridoxal 5'-phosphate synthase subunit pdxS or pdxT. Therefore, where in the claims it is stated “vitamin B6 synthetic pathway”, this may include the DXP-independent pathway. Therefore, the non-functional gene may be epd, pdxB, serC, pdxA, dxs, pdxJ, pdxH, pdxS or pdxT.
  • a bacterium wherein the bacterium is a therapeutic bacterium (or a bacterium comprising an expression cassette for producing a therapeutic molecule) deficient in: a) at least two functional genes in the redox detoxification pathway, wherein the redox detoxification pathway comprises sodA, sodB and sodC; or b) at least one functional gene in the vitamin B1 synthetic pathway, wherein the vitamin B1 synthetic pathway comprises thiE or thiE, and at least one functional gene in the vitamin B6 synthetic pathway (wherein the vitamin B6 synthetic pathway comprises epd, pdxB, serC, pdx/X, dxs, pdxJ or pdxH, optionally epd, pdxB, serC, pdx/X, dxs, pdxJ, pdxH, pdxS or pdxT.
  • the vitamin B6 synthetic pathway comprises epd, pdxB, serC, pd
  • the bacteria may be deficient in: a) at least two functional genes from the list comprising sodA, sodB and sodC; or b) at least one functional gene from the list comprising thiE or thiL, and at least one functional gene from the list comprising: epd, pdxB, serC, pdx/X, dxs, pdxJ or pdxH, optionally epd, pdxB, serC, pdx/X, dxs, pdxJ, pdxH, pdxS or pdxT
  • the biocontained strain requires there to be two non-functional genes, the non-functional genes from two different metabolic pathways. Therefore, one of the above vitamin B6 defunct genes must be combined with a non-functional gene from another pathway. For example, the vitamin B1 synthetic pathway.
  • auxotrophs are conditional lethal mutants in which survival of the bacterium is dependent on the availability of a particular nutrient in its environment.
  • the vitamin B1 synthetic pathway is shown in Figure 7.
  • the bacteria may be deficient in any gene from this pathway.
  • the non-functional gene may be thiE or thiL.
  • the non-functional gene is thiL.
  • the resulting bacterium are therefore auxotrophs for vitamin B1.
  • thiL and pdxH are both non-functional.
  • the superoxide radicals degradation pathway is shown in Figure 8. This catalyzes the degradation of harmful superoxide into oxygen in the bacteria.
  • the bacteria may be deficient in any gene from this pathway.
  • the non-functional gene may be sodA or sodB.
  • sodA and sodB are non-functional.
  • SodC may still remain functional.
  • SodA and sodB may be the only deficient genes in this pathway.
  • Other combinations of inoperable genes are possible.
  • sodA and sodB genes may be knocked out/otherwise rendered inoperable in combination with pdxH/thiL.
  • sodA, sodB and pdxH may be non-functional.
  • sodA, sodB and thiL may be non-functional.
  • sodA, sodB, pdxH and thiL may be nonfunctional.
  • genes may also be rendered non-functional in addition to any of the combinations.
  • other genes which may be rendered inoperable and combined with any of the above double knockouts include any one or more of the following: hemF, hemN, chuA, ahpC and/or sufA.
  • these may be combined with the sodA/sodB knockout.
  • SodA and/or sodB may also be rendered non-functional in combination with at least one non-functional gene from the vitamin B1 synthetic pathway and/or vitamin B6 pathway.
  • a sequence listing is provided for the genes described above in the vitamin B1 an B6 pathways, and the redox detoxification pathway.
  • the genes provided are from E. coli Nissle.
  • the genes which are mutated; or partially or fully deleted or otherwise rendered inoperable may have at least 70% sequence identity to the SEQ ID NO.s provided. For example, at least 70, 75, 80, 85, 90, 95 or 100% sequence identity.
  • Sequence identity may be calculated using any suitable software such as BLAST (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool.” J. Mol. Biol. 215:403-410.)
  • a pharmaceutical formulation includes excipients to preserve the activity or to deliver the cell to the gut.
  • the formulation is an oral formulation.
  • the microbial cell i.e. bacterium
  • the microbial cell may be formulated to preserve its activity and/or for delivery to the gut via an oral tablet or capsule or the like.
  • the microbial cell may be lyophilized and include a lyoprotectant.
  • the formulation may alternatively or additionally include any other excipient required to preserve the activity of the cell.
  • the formulation may be in an oral dosage form with a coating which allows delivery to the gut, for example an enteric coating.
  • the bacteria can be used to express a therapeutic molecule (medicament), or one or more enzymes which catalyzes one or more reactions to produce a therapeutic molecule.
  • the therapeutic molecule can be produced in an amount effective to treat the disease or ameliorate the symptoms of the disease.
  • the expression cassette when the therapeutic molecule is L-DOPA may encode a eukaryotic tyrosine hydroxylase (SEQ ID NO 36, 38, 40 or 42). Additionally, the expression cassette (or an additional expression cassette) may include further genes which express cofactors. For example, the expression cassette may also include FoIX, FolE, FolM and/or phhB. Preferably the expression cassette contains the FolE and FolM genes.
  • the eukaryotic tyrosine hydroxylase (TyrOH) is a member of the biopterin-dependent aromatic amino acid hydroxylase family of non-heme, iron(l Independent enzymes. TyrOH catalyzes the conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA) as shown in Figure 9.
  • the tyrosine hydroxylase of the invention may belong to EC 1.14.16.2.
  • the enzyme may be an animal enzyme.
  • the sequence of the full length rat tyrosine hydroxylase is as follows:
  • the above sequence is SEQ ID NO. 36.
  • the tyrosine hydroxylase may have at least 70, 75, 80, 85, 90, 95, 97 or 100% sequence identity with SEQ ID NO. 36.
  • the enzyme may be truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 (for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids from the N and/or C termini of the construct.
  • the tyrosine hydroxylase may be a mutant, i.e. the enzyme differs from the full length wild type enzyme sequence.
  • the wild type full length rat enzyme comprises:
  • the mutant may not comprise the regulatory domain.
  • the entire regulatory domain may be deleted or only part of the regulatory domain may be deleted.
  • Truncation may be at any point in the regulatory domain to reduce the complexity of the protein for expression in a microbial cell and/or to decrease negative feedback by dopamine for the dopamine-producing microbial cell.
  • the skilled person would be aware of suitable points to truncate the regulatory domain whilst maintaining the activity of the enzyme guided by the crystal structure (Goodwill, K., Sabatier, C., Marks, C. et al. Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for inherited neurodegenerative diseases. Nat Struct Mol Biol 4, 578-585 (1997).
  • the tyrosine hydroxylase may comprise the catalytic domain (and not the regulatory domain or tetramer domain); or the catalytic domain and the tetramer domain (and not the regulatory domain). These domains may comprise the above amino acids sequences or have at least 70, 75, 80, 85, 90, 95, 99 or 100% sequence identity with the above amino acid sequences, and optionally be further truncated to the core secondary structure elements to provide function, for example by removing 1-20 (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acids from the N and/or C termini of the constructs.
  • 1-20 for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
  • the truncated enzyme may comprise the catalytic and tetramer domains, amino acids:
  • the truncated enzyme may comprise the catalytic domain only: SAREDKVPWFPRKVSELDKCHHLVTKFDPDLDLDHPGFSDQVYRQRRKLIAEIAFQYKHGE PIPHVEYTAEEIATWKEVYVTLKGLYATHACREHLEGFQLLERYCGYREDSIPQLEDVSRFL KERTGFQLRPVAGLLSARDFLASLAFRVFQCTQYIRHASSPMHSPEPDCCHELLGHVPMLA DRTFAQFSQDIGLASLGASDEEIEKLSTVYWFTVEFGLCKQNGELKAYGAGLLSSYGELLHS LSEEPEVRAFDPDTAAVQPYQDQTYQPVYFVSESFNDAKDKLRNYASRIQRPF (amino acids 158-456 of SEQ ID NO. 36; SEQ ID NO. 81).
  • the truncated enzyme may be amino acids 1-301 of SEQ ID NO. 38.
  • the tyrosine hydroxylase may be any sequence having at least 70, 75, 80, 85, 90 or 95% sequence identity to the above truncated forms.
  • the enzyme may additionally be truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 (for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids from the N and/or C termini of the construct.
  • the tyrosine hydroxylase may alternatively or additionally comprise a mutation in the catalytic domain.
  • the mutation may be in amino acids 177-198 of SEQ ID NO. 36. These amino acids form a loop as shown by the crystal structure of the enzyme.
  • the amino acid mutated in this loop may be at position 196.
  • the mutant may be Ser 196Glu or Ser196Leu. These are shown below in the rat full length enzyme, and truncated enzyme.
  • the mutation in the truncated form corresponds to position 41 , optionally to Glu/Leu (Ser 40 without the start codon).
  • the tyrosine hydroxylase may comprise any of the truncated forms above and additionally comprise a mutation in the loop: CHHLVTKFDPDLDLDHPGFSDQ (SEQ ID NO. 76), optionally at the underlined serine position.
  • the mutant may be SEQ ID NO. 40 or 42, or a mutant with at least 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO. 40 or 42.
  • the tyrosine hydroxylase may have at least 70, 75, 80, 85, 90, 95 or 100% sequence identity with any of the above mutant forms. Additionally, the mutant may be further truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 amino acids (for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20) from the N and/or C termini of the constructs.
  • 1 to 20 amino acids for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20
  • the inventors have surprisingly found that the above mutants (with the mutation at position 196 in the full length sequence and position 41 in the truncated sequence without the regulatory domain) produced less L-DOPA, for example 5, 10, 15 or 20% less L-DOPA compared to the wild-type. This may for example allow modulation of the L-DOPA released.
  • the GTP cyclohydrolase I may belong to E.C. 3.5.4.16.
  • the GTP cyclohydrolase I may have at least 70, 75, 80, 85, 90, 95 or 100% sequence identity with SEQ ID NO. 44.
  • the enzyme may additionally be truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 (for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids from the N and/or C termini of the construct.
  • the mutation may increase hydroxylation of the tyrosine hydroxylase by at least 120% as compared to the native or wild-type unmutated enzyme (under the same conditions).
  • the mutation may be at any one of the following positions in SEQ ID NO. 44:
  • D97-E112, K121-D130, N170-H180, S193-L200 and S207-N222 For example, D97, M99, T101 , V102, A125, K129, N170, V179, T196, T198 (excluding T198P), S199, L200, S207, H212, E213, F214, L215 and H221.
  • the mutation may be selected from: D97V, D97L, D97A, D97T, M99C, M99T, M99V, M99L, M99I, T1011, T101V, T101 L, V102M, N170K, N170D, N170L, V179A, V179M, T196I, T196V, T196L, T198I, T198V, T198S, T198L, S199Y, S199F, L200P, L200C, L200S, L200A, S207R, S207K, S207M, H212R, H212K, E213K, E213R, F214A, F214G, F214S, L215P, L215Q, L215N, L215D, L215T, L215S, L215G, L215A, L215C, L215F, L215M, H221 R and H221K.
  • the mutant may also comprise any combination of these mutations.
  • the GTP cyclohydrolase I mutant may have at least 70% sequence identity with SEQ ID NO. 44, and comprise any one or more of the above mutations.
  • the GTP mutant may be the endogenous, native GTP cyclohydrolase which is mutated i.e. not an additional recombinant copy.
  • the microbial cell may over-express (compared to the wild-type under the same conditions) any nucleic acid encoding:
  • SEQ ID NO. 52 4a-hydroxytetrahydrobiopterin dehydratase (SEQ ID NO. 52): phhB (SEQ ID NO. 51); and/or dihydroneopterin triphosphate 2’-epimerase (SEQ ID NO. 54): FoIX (SEQ ID NO. 53); and/or dihydromonapterin reductase (SEQ ID NO. 48): FolM (SEQ ID NO. 47)
  • the nucleic acid may also be any encoding enzymes with these activities and having at least 70, 75, 80, 85, 90, 95 or 100% sequence identity with the above SEQ ID NO.s.
  • the enzymes may additionally be truncated to the core secondary structure elements to provide function, for example by removing 1 to 20 (for example 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids from the N and/or C termini of the constructs.
  • the microbial cell may have increased activity of FolE and/or FolM.
  • the microbial cell comprises a recombinant nucleic acid encoding a eukaryotic tyrosine hydroxylase (for example, a tyrosine hydrolase with at least 70% sequence identity to SEQ ID NO. 38) and upregulated FolE and/or FolM. This may be by additional recombinant FolE and/or FolM being added to the cell.
  • the FolE enzyme may be mutated as described above.
  • the microbial cell comprises a recombinant nucleic acid encoding a eukaryotic tyrosine hydroxylase (for example, a tyrosine hydrolase with at least 70% sequence identity to SEQ ID NO. 38) and utilizes the endogenous FolE and FolM cofactors.
  • a eukaryotic tyrosine hydroxylase for example, a tyrosine hydrolase with at least 70% sequence identity to SEQ ID NO. 38
  • the FolE enzyme may be mutated as described above.
  • tyrosine hydroxylase (or example, a tyrosine hydrolase with at least 70% sequence identity to SEQ ID NO. 4) may be under a promoter comprising or consisting of consensus SEQ ID NO. 74.
  • Expression of one or more of the co-factors (for example, FolE and/or FolM) may be under the control of a promoter comprising or consisting of SEQ ID NO. 75.
  • the enzymes (and optionally the promoters described above) are preferably integrated into the genome of the cell.
  • Upregulating expression may be via a recombinant nucleic acid, for example an additional copy of the gene on a plasmid or integrated into the genome, or alternatively via upregulating the endogenous sequence.
  • Vitamin B1 synthetic pathway
  • Vitamin B6 synthetic pathway
  • Figure 1 shows Growth of auxotroph mutants in vitro.
  • Bacterial growth in LB medium a) without (-) or b) with (+) supplementation of the missing nutrients, under aerobic conditions; c) Growth in LB medium without supplementation under anaerobic conditions; d) Growth in SYNTHES synthetic wastewater medium, composed to accurately reflect the average abundance of micro- and macronutrients in wastewater; e) Growth in OECD medium, a less complex synthetic medium recommended by the OECD to simulate wastewater conditions (OECD, 2001); f) Summary of the final biomass yield achieved in d) and e), compared to the wild-type. All data are shown as mean +/- standard deviation from at least 3 biological replicates. Figure 2 shows Survival of auxotrophic strains in simulated sewage conditions.
  • auxotroph strains depend on nutrients provided by the faeces (and residing microbiota), whereas the wild-type actually performs better at higher dilutions, due to less competition for the available nutrients from the other bacteria.
  • FIG 4 shows Abundance of auxotroph strains in mouse Gl tract 8 days after single gavage. Data was collected on the day of dissection. Same mice were used as in Figure 3.
  • FIG. 6 shows the PLP (Vitamin B6) biosynthetic pathway
  • FIG. 7 shows the TPP (Vitamin B1) biosynthetic pathway
  • Figure 8 shows the Superoxide radicals degradation pathway
  • Figures 9-11 show the L-DOPA pathway.
  • Figure 9 provides an overview of the pathway.
  • Figures 10 and 11 respectively show in more detail the co-factor needed to synthesize L- DOPA and the making of this co-factor.
  • Figure 12 shows the Production of tryptamine in the auxotrophic strains. WT and knockout strains were grown in minimal media supplemented with the missing metabolites.
  • Figure 13 shows the Production of L-DOPA.
  • WT and the double knockout strain ApdxH/thiL were grown in a plate-reader in media adjusted to different pH from 3,5-7 with 5% oxygen.
  • L- DOPA concentration measured by HPLC is shown on the y-axis.
  • N 3 biological replicates per group, mean +/- standard deviation is shown.
  • Figure 14 shows the Colonization of auxotrophic E.coli Nissle strains in an NMRI mouse model without antibiotic treatment.
  • CFU counts were measured in the faeces at indicated time points and in gut content at study endpoint day 11.
  • Figure 15 shows Colonization over the first 24 hours in faeces after first gavage of 10 11 CFU per animal for the individual strains compared to the WT.
  • the y-axis gives the abundance of the bacterial strain in CFU/g faeces normalized to the sample weight, limit of detection is 75 CFU/g, x-axis shows the measured timepoints.
  • N 5-6 biological replicates per group, mean +/- standard deviation is shown.
  • Figure 17 shows Colonization of the individual auxotrophic strains in faeces over 11 days compared to the WT strain as described for figure 15.
  • N 5-6 biological replicates per group, mean +/- standard deviation is shown.
  • Example 1 Making of the auxotrophic cell line
  • Knockouts of essential genes were performed using CRISPR/Cas9 as described previously (Hao Luo et al., ACS Synthetic Biology 2020 9 (3), 494-499 DOI: 10.1021/acssynbio.9b00488). Briefly, a two- plasmid system consisting of an inducible cas9/A-Red expression plasmid and a guide RNA (gRNA) plasmid were used to introduce double-strand breaks at the desired genomic loci. gRNAs were designed using CRISPy-web after uploading the EcN genome sequence (GenBank: CP007799.1).
  • Templates for homologous recombination at the cut site were generated as follows: loci were amplified using oligos binding 500 bp up and downstream of the gene of interest and then fused using overlap-extension PCR to generate dsDNA products of approximately 1 kb. The resulting dsDNA fragments were purified and co-transformed with the gRNA plasmid to generate markerless gene knockouts in EcN. CRISPR/Cas9 and gRNA expression plasmids were cured from the strains as described previously and the sensitivity to used antibiotic markers was confirmed by plating on solid media +/- antibiotics.
  • dapk mutants Existing biocontainment cell lines include dapk mutants. These cells lack a functional dapk gene.
  • the dapk gene encoding 4-hydroxy-tetrahydropicolinate synthase, is involved in synthesizing lysine and diaminopimelic acid (required for cell wall biosynthesis) and therefore makes the bacteria dependent on exogenous diaminopimelate (DAP) for cell wall biosynthesis and growth.
  • DAP diaminopimelate
  • Further lysine auxotrophs include the /ysA mutant.
  • the LysA protein catalyzes the last step in the lysine biosynthetic pathway forming lysine.
  • the protein expressed from the dapk gene catalyses one of the initial steps in the lysine and diaminopimelic acid biosynthetic pathway.
  • a further known biocontainment cell line is the thyk mutant. These cells lack a functional thyk gene.
  • the thyk gene is involved in synthesis of thymine, a building block of DNA. Therefore thyk, like lysk and dapk, is another essential gene.
  • Bacterial growth curves Three single colonies were picked from each strain, grown in 300 pl LB+ medium (composition see below) in 96 deep-well plates and shaken at 250 rpm at 37°C for 16 hours. The cells were pelleted at 4000 x g for 10 minutes, resuspended in PBS, this washing step was repeated 2 more times. Cells were then again resuspended in PBS and the main culture was inoculated by diluting this preculture 1 :100 into 200 pl of the different media detailed below. Cells growth was followed at 37°C with 700 rpm orbital shaking using the BioTekTM ELx800 plate reader by measuring the optical density at 630nm every 10 minutes for 24 h. For anaerobic growth, a plate reader placed in an anaerobic chamber was used.
  • LB- medium contained 5 g/L yeast extract, 10 g/L tryptone and 10 g/L NaCI. LB+ medium additionally contained 1 pM TPP, 12 mg/L PLP, 100 mg/L diaminopimelic acid (DAP), 2 mM thymine.
  • SYNTHES medium was prepared according to Aiyuk S, Verstraete W (2004) Sedimentological evolution in an LIASB treating SYNTHES, a new representative synthetic sewage, at low loading rates. Bioresour Technol 93(3):269-278. doi: 10.1016/ j. biotech.2003.11.006. The medium was prepared using tap water, pH was adjusted to 7.0 and the medium was autoclaved before use.
  • OECD synthetic wastewater medium was prepared according to OECD (2001), Test No. 303: Simulation Test - Aerobic Sewage Treatment -- A: Activated Sludge Units; B: Biofilms, OECD Guidelines for the Testing of Chemicals, Section 3, OECD Publishing, Paris.
  • LB Lysogeny broth
  • Fig 1a an increase in lag time
  • Fig 1b a decrease in the maximum specific growth rate
  • the two novel knockout combinations ApdxH/thiL and AsodA/B show no growth under wastewater conditions, which was also confirmed by plating cells onto solid SYNTHES media and incubating for 3 days at 37C, after which no visible growth was observed for the knockouts, while the wild-type had formed full colonies (data not shown).
  • Example 3 Further in vitro testing in simulated sewage systems supports lack of survival of double knock-outs outside of the gut
  • Washed cells of auxotroph strains were added to the wastewater samples at a density of 10 8 CFU/mL and incubated for 25 days at room temperature in 96-deep-well plates with a loosely fitted lid to limit evaporation but allow some gas transfer. At the indicated time points, samples were taken, diluted in SYNTHES medium and plated on LB+ medium to count viable colonies (CFUs). Total doublings of the auxotroph strains were calculated from the known initial and the maximum cell density reached during the experiment.
  • Example 4 ln vivo testing shows double knock-outs are able to survive in mammalian gut and supply therapeutic molecule at a constant concentration
  • AMTs In order to produce and deliver small molecule therapeutics effectively, AMTs need to be able to survive and remain metabolically active for a certain amount of time inside the patient.
  • Example 5 Testing small molecule production in novel strains shows consistent release of therapeutic molecule
  • a requirement for effectiveness of small molecule-delivering AMTs is their ability to produce the desired compounds in vivo, despite the essential gene knockout(s). While it is challenging to simulate Gl tract conditions in vitro, testing production under lab conditions enables strain comparison under more controlled conditions and known nutrient availability. Therefore, as a model compound for tyrosine and tryptophan derivate pathways we tested production of serotonin (5-hydroxytryptamine, 5-HT), an important hormone and neurotransmitter produced in the mammalian gut and nervous system, in our engineered and biocontained AMTs, as a representative molecule that requires active amino acid and cofactor metabolism, as well as oxygen usage, when produced in bacteria.
  • serotonin (5-hydroxytryptamine, 5-HT
  • strains were transformed with plasmid pMUT27 which confers serotonin production capability.
  • Cells were grown in a modified M9+ medium containing 1x M9 salts (M6030, Sigma Aldrich), 0.2% (w/v) glucose, 0.1 % (w/v) casamino acids (Cat. No. C2000, Teknova), 1 mM MgSCL, 50 pM FeCh, 0.2% (v/v) 2YT medium, and 50 mg/L of L-tryptophan. All auxotrophy supplements were added at the concentrations described in Example 2. Kanamycin was added at a final concentration of 50 mg/L.
  • LC-MS liquid chromatography mass spectrometry
  • the selection of gene knockouts can impact metabolite production, such as increased production of the main product ( 5-HT) compared to wildtype and the complete loss of the by-product tryptamine (TRM), and reduction of phenethylamine (PEA) formation in the ApdxH and ApdxH/thiL strains.
  • metabolite production such as increased production of the main product ( 5-HT) compared to wildtype and the complete loss of the by-product tryptamine (TRM), and reduction of phenethylamine (PEA) formation in the ApdxH and ApdxH/thiL strains.
  • TDC heterologous enzyme tryptophan decarboxylase
  • serotonin production requires pyridoxal phosphate for activity, shifting the balance favourably towards serotonin in these strains (Fig 5).
  • DAP and Thymidine are needed in relatively high concentrations to supplement the KO, these need to be added to the fermenter when growing the biomass, potentially adding significant cost (DAP: 200 USD 1 5g, need 100 mg/L, Thymidine 80 USD 1 5g, need 500 mg/L; PLP: 120 USD / 5g, need 12 mg/L; TPP: 40 USD / 5g, need 0.5 mg/L).
  • DAP+THY would add about 12 USD/L broth
  • PLP+TPP would add 0,3 USD/L.
  • socW/B KO no complementation of the broth is required if grown micro- or anaerobic.
  • Example 5 shows consistent in vitro production of serotonin from the biocontained strains and increased serotonin production in the ApdxH and ApdxH/thiL knockout strains.
  • this example we support this data showing production of a second tryptophan derivative: tryptamine.
  • strains were transformed with plasmid pMUT28 which confers tryptamine production capability as product.
  • Cells were grown in a modified M9+ medium as described in example 5 with kanamycin added at a final concentration of 50 mg/L. All auxotrophy supplements were added at the concentrations described in example 2.
  • Three single colonies were picked from each strain and grown as in example 5, that includes the pre-cultures and production cultures. The production cultures supernatant were separated and the samples analysed by HPLC with an Agilent Zorbax Eclipse Plus C18 3.0 x 100mm, 3,5 pm column kept at 30°C.
  • the flow rate was 1.0 mL/min with 0.05% Acetic acid (A) and acetonitrile (B) as mobile phase.
  • the gradient started at 5% B and followed a gradient to 70% over 8 min, and stayed at 70% B for 1 min. All data shown are mean +/- SD with the 3 biological replicates.
  • Example 5 and 6 show consistent and increased production from strains producing tryptophan derivatives (5-HT and tryptamine as main product respectively) in combination with the ApdxH and ApdxH/thiL knockout.
  • tryptophan derivatives (5-HT and tryptamine as main product respectively)
  • ApdxH and ApdxH/thiL knockout 5-HT and tryptamine as main product respectively
  • another aromatic amino acid production pathway was tested giving the ApdxH/thiL double knock strain the capability for production of the L-tyrosine derivative L-DOPA.
  • strains were transformed with plasmid pMUT-HM181_5.6 which confers L-DOPA production capability.
  • Cells were grown in a modified M9+ medium containing 1x M9 salts (M6030, Sigma Aldrich), 0.2% (w/v) glucose, 1 mM MgSO4, 50 pM FeCh, 0.2% (v/v) 2YT medium. All auxotrophy supplements were added at the concentrations described in example 2. Kanamycin was added at a final concentration of 50 mg/L. The media was adjusted to pH 3,5 to 7 in different tubes with 0,5 intervals. Three single colonies were picked from each strain and grown as pre-culture as in example 5.
  • the main culture was inoculated by diluting the preculture 1 :100 into fresh medium of total volume of 200pl.
  • the cells were grown for 24 hours in a Synergi H1 plate reader, where the oxygen level was set to 5% using a BioTekTM CO2 and O2 gas controller. Afterwards, the culture was spun down and the supernatant was separated. The samples were subsequently run on a HPLC.
  • the system used an Cortecs UPLC T3 2.1 x 150mm, 1.6 pm column kept at 30°C.
  • the flow rate was 0,3 mL/min with 10mM ammonium formate (A) and acetonitrile (B) as mobile phase.
  • the gradient started at 0% B and followed a gradient to 70% B from 3,7 min to 4 min, and stayed at 70%B for 1 min. All data shown are mean +/- SD with the 3 biological replicates.
  • the data shows production of L-DOPA from both the WT and ApdxH/thiL knockout strain.
  • a decrease in L-DOPA levels occur with decreasing pH condition.
  • the observed pH range with increased production of the ApdxH/thiL double knockout is highly relevant for production of the AMT under human gut conditions, where the pH gradually increases in the small intestine from pH 6 to about pH 7.4, drops to 5.7 in the caecum and again gradually increases reaching pH 6.7 in the rectum.
  • Example 8 The double knockouts ApdxHlthiL and AsodA/B show survival in mammalian gut up to 11 days without antibiotics, and better survival than single knockouts
  • the AMTs To produce and deliver therapeutic molecules the AMTs must survive and remain in the gut of the patient over a certain period of time. However antibiotic treatment before the first dosing to free up a niche for the E.coli strains in the gut microbiota is not possible in a clinical setting. Also, it is desirable with regard to biosafety that the colonization of the strains should decrease and wash out after treatment with the AMT is stopped.
  • bacteria were grown in 2YT media + supplements (see Example 2) for 16h, washed and resuspended in PBS to 10 12 CFU/ml, of which 100 pL were gavaged (10 11 CFU). Faecal samples were collected for quantification of bacteria 2 times after the first gavage (5- and 8-hours) then in the morning on day 1-5, and day 7, 9 and 11. All samples were dissolved in PBS, 10 fold dilution rows (10 -1 to 10 -11 ) in PBS were generated and plated in duplicates on selective LB media with supplements containing 50mg/L kanamycin. At dissection (day 11) the gut content was collected and processed similar to the faecal samples.
  • the gut was divided in small intestine comprising duodenum, jejunum and ileum; and cecum and colon. All samples were weighed, colonies were counted after 24 hours incubation at 37 °C, and the count were normalized to volume of PBS (1mL) and weight of the faeces or gut content.

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Abstract

L'invention concerne une bactérie déficiente en au moins deux gènes fonctionnels dans la voie redox; ou en au moins un gène fonctionnel dans la voie de synthèse de la vitamine B1 et en au moins un gène fonctionnel dans la voie biologique de la vitamine B6. L'invention concerne également la bactérie comprenant une cassette d'expression pour la production d'une molécule thérapeutique. L'invention concerne également un kit avec la bactérie et un plasmide; ainsi qu'une formulation pharmaceutique comprenant la bactérie. L'invention concerne également l'utilisation de la bactérie en tant que médicament, par exemple pour le traitement de la maladie de Parkinson.
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