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WO2018178969A1 - Organismes transformés à cellules urticantes exprimant une protéine exogène d'intérêt - Google Patents

Organismes transformés à cellules urticantes exprimant une protéine exogène d'intérêt Download PDF

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Publication number
WO2018178969A1
WO2018178969A1 PCT/IL2018/050294 IL2018050294W WO2018178969A1 WO 2018178969 A1 WO2018178969 A1 WO 2018178969A1 IL 2018050294 W IL2018050294 W IL 2018050294W WO 2018178969 A1 WO2018178969 A1 WO 2018178969A1
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Prior art keywords
protein
interest
organism
stinging
transformed
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English (en)
Inventor
Yehu MORAN
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Yissum Research Development Co of Hebrew University of Jerusalem
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Yissum Research Development Co of Hebrew University of Jerusalem
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/60—New or modified breeds of invertebrates
    • A01K67/61—Genetically modified invertebrates, e.g. transgenic or polyploid
    • 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
    • A61K35/56—Materials from animals other than mammals
    • A61K35/614—Cnidaria, e.g. sea anemones, corals, coral animals or jellyfish
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/70—Invertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention relates to a transformed organism having stinging cells that express an exogenous protein of interest under the control of an endogenous promoter.
  • the invention further relates to a method for transforming an organism having stinging cells to produce an exogenous protein of interest, as well as to a method for delivery of an exogenous protein of interest to a mammal.
  • Cnidaria is a diverse phylum of animals (e.g., sea anemones, corals, hydroids and jellyfish) that are characterized by a unique intracellular structure, called cnidocyst, cnida or stinging organelle.
  • This organelle which is found within specialized neuronal cells known as cnidocytes (stinging cells), is a product of extensive Golgi secretions and serves as a microscopic weapon that enables cnidarians to inject venom to their prey and/or predators.
  • Cnidocysts can be divided into three main categories: i) nematocysts, the dart-shaped cnidae with spines on hollow tubules that are used for prey piercing and venom injection; ii) spirocysts, the elastic cnidae used for prey entanglement; and iii) ptychocysts, the sticky cnidae that are used for adherence to prey and for tube construction in some sea anemones.
  • the stinging organelle consists of a complex capsule polymer composed of cysteine-rich peptides, such as minicollagens and nematocyst outer wall antigen (NOWA). Furthermore, the capsule elongates at its end into a tubule comprised of a polymer of peptides, including minicollagens, nematogalectins, and other structural proteins. The tubule invaginates into the capsule during maturation and remains tightly coiled until activated during prey capture or defense, which results in the uncoiling of the tubule and discharge of the content of the capsule.
  • cysteine-rich peptides such as minicollagens and nematocyst outer wall antigen (NOWA).
  • the capsule elongates at its end into a tubule comprised of a polymer of peptides, including minicollagens, nematogalectins, and other structural proteins.
  • the tubule invaginates into the capsule during maturation and remains tightly
  • Nematostella vectensis is a species of small sea anemone characterized by nematocysts which can create tiny pores in mammalian skin that enable drug delivery. As this procedure is practically painless it has an extraordinary potential in the pharma industry.
  • EP 1956894 concerns a method for preconditioning tissue, such as skin, prior to delivery of active agent into and/or through tissue, by administering stinging capsule/cell onto the tissue and discharging the stinging capsule/cell to enhance transdermal/dermal, transmembranal, transmucosal or transcuticular permeability, and subsequently applying the active agent to the tissue.
  • WO 2006/048865 discloses a dry composition of matter comprising dehydrated stinging capsules and methods of producing and using same, for delivery of an active agent to a tissue. Nonetheless, the method of delivery employed by WO 2006/048865 comprises the modification of isolated stinging capsules to include the active agent by means of diffusion, electroporation, liposome fusion or microinjection into the capsule. Thus, this kind of preparation process of the capsule may compromise the integrity of the capsule, which may reduce the efficiency of the capsule to deliver the active agent. Furthermore, repetition of the preparation process is required for every batch of isolated capsules, which is time, effort and fund consuming.
  • US 8,337,868 discloses stinging cells expressing an exogenous polynucleotide coding for a therapeutic, diagnostic or a cosmetic agent, and methods, compositions and devices utilizing such stinging cells or capsules for delivering the therapeutic, diagnostic or cosmetic agent to a tissue.
  • the expression of the exogenous polynucleotide is effected by transfection of an organism having stinging cells with a construct comprising an exogenous sequence coding for the agent, under the control of an exogenous promoter, and then isolation of stinging cells or capsules from the transformed organism. It is an object of the present invention to provide transformed cnidarian organism, expressing an exogenous protein of interest under control of an endogenous promoter.
  • the present invention relates to a transformed organism of a phylum selected from the group consisting of Cnidaria and Myxozoa, having an exogenous sequence coding for a protein of interest under the expression control of an endogenous stinging cell-specific gene control element.
  • said control element is present in its original genomic location.
  • the transformed organism is of a class selected from a group consisting of Anthozoa, Hydrozoa and Scyphozoa. Specifically, the transformed organism is Nematostella vectensis.
  • the stinging cell is selected from the group consisting of: a cnidocyte, a nematocyte, a spirocyte and a ptychocyte.
  • the protein of interest is a protein that is delivered to a mammal for a therapeutic, diagnostic or cosmetic purpose.
  • the protein of interest is selected from the group consisting of a drug, vaccine, hormone, enzyme, antibody or label.
  • the exogenous sequence coding for the protein of interest is fused to a signal sequence encoding a signal peptide.
  • the signal peptide is derived from a cnidarian minicollagen, nematogalectin or toxin.
  • said minicollagen is NEP3.
  • the exogenous sequence coding for the protein of interest is conjugated to a sequence coding for a detectable label protein.
  • the exogenous sequence coding for the protein of interest and the sequence coding for the detectable label protein are linked by a sequence coding for a cleavable amino acid sequence.
  • the cleavable amino acid sequence is the viral peptide P2A.
  • the endogenous stinging cell-specific gene control element is the promoter of NvNcol3 gene.
  • the present invention relates to a method for expressing an exogenous protein of interest under the control of an endogenous gene control element in a cnidarian organism, comprising:
  • step (b) comprises co-transferring the transforming vector with one or more additive or molecule required for integration of the transforming sequence into the genome of the organism at a specific location, under the expression control of an endogenous stinging cell-specific gene control element.
  • transferring of the transforming vector to the zygote of the organism is carried out by microinjection.
  • said integration of the transforming sequence into the genome of the organism at a specific location is achieved by CRISPR- Cas9 technology.
  • the present invention relates to a stinging cell expressing a protein of interest, wherein the stinging cells is isolated from the transformed organism of the invention.
  • a stinging capsule containing a protein of interest wherein the stinging capsule is isolated from the stinging cell expressing a protein of interest according to the invention.
  • the present invention further encompasses a method for producing a line of transformed cnidarian organisms expressing an exogenous protein of interest, comprising:
  • the present invention related to a pharmaceutical composition
  • a pharmaceutical composition comprising, as an active ingredient, at least one stinging capsule isolated from the transformed organism of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, a gel, oil and a semisolid formulation.
  • the present invention relates to a delivery device comprising: (a) at least one stinging capsule containing a protein of interest according to the invention; and (b) a support.
  • the support is selected from the group consisting of a patch, a foil, a plaster and a film.
  • the at least one stinging capsule is secured to the support by biological glue, polylysine, or a mesh support.
  • the delivery device further comprises a mechanism for triggering the activation of the at least one stinging capsule, selected from the group consisting of a mechanical triggering mechanism, a chemical triggering mechanism and an electrical triggering mechanism.
  • the present invention relates to a method for transdermal delivery of a protein of interest to a mammal, comprising the steps of:
  • the present invention relates to a method for transdermal delivery of a protein of interest to a mammal, comprising the steps of:
  • Figs. 1A-1B show microinjection of a transforming vector into a N. vecetensis zygote.
  • Fig. 1A is an illustration of the transforming vector in the form of a plasmid, which was microinjected into N. vecetensis zygotes and resulted in genomic integration of memOrange2 into the native NvNcol3 locus.
  • the transforming vector includes two homology arms, at positions 1380459-1381924 and 1381925-1383035 in scaffold 23 of the genome of N. vectensis, spanning the memOrange2 gene (coding for mOrange2 fluorescent protein with a C-terminal RAS-derived membrane tag).
  • Fig. IB shows a N. vecetensis zygote before and after injection of a mixture containing a single guide RNA of SEQ ID NO: 1 (250 ng/ ⁇ ), Cas9 recombinant protein with nuclear localization signal (500 ng/ ⁇ ) and the transforming vector of Fig. 1A.
  • the zygote was held with a holding capillary and injected with a pulled glass needle. Fluorescent dye bound to dextran was used as a tracer. The average diameter of a zygote is roughly 250 ⁇ .
  • A ampicillin resistance sequence
  • Al Ascl restriction enzyme
  • Bl before injection
  • G gene
  • HA homology arm
  • I after injection
  • l-S l-Scel restriction enzyme
  • K kanamycin resistance sequence
  • mem02 memOrange2
  • IP insertion point
  • PI Pad restriction enzyme
  • SI Sbfl restriction enzyme
  • TF transforming plasmid
  • Fig. 2 shows the expression of memOrange2 in various developmental stages (planula, primary polyp, and adult) of the injected parent generation and the first filial generation of N. vecetensis. Rectangles indicate the tentacle of the polyp having very strong expression of memOrange2.
  • Figs. 3A-3F show the expression of memOrange2 in the first filial generation (Fl) of transformed N. vecetensis carrying a memOrange2 knock-in gene at the minicollagen-3 (NvNcol3) locus.
  • Fig. 3A is a representative image out of live imaging analysis under fluorescent light of an adult transformed N. vecetensis of Fl generation.
  • Fig. 3B is a representative image out of live imaging analysis under fluorescent light of a tentacle of an adult transformed N. vecetensis of Fl generation
  • Fig. 3C shows a white light image of an isolated nematocyst from Fl generation of transformed N. vecetensis.
  • Fig. 3D shows a fluorescent image of the isolated nematocyst shown in Fig. 3A.
  • Fig. 3E shows RNA expression of memOrangel and NvNcol3 genes, revealed by double Fluorescent in situ hybridization (FISH) analysis, in an early planula of transformed N. vecetensis of Fl generation.
  • FISH Fluorescent in situ hybridization
  • Fig. 3F shows protein expression of memOrange2 and NvNcol3, revealed by immunostaining, in a tentacle of a polyp transformed N. vecetensis of Fl generation.
  • Me merge
  • mem02 memOrange2
  • NC NvNcol3
  • the present invention provides a transgenic cnidarian organism able to express an exogenous protein of interest.
  • the provision of a transformed organism, or line of organisms, with stinging cells according to the invention addresses the need for producing stinging cells expressing high levels of an exogenous protein of interest.
  • the heterologous (foreign) gene expression in the stinging cells of the transformed organism allows the production of the protein of interest with high yield and efficiency.
  • the present invention also provides a method to deliver the exogenous protein of interest to a mammal, by application of at least one capsule isolated from the stinging cells of the transformed organism.
  • US Patent No. 8,337,868 discloses the expression of foreign sequences in an organism of a phylum selected from the group consisting of Cnidaria, Dinoflagellata and Myxozoa, preferably from a class selected from the group consisting of Anthozoa, Hydrozoa and Scyphozoa, by transfecting the organism with an expression construct where the sequence coding for the foreign protein is under the expression control of an exogenous control element (promoter).
  • promoter an exogenous control element
  • the present invention concerns a transformed organism having an exogenous sequence coding for a protein of interest, under the expression control of an endogenous stinging cell-specific gene control element.
  • the control element is present in its original genomic location.
  • the organism of the invention is a member of a phylum selected from the group consisting of Cnidaria and Myxozoa, preferably of a class selected from the group consisting of Anthozoa, Hydrozoa and Scyphozoa.
  • Myxozoa is a group of modified cnidarians that have undergone evolution from a free swimming, self-sufficient jellyfish-like creature into a form of obligate parasites aquatic animals. Accordingly, the term "cnidarian” as used herein refers to animals of both Cnidaria and Myxozoa phyla.
  • the organism is a member of Cnidaria, a phylum which encompasses Anthozoa (e.g., sea anemones, corals, sea pens), Scyphozoa (e.g., jellyfish), Cubozoa (e.g., box jellies) and Hydrozoa.
  • the organism is the sea anemone Nematostella vectensis (hereinafter: "N. vectensis").
  • N. vectensis is transformed according to the invention, there is no requirement for any form of endogenous toxin neutralization (e.g., expression of an exogenous sequence, such as an antisense sequence, for the toxin or a sequence coding for an inactivating enzyme of the toxin) because the naturally expressed toxins of N. vectensis have poor, or even lack activity, on vertebrates. Moreover, the naturally expressed toxins of N. vectensis are not painful even when injected to humans transdermally.
  • endogenous toxin neutralization e.g., expression of an exogenous sequence, such as an antisense sequence, for the toxin or a sequence coding for an inactivating enzyme of the toxin
  • organisms having active endogenous toxins can also be utilized by the present invention, provided inactivation of the endogenous toxin is effected prior to use.
  • inactivation can be effected via one of several methods, including but not limited to, temperature or chemical denaturation, enzymatic inactivation and ligand inactivation (e.g., Fab fragment of an antibody).
  • Inactivation of the endogenous toxins can also be effected by transforming the organism with a polynucleotide sequence coding for a polynucleotide capable of inhibiting toxin synthesis (e.g. antisense or ribozyme), or coding for an enzyme or an antibody, which is capable of inactivating the endogenous toxin protein.
  • the inactivating polynucleotide sequence can be introduced into the stinging cell of the organism together with the exogenous sequence coding for a protein of interest.
  • co-transformation is effected using a single expression construct expressing both polynucleotides.
  • transformed organism and transgenic organism refer to an organism whose genetic material has been altered by addition of exogenous genetic material, by the use of any genetic engineering technique.
  • the "stinging cell” is selected from the group consisting of a cnidocyte, a nematocyte, a spirocyte and a ptychocyte. In a specific embodiment of the invention, the stinging cell is a nematocyte.
  • express or "expression” when used in context of the exogenous polynucleotide coding for a protein of interest refers to generation of a polynucleotide (transcript) or a polypeptide product.
  • gene control element refers to a nucleic acid sequence that directs transcription of a nucleic acid.
  • the gene control element can be a promoter, such as a constitutive or an inducible promoter, or an enhancer.
  • the expression control sequence is operably linked to the nucleic acid sequence to be transcribed.
  • endogenous stinging cell-specific gene control element refers to a gene control element that is found in the genome of the transformed organism, and is active only in the stinging cells of the organism.
  • endogenous refers to a nucleic acid sequence or a polypeptide that originates from the specific organism undergoing the transformation according to the invention.
  • the endogenous gene control element of N. vectensis is used.
  • the endogenous stinging cell-specific gene control element can be inserted into the organism along with the exogenous sequence coding for a protein of interest.
  • the exogenous sequence coding for a protein of interest is inserted into the cell and integrated into the genome in a location allowing its expression according to an endogenous gene control element that is present in its original genomic location. Accordingly, the integration of the transforming sequence into the genome of the organism may take place at a random location or at a specific (targeted) location.
  • the endogenous gene control element is present in its original genomic location within the stinging cell.
  • the "exogenous sequence" coding for a protein of interest is a DNA polynucleotide sequence that is not found in the genome of the transformed organism. The insertion of the exogenous sequence into a cell of the organism leads to the synthesis of the protein of interest through transcription and translation processes that occur within the cell.
  • polynucleotide and “nucleic acid sequence” as used interchangeably herein, refer to chains of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase.
  • the "protein of interest” is a protein that is delivered to a mammal for any of a therapeutic, diagnostic or cosmetic purpose.
  • the delivery of the protein of interest to a mammal is preferably a transdermal delivery, through the skin of the mammal.
  • the protein of interest is selected from a group consisting of a drug, vaccine, hormone, enzyme, antibody or label.
  • the protein of interest is of small to medium size (i.e., up to 100 kDa).
  • the protein of interest may also be a glycoprotein of small to medium size.
  • the term "mammal”, as used herein, refers to a human, a farm animal, a sport animal, a pet, a primate, a horse, a dog, a cat, a mouse and a rat.
  • Examples of a protein of interest to be delivered to a mammal for therapeutic purposes according to the invention include, but not limited to, a polypeptide (such as peptide hormones, antibodies or antibody fragments), an enzyme, a structural protein and an antisense or ribozyme transcripts which can be directed at specific target sequences (e.g., transcripts of tumor associated genes) to thereby downregulate activity thereof and exert a therapeutic effect.
  • a polypeptide such as peptide hormones, antibodies or antibody fragments
  • an enzyme e.g., an enzyme
  • a structural protein e.g., an enzyme
  • an antisense or ribozyme transcripts which can be directed at specific target sequences (e.g., transcripts of tumor associated genes) to thereby downregulate activity thereof and exert a therapeutic effect.
  • Protective protein antigens for vaccination may also be expressed in the stinging cells according to the present invention.
  • the protein of interest may also be a prodrug, which can be activated prior
  • prodrug refers to an agent which is inactive, but which is convertible into an active form via enzymatic, chemical or physical activators.
  • a prodrug for example an enzyme
  • an activator compound for example an ion
  • specific enzymes, molecules or pH conditions present in the target tissues can activate the prodrug.
  • Non-limiting examples of a protein of interest to be delivered to a mammal for therapeutic purposes according to the invention are selected from: Interferon beta-1 (Genbank accession P01574.1) that is widely used for the treatment of multiple sclerosis, Interferon alpha-2a (GenBank accession AET86951.1) and alpha-2b (GenBank accession JN591570.1) that can be used as drugs for battling hepatitis C infection and melanoma, and exenatide (GenBank accession P26349), a peptide drug for treating diabetes type II.
  • Interferon beta-1 Genbank accession P01574.1
  • Interferon alpha-2a GenBank accession AET86951.1
  • alpha-2b GenBank accession JN591570.1
  • exenatide GenBank accession P26349
  • GCSF glycoprotein Granulocyte-Colony Stimulating Factor
  • GCSF glycoprotein Granulocyte-Colony Stimulating Factor
  • the protein of interest can be a peptide or a protein immunogen, such as Hepatitis B surface antigen (HBsAg; GenBank accession ACJ66227.1), so that delivery of said immunogen by use of the transformed organism according to the invention facilitates pain-free vaccination.
  • HBsAg Hepatitis B surface antigen
  • Non-limiting examples of a protein of interest to be delivered to a mammal for diagnostic purposes according to the invention are selected from the group consisting of a probe, a ligand, an antibody, a receptor and a receptor analog.
  • Examples of a protein of interest to be delivered to a mammal for cosmetic purposes according to the invention include, but not limited to an anti-wrinkling agent, an antiacne agent, an exfoliant, a hair follicle stimulating agent and a hair follicle suppressing agent, a protease (such as collagenase, vibriolysin, for burn debridement, exfoliation, acne and abnormal skin conditions), TGF- ⁇ Rll agonists and antagonists for stimulation or suppression of hair growth and a-interferon for care of aged or damaged skin and cosmetically used toxins such as the Botulinum toxin (GenBank accession AF464912).
  • amino acid sequence refers to chains of amino acids of any length.
  • the chain may be linear or branched.
  • the chain may comprise modified amino acids, and/or may be interrupted by non-amino acids.
  • the terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • the polypeptides can occur as single chains or associated chains.
  • the exogenous sequence coding for the protein of interest is fused to a signal sequence encoding a signal peptide. Consequently, the transformed organism of the invention expresses the protein of interest, which is fused to a signal peptide.
  • the signal peptide enables the transport of the protein of interest from the Golgi into the capsule of the stinging cell.
  • the protein of interest expressed in the stinging cells of the transformed organism of the invention is accumulated and stored in the stinging capsule.
  • a suitable signal peptide according to the invention is a signal peptide derived from a cnidarian minicollagen, nematogalectin or toxin.
  • the signal peptide is derived from NEP3, which is a marker protein of cnidocytes (NCBI accession number XP_001640559).
  • the exogenous sequence coding for the protein of interest is conjugated to a sequence coding for a detectable label protein. Consequently, the protein of interest expressed by the transformed organism is conjugated to a detectable label protein, in order to enable easy selection and separation of transformed organisms from non-transformed organisms.
  • a detectable label protein may be a florescent protein.
  • detectable label proteins suitable for the invention are selected from: DsRed, Tl, Dimer2, mRFPl, mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry and mCherry.
  • the detectable label protein is mOrange2.
  • the exogenous sequence coding for the protein of interest and the sequence coding for the detectable label protein are linked by a sequence coding for a cleavable amino acid sequence. Consequently, the protein of interest and the detectable label protein are linked by a cleavable amino acid sequence.
  • the cleavable amino acid sequence enables the release of the protein of interest from the detectable label protein immediately upon translation.
  • the cleavable amino acid sequence is an amino acid sequence that can be cleaved by one of the endogenous enzymes of the transformed organism, or alternatively, an amino acid sequence that can cleave itself (hereinafter "self-cleavable").
  • self-cleavable amino acid sequence are selected from, but not limited to, the viral peptide P2A and internal ribosome entry site (IRES).
  • the cleavable amino acid sequence is P2A, which is an efficient self-cleavable peptide sequence that cleaves itself immediately upon translation.
  • the protein expressed in the transgenic organism according to the invention comprises the protein of interest fused to a signal peptide and, optionally, further conjugated to a detectable label protein by a cleavable amino acid sequence.
  • the protein of interest is expressed under the control of an endogenous stingi ng cell-specific gene control element.
  • the endogenous stinging cell-specific gene control element is the promoter of NvNcol3 gene.
  • Another aspect of the invention provides a method for transforming a cnidarian organism, comprising: inserting a transforming sequence into a vector to obtain a transforming vector; and transferring the transforming vector into a zygote of the organism, thereby obtaining a transformed organism expressing an exogenous protein of interest.
  • the invention provides a method for expressing an exogenous protein of interest under the control of an endogenous gene control element in a cnidarian organism, comprising:
  • step (a) of the method for expressing an exogenous protein of interest in a cnidarian organism according to the invention the transforming sequence is inserted into a vector.
  • the insertion of a transforming sequence into a vector results in a transforming vector.
  • vector refers to a replicable polynucleotide construct, which is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a cell.
  • vectors include, but are not limited to, viral vectors (e.g., adenoviruses, adeno-associated viruses, retroviruses), naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes.
  • Vector components may generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes and one or more gene control elements (such as a promoter, enhancer and terminator). For expression (i.e., translation), one or more translational controlling elements may also be required, such as ribosome binding sites, translation initiation sites, and stop codons.
  • the vector may also contain target sequences for restriction enzymes, and one or more sequences that would confer resistance to one or more antibiotic agent (such as ampicillin and kanamycin) for selection purposes. Suitable vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art.
  • cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting stinging cells expressing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.
  • the vector can be any high copy number plasmid, for example the pUC series or pBluescript.
  • a linear DNA fragment can also be used.
  • the plasmid serves as a carrier for the exogenous DNA sequence coding for a protein of interest, and allows the integration of the functional relevant part, i.e., the protein of interest spanned by the homology arms, into the genome.
  • homology arm refers to a nucleic acid sequence which is identical to a polynucleotide sequence in the genome of the organism to be transformed. Accordingly, the homology arms spanning the exogenous DNA sequence coding for a protein of interest serve as genomic coordinates for the integration of the transforming sequence at a specific location in the genome of the organism.
  • transforming sequence refers to an exogenous DNA sequence coding for a protein of interest fused to a signal DNA sequence coding for a signal peptide.
  • signal peptide refers to an amino acid sequence, usually present at the N-terminus of a protein, which prompts the stinging cell to translocate the protein to the stinging capsule (nematocyst).
  • signal sequence refers to a polynucleotide that encodes for a signal peptide.
  • the signal peptide transports the protein of interest from the Golgi of the cell to the stinging capsule (nematocyst).
  • the signal sequence may be from the same species of the organism that is transformed or may be from a different species, as long as it can transport the protein attached to it into the stinging capsule.
  • a suitable signal DNA sequence according to the invention is a signal sequence that codes for a signal peptide of a cnidarian minicollagen, nematogalectin or toxin.
  • the signal sequence codes for the signal peptide of NEP3, which is a marker protein of cnidocytes.
  • the transforming DNA sequence according to the invention optionally comprises the exogenous DNA sequence coding for the protein of interest conjugated to a DNA sequence coding for a detectable label protein (also termed "reporter gene” or “marker”).
  • the reporter gene is fused to the exogenous sequence coding for the protein of interest directly, or indirectly (e.g., in conjugation with the signal sequence fused to the exogenous sequence).
  • the exogenous sequence coding for the protein of interest and the sequence coding for the detectable label protein are linked by a sequence coding for a cleavable amino acid sequence.
  • the following nucleic acid sequence (denoted herein as SEQ ID NO: 2) is a non-limiting example of a transforming sequence, wherein the protein of interest is Interferon beta- la.
  • the transforming sequence also comprises an endogenous stinging cell-specific gene control element.
  • said gene control element is provided in its original genomic location in the genome of the organism, its sequence can be absent from the transforming sequence.
  • a transforming sequence that lacks a gene control element is required to integrate into the genome of the organism at a specific location, in a manner which would be operable to express the exogenous sequence coding for the protein of interest under the control of an endogenous genomic control element.
  • the endogenous stinging cell-specific gene control element is the nematocyte-specific promoter of the NvNcol3 gene (the sequence thereof is known in the art), which codes for minicollagen-3, a highly-expressed structural component of the nematocyst capsule.
  • the transforming sequence according to the invention can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence.
  • step (b) of the method for expressing an exogenous protein of interest under the control of an endogenous gene control element in a cnidarian organism the transforming vector that is obtained in step (a) is transferred into a zygote of the organism to be transformed, by any suitable method known in the art, such as microinjection, electroporation, viral infection, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances, microprojectile bombardment, lipofection.
  • the choice of introducing vectors or polynucleotides may depend on features of the host cell.
  • the transforming vector is transferred to the zygote of the organism to be transformed by microinjection.
  • the transferring of the transforming vector to the zygote of the organism is carried out by microinjection.
  • microinjection refers to injecting a substance into a living cell, by using a glass micropipette. The microinjection is carried out under the magnification of a microscope.
  • the transforming vector is transferred to the zygote together with one or more additive or molecule (e.g., RNA, DNA and protein) which is required for a successful integration of the transforming sequence into the organism's genome.
  • additive or molecule e.g., RNA, DNA and protein
  • the integration of the transforming sequence into the genome of the organism may take place at a random location or at a specific (targeted) location.
  • the integration of the exogenous polynucleotide at the targeted location in the genome, for example, under the control of an endogenous promoter in its original genomic location may take place by any routinely used site-specific mutagenesis technique.
  • step (b) of the method for expressing an exogenous protein of interest under the control of an endogenous gene control element in a cnidarian organism comprises co-transferring the transforming vector with one or more additive or molecule required for integration of the transforming sequence into the genome of the organism at a specific location, under the expression control of an endogenous stinging cell-specific gene control element.
  • the integration at a targeted location is achieved by the use of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR- associated protein 9) technology.
  • CRISPR-Cas9 Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR- associated protein 9
  • a mixture containing Cas9 nuclease and two RNA molecules is delivered into a cell in order to cut the cell's genome at a desired location.
  • the two RNA molecules are synthetic RNA sequences, one of which is a user-defined nucleotide spacer that is complementary to the target genomic sequence (guide RNA, gRNA) and the other is a scaffold sequence which binds Cas9 (trans-activating CRISPR RNA, tracrRNA).
  • the two RNA molecules can be linked together to form a single guide RNA (sgRNA) sequence.
  • sgRNA single guide RNA
  • the transforming vector is transferred to the zygote of the organism together with reagents required for the CRISPR-Cas9 gene editing system, i.e., gRNA and tracrRNA, either as separate molecules or joined together as a single guide RNA, and Cas9 recombinant protein with a nuclear localization signal (NLS).
  • reagents required for the CRISPR-Cas9 gene editing system i.e., gRNA and tracrRNA, either as separate molecules or joined together as a single guide RNA, and Cas9 recombinant protein with a nuclear localization signal (NLS).
  • nucleic acid sequence (denoted herein as SEQ ID NO: 1) is a non-limiting example of a single guide RNA, suitable for targeting the transforming sequence to be placed under the expression control of the promoter of the NvNcol3 gene, which is present in stinging cells of N. vectensis in its original genomic location.
  • Positions 3-22 in SEQ ID NO: 1 are the gRNA and positions 23-101 are the tracrRNA.
  • Any commercially available Cas9 recombinant protein conjugated to a nuclear localization signal is suitable according to the invention.
  • Cas9 recombinant protein with nuclear localization signal can also be synthesized by a person of skill in the art according to a well-known sequence.
  • the NLS may be located at the C-terminus or the N-terminus of the Cas9 protein.
  • steps (a) and (b) of the method according to the invention using a transforming vector devoid of a gene control element, results in an exogenous sequence coding for a protein of interest placed under the expression control of an endogenous stinging cell-specific gene control element (promoter), wherein said control element is present in its original genomic location.
  • promoter cell-specific gene control element
  • the action of integrating the exogenous sequence coding for the protein of interest into the genome of the organism under the expression control of the endogenous stinging cell-specific gene control element involves inserting ("knocking-in") an exogenous polynucleotide sequence of a gene coding for the protein of interest into a native locus of a highly-expressed gene (e.g. a gene coding for a structural component) that is endogenous to the transformed organism. Consequently, the expression of the exogenous sequence is regulated by the control element of the endogenous gene.
  • the endogenous control element is present in its original genomic location within the DNA.
  • the organism developed from the zygote to which a transforming vector was transferred is a transformed organism that expresses the protein of interest in its stinging cells.
  • the protein of interest is accumulated and stored in the stinging capsules (organelles) of the transgenic organism.
  • the transformed organism obtained by the method of the invention can be further bred to produce filial generations in a line of transformed organisms.
  • the present invention provides a stinging cell expressing a protein of interest.
  • the stinging cell is isolated from the transformed organism obtained according to the method of the invention by any isolation process known in the art.
  • the present invention provides a stinging capsule containing a protein of interest, wherein the stinging capsule is isolated from the stinging cells of the transgenic organism.
  • a further aspect of the invention provides a method for producing a line of transformed cnidarian organisms, which express an exogenous protein of interest, comprising:
  • the present inventors have discovered that the filial generations in the line of transformed organisms, for example the first filial generation (Fl), express the protein of interest at particularly high levels.
  • the filial generations in the line of transformed organisms for example the first filial generation (Fl)
  • Fl first filial generation
  • a pharmaceutical composition comprising, as an active ingredient, at least one stinging capsule containing a protein of interest and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, a gel, oil and a semisolid formulation.
  • the at least one capsule (also referred to herein as "organelle”) is isolated from a stinging cell of a transgenic organism that expresses an exogenous polynucleotide coding for the polypeptide of interest under the expression control of an endogenous gene control element according to the present invention.
  • the isolation of the capsule may be carried out according to known methods in the art.
  • the capsule is lyophilized and can be maintained as dry powder, which is stable up to several years.
  • a delivery device comprising: (a) at least one stinging capsule containing a protein of interest; and (b) a support.
  • the at least one stinging capsule is placed on the support, and the support is then applied to an outer surface of a mammalian tissue.
  • the support is selected from the group consisting of a patch, a foil, a plaster and a film.
  • the at least one stinging capsule can be secured to the support by, for example, biological glue (e.g. BIOBONDTM), polylysine, a mesh support, or any other acceptable attaching material.
  • biological glue e.g. BIOBONDTM
  • polylysine e.g. polylysine
  • mesh support e.g. a mesh support
  • any other acceptable attaching material e.g. BIOBONDTM
  • the at least one stinging capsule is activated to release the protein of interest into the tissue.
  • the mammalian tissue is the skin.
  • the device comprises a mechanism for triggering the activation of the stinging capsule, the mechanism being selected from the group consisting of a mechanical triggering mechanism, a chemical triggering mechanism and an electrical triggering mechanism.
  • a non-limiting example for a mechanical triggering mechanism is an exertion of pressure on the support subsequent to the application of device to the tissue.
  • the exertion of pressure forces contact between the at least one stinging capsule and the tissue, thereby activating discharge of the capsule's content.
  • Non-limiting examples for chemical triggering mechanism are water and aqueous solutions of any one of sodium thiocyanate (NaSCN), sodium citrate, ethylene glycol tetraacetic acid (EGTA), and pentasodium triphosphate.
  • NaSCN sodium thiocyanate
  • EGTA ethylene glycol tetraacetic acid
  • pentasodium triphosphate pentasodium triphosphate.
  • the chemical substances can be applied prior to, or following, application of the device to the tissue. Chemical activation of discharge is advantageous since it allows for simultaneous discharge of most if not all of the stinging capsules upon the device.
  • the chemical triggering mechanism is 1% pentasodium triphosphate at a pH between of 8 and 10.
  • a non-limiting example for an electrical triggering mechanism is applying an electrical pulse to the device.
  • the electrical pulse is approximately 20-30 Volts for 30 microseconds.
  • a method for transdermal delivery of a protein of interest to a mammal comprising the steps of:
  • the capsule lotion can be applied to the skin either directly or indirectly by the use of a support as specified above.
  • the anhydrous lotion is a gel consisting of 2% hydroxypropylcellulose in absolute ethanol.
  • the aqueous solution is 1% pentasodium triphosphate at a pH between of 8 and 10.
  • the method for transdermal delivery of a protein of interest to a mammal may comprise the steps of:
  • the at least one stinging capsule can be secured to the support by, for example, biological glue (e.g. BIOBONDTM), polylysine, a mesh support, or any other acceptable attaching material.
  • biological glue e.g. BIOBONDTM
  • polylysine e.g. polylysine
  • mesh support e.g. a mesh support
  • any other acceptable attaching material e.g. BIOBONDTM
  • the step of triggering the discharge of the protein of interest from the at least one stinging capsule is carried out by a triggering mechanism selected from the group consisting of a mechanical triggering mechanism, a chemical triggering mechanism and an electrical triggering mechanism as described above.
  • the transformed organism is a member of a line of transgenic organisms produced as described above.
  • Nematostella vectensis polyps were grown in 16 %o sea salt water at 17 °C. Polyps were fed with Artemia saiina nauplii three times a week.
  • CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR- associated protein 9 type II technology was applied for genome engineering of N. vectensis. For generating a transformed line of N. vectensis expressing memOrange2 under the native regulatory region of NvNcol3, a fertilized N.
  • vectensis zygote was microinjected with a mixture that included a single guide RNA (250 ng/ ⁇ ) of SEQ ID NO: 1, Cas9 recombinant protein with nuclear localization signal (500 ng/ ⁇ ; PNA Bio, USA) and a transforming vector that included two homology arms (at positions 1,380,459- 1,381,924 and 1,381,925-1,383,035 in scaffold 23 of the N. vectensis genome) spanning the memOrange2 gene (Fig. 1A).
  • a pulled glass needle was used to microinject the mixture containing the transforming plasmid into a zygote which was held with a holding capillary as shown in Fig. IB.
  • the expression of the memOrange2 transgene in injected zygotes and embryos was monitored under a Nikon SMZ18 fluorescent stereomicroscope equipped with a Nikon Ds-OJ2 monochrome camera and Elements BR software (Nikon, Japan).
  • microinjected embryos were then grown at 22 °C for four months and were fed with Artemia saiina nauplii until sexual maturity was reached. After reaching sexual maturity, N. vectensis polyps were induced to spawn and the gametes of each injected anemone were mixed with those of a wild type anemone of the opposite sex.
  • Transformed Fl embryos were detected 72 hours after fertilization by scanning for mOrange2 expression in nematocytes under a Nikon SMZ18 fluorescent stereomicroscope equipped with a Nikon Ds-Qi2 monochrome camera and Elements BR software (Nikon, Japan).
  • N. vectensis were collected and kept frozen before capsule extraction.
  • the anemones were homogenized in 12.5 ppt artificial seawater, followed by two centrifugations in Percoll gradients, differentiating between the relatively dense intact capsules and the discharged capsules and the cell debris.
  • the isolated purified capsules were washed with decreasing salinity of NaCI and CaCI 2 to a final concentration of 15 mM NaCI and 0.2 mM CaCI 2 and immediately freeze-dried.
  • the microcapsules were kept in powder form at 2 to 8 °C until use.
  • CRISPR/Cas9 technology was used to knock-in the reporter gene into the genomic locus of the NvNcol3 gene that codes for minicollagen-3 protein, which is a major structural protein of the cnidocyst capsule wall and, hence, is constitutively and abundantly expressed in the cnidocyte.
  • the reporter gene codes for mOrange2 fluorescent protein with a C-terminal RAS-derived membrane tag (memOrange2).
  • the microinjected embryos started expressing the fluorescent protein in their cnidocytes 72 hours post fertilization (hpf). As shown in Fig.
  • Figs. 3A-3B show that Fl generation of transformed N. vectensis expressing memOrange2 under the control of the native NvNcol3 gene successfully and uniformly expresses memOrange2 in their nematocytes. Moreover, as shown in Figs. 3C and 3D, significant quantities of memOrange2 are observed in the capsule and the tubule of the nematocyst. These results indicate that inducing the expression of medium-sized proteins by stinging cells via the approach is feasible.

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Abstract

La présente invention concerne un organisme transformé présentant des cellules urticantes qui expriment une protéine exogène d'intérêt sous le contrôle d'un promoteur endogène. L'invention concerne également un procédé de transformation d'un organisme présentant des cellules urticantes pour produire une protéine exogène d'intérêt, ainsi qu'un procédé d'administration d'une protéine exogène d'intérêt à un mammifère.
PCT/IL2018/050294 2017-03-28 2018-03-13 Organismes transformés à cellules urticantes exprimant une protéine exogène d'intérêt Ceased WO2018178969A1 (fr)

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Citations (3)

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WO2003079967A2 (fr) * 2002-03-26 2003-10-02 Nanocyte Inc. Cellules urticantes exprimant un polynucleotide exogene qui code pour un agent therapeutique, diagnostic ou cosmetique, et procedes, compositions et dispositifs faisant intervenir l'utilisation de ces cellules urticantes ou de capsules derivees de celles-ci pour administrer l'agent therapeutique, diagnostic ou cosmetique a
WO2006048865A2 (fr) 2004-11-05 2006-05-11 Nanocyte Inc. Compositions et trousses contenant des gelules piqueuses deshydratees et leurs methodes de preparation et d'utilisation
EP1956894A2 (fr) 2005-04-19 2008-08-20 Nanocyte Inc. Procedes, compositions et dispositfs utilisant des cellules/capsules urticantes

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WO2003079967A2 (fr) * 2002-03-26 2003-10-02 Nanocyte Inc. Cellules urticantes exprimant un polynucleotide exogene qui code pour un agent therapeutique, diagnostic ou cosmetique, et procedes, compositions et dispositifs faisant intervenir l'utilisation de ces cellules urticantes ou de capsules derivees de celles-ci pour administrer l'agent therapeutique, diagnostic ou cosmetique a
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