WO2018178969A1

WO2018178969A1 - Transformed organisms with stinging cells expressing an exogenous protein of interest

Info

Publication number: WO2018178969A1
Application number: PCT/IL2018/050294
Authority: WO
Inventors: Yehu MORAN
Original assignee: Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
Priority date: 2017-03-28
Filing date: 2018-03-13
Publication date: 2018-10-04

Abstract

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.

Description

TRANSFORMED ORGANISMS WITH STINGING CELLS EXPRESSING

AN EXOGENOUS PROTEIN OF INTEREST

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

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. 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.

It is a further object of the invention to provide a method for expressing an exogenous protein of interest under control of an endogenous promoter in a transformed cnidarian organism.

It is a still further object of the invention to provide a stinging cell expressing a protein of interest under control of an endogenous promoter and pharmaceutical composition comprising the same.

It is another object of the invention to provide a device for delivering a target protein to a mammal, comprising at least one stinging capsule containing a target protein isolated from a transformed organism.

It is yet another object of the invention to provide a method for delivering a target protein to a mammal.

Other objects and advantages of the invention will become apparent as the description proceeds.

SUMMARY OF THE INVENTION

In one aspect, 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.

In some embodiments, said control element is present in its original genomic location. According to one embodiment, the transformed organism is of a class selected from a group consisting of Anthozoa, Hydrozoa and Scyphozoa. Specifically, the transformed organism is Nematostella vectensis.

According to a further embodiment, the stinging cell is selected from the group consisting of: a cnidocyte, a nematocyte, a spirocyte and a ptychocyte.

According to a still further embodiment, the protein of interest is a protein that is delivered to a mammal for a therapeutic, diagnostic or cosmetic purpose. According to another embodiment, the protein of interest is selected from the group consisting of a drug, vaccine, hormone, enzyme, antibody or label.

In another embodiment of the invention, the exogenous sequence coding for the protein of interest is fused to a signal sequence encoding a signal peptide.

According to one embodiment, the signal peptide is derived from a cnidarian minicollagen, nematogalectin or toxin. According to a specific embodiment, said minicollagen is NEP3.

In some embodiments, the exogenous sequence coding for the protein of interest is conjugated to a sequence coding for a detectable label protein.

According to one embodiment of the invention, 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. In a specific embodiment, the cleavable amino acid sequence is the viral peptide P2A.

According to a specific embodiment, the endogenous stinging cell-specific gene control element is the promoter of NvNcol3 gene. In a further aspect, 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:

(a) inserting a transforming sequence into a vector to obtain a transforming vector; and

(b) transferring the transforming vector into a zygote of the organism.

In some embodiment, 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.

According to a specific embodiment, transferring of the transforming vector to the zygote of the organism is carried out by microinjection.

According to another specific embodiment, said integration of the transforming sequence into the genome of the organism at a specific location is achieved by CRISPR- Cas9 technology.

In a still further aspect, 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.

In another aspect, there is provided 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:

(I) transforming a cnidarian organism according to the method of any one of claims 15-18 to obtain a parent generation (F0) transformed organism;

(II) growing the transformed organism to a sexually mature transformed organism;

(III) inducing the sexually mature transformed organism to spawn gametes;

(IV) mixing the gametes spawned by the transformed organism with gametes of a wild-type organism of the same species of the opposite sex to obtain a first filial generation (Fl) of transformed organisms; and optionally

(V) repeating steps (ll)-(IV) to obtain further filial generations of transformed organisms.

In another aspect, the present invention related to 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.

According to one embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, a gel, oil and a semisolid formulation.

In a further aspect, 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.

According to an embodiment of the invention, the support is selected from the group consisting of a patch, a foil, a plaster and a film. According to another embodiment, the at least one stinging capsule is secured to the support by biological glue, polylysine, or a mesh support.

According to a further embodiment, 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.

In a further aspect, the present invention relates to a method for transdermal delivery of a protein of interest to a mammal, comprising the steps of:

(i) transforming an organism according to the method for expressing an exogenous protein of interest under the control of an endogenous gene control element in a cnidarian organism according to the invention;

(ii) isolating at least one stinging capsule containing the protein of interest from the transformed organism;

(iii) lyophilizing the at least one stinging capsule to obtain a capsule powder;

(iv) mixing the capsule powder with an anhydrous lotion to obtain a capsule lotion;

(v) applying the capsule lotion to the skin; and

(vi) applying an aqueous solution to the skin, thereby triggering the discharge of the protein of interest from the at least one capsule into the mammal.

In a still further aspect, the present invention relates to a method for transdermal delivery of a protein of interest to a mammal, comprising the steps of:

(iii) securing the at least one stinging capsule to a support; (iv) applying the at least one stinging capsule containing the protein of interest to the skin; and

(v) triggering a discharge of the protein of interest from the at least one stinging capsule.

BRIEF DESCRIPTION OF THE DRAWINGS

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/μΙ), Cas9 recombinant protein with nuclear localization signal (500 ng/μΙ) 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 μιτι.

Abbreviations: A (ampicillin resistance sequence); Al (Ascl restriction enzyme); Bl (before injection); G (genome); 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. Abbreviations: Ad (adult); FO (injected parent generation); Fl (first filial generation); PI (planula); Po (primary polyp).

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.

Fig. 3F shows protein expression of memOrange2 and NvNcol3, revealed by immunostaining, in a tentacle of a polyp transformed N. vecetensis of Fl generation. Abbreviations: Me (merge); mem02 (memOrange2); NC (NvNcol3).

DETAILED DESCRIPTION OF THE INVENTION

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).

Conversely, the present inventors have discovered that placing an exogenous sequence coding for a protein of interest under the control of an endogenous control element gives much better results in terms of yield and expression specificity.

By a first aspect, 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. According to a specific embodiment, 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. In a specific embodiment of the invention, 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.

In another specific embodiment of the invention, the organism is the sea anemone Nematostella vectensis (hereinafter: "N. vectensis").

It should be noted that in case 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.

Alternatively, organisms having active endogenous toxins can also be utilized by the present invention, provided inactivation of the endogenous toxin is effected prior to use. Such 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. Optionally, such co-transformation is effected using a single expression construct expressing both polynucleotides. The terms "transformed organism" and "transgenic organism" as used interchangeably herein, refer to an organism whose genetic material has been altered by addition of exogenous genetic material, by the use of any genetic engineering technique.

As used herein, 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.

As used herein, the term "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.

The term "gene control element" as used herein 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. As used herein the term "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.

The term "endogenous" refers to a nucleic acid sequence or a polypeptide that originates from the specific organism undergoing the transformation according to the invention. For example, when N. vectensis is selected to overexpress the protein of interest, the endogenous gene control element of N. vectensis is used.

According to the present invention, 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. Alternatively, only 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.

In a specific embodiment, the endogenous gene control element is present in its original genomic location within the stinging cell.

As used herein, 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.

The terms "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.

As used herein, 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. Typically, the protein of interest is selected from a group consisting of a drug, vaccine, hormone, enzyme, antibody or label.

According to a specific embodiment, 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. 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 to, during, or following discharge of the stinging cell.

As used herein, the term "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) can be activated just prior to stinging cell discharge by providing an activator compound (for example an ion), which can be diffused or pumped (during discharge) into the cell or capsule. Alternatively, 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. Another example is the glycoprotein Granulocyte-Colony Stimulating Factor (GCSF; GenBank accession NP_000750.1), which is used for stimulating the production of white blood cells after chemotherapy, and in some cases before blood donation. Additionally, 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.

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-β 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).

The terms "amino acid sequence", "polypeptide", "peptide" and "protein", as used interchangeably herein, refer 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. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is to be understood that the polypeptides can occur as single chains or associated chains. According to the invention, 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. Upon synthesis, the signal peptide enables the transport of the protein of interest from the Golgi into the capsule of the stinging cell. Thus, the protein of interest expressed in the stinging cells of the transformed organism of the invention is accumulated and stored in the stinging capsule.

By a non-limiting example, a suitable signal peptide according to the invention is a signal peptide derived from a cnidarian minicollagen, nematogalectin or toxin. According to a specific embodiment, the signal peptide is derived from NEP3, which is a marker protein of cnidocytes (NCBI accession number XP_001640559).

According to one embodiment of the invention, 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. Non-limiting examples of detectable label proteins suitable for the invention are selected from: DsRed, Tl, Dimer2, mRFPl, mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry and mCherry. In a specific embodiment of the invention, the detectable label protein is mOrange2.

According to another specific embodiment of the invention, 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"). Examples of a self-cleavable amino acid sequence are selected from, but not limited to, the viral peptide P2A and internal ribosome entry site (IRES).

Accordingly, in a specific embodiment of the invention, 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. In a specific embodiment, 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. In another aspect, 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:

(b) transferring the transforming vector into a zygote of the organism.

In 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.

The terms "vector" and "expression construct", as used interchangeably herein, refer 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. Examples of 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. While the 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.

According to one embodiment of the invention, the vector can be any high copy number plasmid, for example the pUC series or pBluescript. According to other embodiments, a linear DNA fragment can also be used.

It should be appreciated that in some embodiments, 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.

The term "homology arm" as used herein, 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.

The term "transforming sequence" as used herein refers to an exogenous DNA sequence coding for a protein of interest fused to a signal DNA sequence coding for a signal peptide. The term "signal peptide", as used herein 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). The "signal sequence" refers to a polynucleotide that encodes for a signal peptide.

Hence, upon synthesis of the protein of interest in the stinging cell, 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.

By a non-limiting example, 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. According to a specific embodiment, 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).

According to one embodiment of the invention, 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.

ATG GTG AG C A AG GGCGAGGAG A ATA AC ATG G C A ATC ATCA AG GAG TTC ATG AG ATTCA AG GT GAGAATGGAGGGCTCAGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCAGA CCATACGAGGGCTTTCAGACAGCAAAGCTGAAGGTGACAAAGGGTGGCCCACTGCCATTCGCA TGGGACATCCTGTCCCCACATTTCACATACGGCTCAAAGGCATACGTGAAGCACCCAGCAGACA TCCCAGACTACTTCAAGCTGTCATTCCCAGAGGGCTTCAAGTGGGAGAGAGTGATGAACTACGA G G ACG G CG G CGTG G TG AC AGTG AC AC AG G ACTC ATC ACTCC A AG ACG G CG AGTTCATCTAC A A GGTGAAGCTGAGAGGCACAAACTTCCCATCAGACGGCCCAGTGATGCAGAAGAAGACAATGG GCTGGGAGGCATCATCAGAGAGAATGTACCCAGAGGACGGCGCACTGAAGGGCAAGATCAAG ATG AG ACTG A AG CTG AAGGACGGCGG CC ACTAC AC ATC AG AG GTG A AG AC A AC ATA CA AG G C A A AG A AG CC AGTG C AG CTG CCCG G CG C ATAC ATCGTG G AC ATC A AG CTG G ACATC AC ATC AC A CAACGAGGACTACACAATCGTGGAACAGTACGAAAGAGCAGAGGGCAGACACTCAACAGGCG G C ATG G ACG AG CTGTAC A AG G G CG G CTCTG G CG G GTCTG G G G G GTCTG G G AGTG GTG CTACC AACTTTTCCCTCTTGAAACAGGCAGGCGATGTTGAGGAAAATCCTGGACCCATGAAGCTGACAT ACATCCTGCTTATTGCAGTTGTCGGTGTCGCTATAGAGGCCAAAAGCGTCAAGAAATCAAAGGC CCATCATAAAA AAAA AAG G ATGTCTTATAACCTCTTG GGATTCCTG CAAAG GTCCTCAAACTTCC AGTGCCAGAAGTTGCTCTGGCAACTGAATGGCCGACTTGAGTATTGCCTCAAAGACAGGATGA ATTTTG ATATACCG G A AG AG ATA A A AC AG CTG C AG C A ATTTC AG A AG G A AG ACG CG G C ACTTA C A ATATACG A A ATG CTG C AG A ATATATTTG CTATATTC AG G C AG G ATTCTAGTAG TAC AG G CTG GAACGAGACGATCGTTGAGAATTTGTTGGCTAATGTTTACCACCAAATCAACCACTTGAAGACG GTTTTG G AG G A A A AG CTCG A A A AG G AG G ATTTC ACTAG AG G G A A ACTTATG TCCTCCCTG C ACC TG A AG AG GTATTATG GTCG A ATTCTCC ACTATCTTA A AG CA A A AG A ATACTCTC ATTGTG CTTG G ACTATTGTAAGGGTTGAAATCCTGAGAAACTTCTACTTTATTAATAGGCTGACAGGTTACCTTCG GAACTAA, wherein positions 1-708 code for mOrange2, positions 709-735 code for a flexible linker, positions 736-801 code for the self-cleavable P2A peptide, positions 802- 897 code for the signal peptide of NEP3 and positions 898-1398 code for I nterferon beta la (898-1398).

According to some embodiments of the invention, the transforming sequence also comprises an endogenous stinging cell-specific gene control element. According to other embodiments, wherein 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. However, 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.

In cases when the transformed organism is N. vectensis, and the stinging cell is a nematocyte or a spirocyte, 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.

In 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 according to the invention, 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.

According to a specific embodiment, the transforming vector is transferred to the zygote of the organism to be transformed by microinjection. Thus, the transferring of the transforming vector to the zygote of the organism is carried out by microinjection.

The term "microinjection" as used herein 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.

According to some embodiments, wherein the transforming sequence is to be integrated into the genome of the organism, 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.

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.

Accordingly, in some embodiments, 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.

In a non-limiting example, 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. According to this technology, 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). In some cases, the two RNA molecules can be linked together to form a single guide RNA (sgRNA) sequence. The technology allows existing genes to be removed and/or mutated, as well as new genes to be added.

Accordingly, 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).

The following 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.

GGCAGUAGUUAGGGCAUCCCGGGUUUUAGAGCUAGAAAUAGCAGUUAAAAUAAGGCUAG U CCG U U A U CAACU U G A AAA AG U GG CACCG AG UCGGUGCUUUU

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. In addition, 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.

The execution of 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.

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 according to the method of the invention 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. Notably, the endogenous control element is present in its original genomic location within the DNA.

As would be appreciated by a person skilled in the art, "knocking-in" an exogenous nucleic acid sequence into a specific genomic location is technically challenging. In most cases and most organisms the procedure fails, especially for polynucleotides longer than a few base pairs. Therefore, a person skilled in the art would generally prefer using standard and simpler techniques that do not require site-specific mutagenesis. However, using the promoter in its original genomic location as described herein, guarantees expression only in stinging cells, thereby avoiding useless expression in other tissues that can inhibit the growth of the organism. In addition, random incorporation of genetic material to the genome might be cytotoxic and thus destructive to the stinging cells.

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.

In a further aspect, 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.

In a still further aspect, 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:

(I) transforming a cnidarian organism according to the method specified above, to obtain a parent generation (F0) transformed organism;

(III) inducing the sexually mature transformed organism to spawn gametes according to any method known in the art; (IV) mixing the gametes spawned by the transformed organism with gametes of a wild-type organism of the same species of the opposite sex to obtain a first filial generation (Fl) of transformed organisms; and optionally

Interestingly, 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.

According to another aspect of the present invention there is provided 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.

Following isolation of the capsule from the stinging cell, the capsule is lyophilized and can be maintained as dry powder, which is stable up to several years.

According to a further aspect of the present invention there is provided 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.

According to one embodiment of the invention, 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. BIOBOND™), polylysine, a mesh support, or any other acceptable attaching material.

According to one embodiment of the invention, following application of the device to the outer surface of the mammalian tissue, the at least one stinging capsule is activated to release the protein of interest into the tissue.

According to a specific embodiment, the mammalian tissue is the skin.

In order to achieve activation of the stinging capsule, 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. 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.

According to a specific embodiment, 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. According to a specific embodiment, the electrical pulse is approximately 20-30 Volts for 30 microseconds.

According to a still further aspect of the present invention there is provided a method for transdermal delivery of a protein of interest to a mammal, the method comprising the steps of:

(i) transforming an organism as described above;

(v) applying the capsule lotion to the skin; and

The capsule lotion can be applied to the skin either directly or indirectly by the use of a support as specified above.

According to a specific embodiment, the anhydrous lotion is a gel consisting of 2% hydroxypropylcellulose in absolute ethanol. According to another specific embodiment, the aqueous solution is 1% pentasodium triphosphate at a pH between of 8 and 10.

Alternatively, the method for transdermal delivery of a protein of interest to a mammal may comprise the steps of:

(i) transforming an organism as described above;

(iii) securing the at least one stinging capsule to a support;

(iv) applying the support to the skin; and

According to the invention, the at least one stinging capsule can be secured to the support by, for example, biological glue (e.g. BIOBOND™), polylysine, a mesh support, or any other acceptable attaching material.

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.

According to a specific embodiment, the transformed organism is a member of a line of transgenic organisms produced as described above.

The invention will now be described with reference to specific examples and materials. The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of specific embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

EXAMPLES

Materials and methods

Sea anemone culture

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.

Transforming Nematostella vectensis to express memOrange2

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/μΙ) of SEQ ID NO: 1, Cas9 recombinant protein with nuclear localization signal (500 ng/μΙ; 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).

The 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).

Isolation of stinging capsules

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 CaCI₂ to a final concentration of 15 mM NaCI and 0.2 mM CaCI₂ and immediately freeze-dried. The microcapsules were kept in powder form at 2 to 8 °C until use.

Example 1:

Successful generation of a transformed line of N. vectensis expressing memOrange2

In order to generate a transformed line of N. vectensis that expresses a fluorescent reporter protein in its cnidocytes (stinging cells), 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. 2, the expression of memOrange 2 in the injected parent generation (F0) was restricted to small-to-medium patches. By contrast, the first filial generation (Fl) exhibited specific and strong expression of memOrange2 in a very wide population of cnidocytes, with extremely strong expression in the tentacles of the polyp. These results indicate that a higher yield of the protein of interest can be obtained in stinging cells of filial generation rather than the injected parent generations.

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.

In addition, utilization of the CRISPR/Cas9 technology exploits the homologous recombination based DNA repair mechanism of the cell to insert the transgene into the native gene and, thus, places it under the control of its native regulatory elements. Hence, the expression pattern of the memOrange2 accurately mirrored the native expression of NvNcol3, both at the mRNA and protein levels, as demonstrated by double fluorescent in situ hybridization (FISH) analysis (Fig. 3E) and immunostaining experiments (Fig. 3F), respectively.

Taken together, the results indicate that stinging cells isolated from transformed organisms, such as N. vectensis, strongly express exogenous medium-sized protein, such as memOrange2, under the control of endogenous NvNcol3 promoter.

Claims

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.

The transformed organism according to claim 1, wherein said control element is present in its original genomic location.

The transformed organism according to claim 1 or 2, wherein the organism is of a class selected from a group consisting of Anthozoa, Hydrozoa and Scyphozoa.

The transformed organism according to any one of claims 1-3, wherein the organism is Nematostella vectensis.

The transformed organism according to claim 1 or 2, wherein the stinging cell is selected from the group consisting of: a cnidocyte, a nematocyte, a spirocyte and a ptychocyte.

The transformed organism according to claim 1 or 2, wherein the protein of interest is a protein that is delivered to a mammal for a therapeutic, diagnostic or cosmetic purpose.

The transformed organism according to claim 6, wherein the protein of interest is selected from the group consisting of a drug, vaccine, hormone, enzyme, antibody or label.

8. The transformed organism according to any one of claims 1-7, wherein the exogenous sequence coding for the protein of interest is fused to a signal sequence encoding a signal peptide.

9. The transformed organism according to claim 8, wherein the signal peptide is derived from a cnidarian minicollagen, nematogalectin or toxin.

10. The transformed organism according to claim 9, wherein said minicollagen is NEP3.

11. The transformed organism according to any one of claims 1-10, wherein the exogenous sequence coding for the protein of interest is conjugated to a sequence coding for a detectable label protein.

12. The transformed organism according to claim 11, wherein 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.

13. The transformed organism according to claim 12, wherein the cleavable amino acid sequence is the viral peptide P2A.

14. The transformed organism according to claim 1 or 2, wherein the endogenous stinging cell-specific gene control element is the promoter of NvNcol3 gene.

15. A method for expressing an exogenous protein of interest under the control of an endogenous gene control element in a cnidarian organism, comprising:

(b) transferring the transforming vector into a zygote of the organism.

16. The method according to claim 15, wherein 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.

17. The method according to claim 15 or 16, wherein transferring of the transforming vector to the zygote of the organism is carried out by microinjection.

18. The method according to claim 16, wherein said integration of the transforming sequence into the genome of the organism at a specific location is achieved by CRISPR-Cas9 technology.

19. A stinging cell expressing a protein of interest, wherein the stinging cells is isolated from the transformed organism of any one of claims 1 to 14.

20. A stinging capsule containing a protein of interest, wherein the stinging capsule is isolated from the stinging cell of claim 19.

21. A method for producing a line of transformed cnidarian organisms expressing an exogenous protein of interest, comprising:

(III) inducing the sexually mature transformed organism to spawn gametes;

(IV) mixing the gametes spawned by the transformed organism with gametes of a wild-type organism of the same species of the opposite sex to obtain a first filial generation (Fl) of transformed organisms; and optionally (V) repeating steps (ll)-(IV) to obtain further filial generations of transformed organisms.

22. A pharmaceutical composition comprising, as an active ingredient, at least one stinging capsule isolated from the transformed organism of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

23. The pharmaceutical composition according to claim 22, wherein the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous solution, a gel, oil and a semisolid formulation.

24. A delivery device comprising: (a) at least one stinging capsule according to claim 20; and (b) a support.

25. The delivery device according to claim 24, wherein the support is selected from the group consisting of a patch, a foil, a plaster and a film.

26. The delivery device according to claim 24 or 25, wherein the at least one stinging capsule is secured to the support by biological glue, polylysine, or a mesh support.

27. The delivery device according to any one of claims 24-26, further comprising 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.

28. A method for transdermal delivery of a protein of interest to a mammal, comprising the steps of:

(i) transforming an organism according to the method according to any one of claims 15-18; (ii) isolating at least one stinging capsule containing the protein of interest from the transformed organism;

(v) applying the capsule lotion to the skin; and

29. A method for transdermal delivery of a protein of interest to a mammal, comprising the steps of:

(i) transforming an organism according to the method according to any one of claims 15-18;

(iii) securing the at least one stinging capsule to a support;

(iv) applying the at least one stinging capsule containing the protein of interest to the skin; and