A genetically modified eukaryotic microalga and injectable composition for symbiotic therapy

Abstract

The present invention relates to a symbiotic therapeutic technique based on a genetically modified eukaryotic microalga (51), preferably photosynthetic, such as for example Chlamydomonas reinhardtii, in which an expression cassette (10) codifying a biologically active peptide and / or protein comprises a secretion coding module (5) of a therapeutic agent to allow the biologically active peptide / protein to be released so that it can cross the cell wall of the microalga (51), to be secreted outside of it and enter directly into the circulation in a subject (53) in which said genetically modified eukaryotic microalga (51) has been subcutaneously or intradermally injected. The microalga can be of a type that naturally: a) lacks flagella; or b) has flagella, in which case a prior genetic modification prevents the formation or inactivates the flagella, so that the microalga remains immobilised at a subject injection site (53). The expression cassette (10) can further comprise at least one further coding module selected from: a homing module (6), for targeting the peptide / protein to a predetermined tissue and / or cell population of the subject (53); a penetrating module (7) for inducing internalisation of the protein within a cell of said subject (53) through the cell membrane or by induced endocytosis; a protection module (9). The invention also relates to a subcutaneously or intradermally injectable composition containing said microalgae, and to a kit for preparing said composition.

Description

TITLEA GENETICALLY MODIFIED EUKARYOTIC MICROALGA AND INJECTABLE COMPOSITION FOR SYMBIOTIC THERAPYDESCRIPTIONScope of the invention

[0001] The present invention relates to a genetically modified eukaryotic microalga for prolonged delivery of a peptidic and / or proteic therapeutic agent into a living organism. The invention relates to an injectable composition containing said microalga.Prior art - Technical problems

[0002] Efficient therapy delivery is a crucial problem in the treatment of human diseases. The cellular mechanisms and molecular pathways that cause debilitating conditions in humans, such as ageing and age-related diseases, are known. Moreover, from experimentation in vitro and in genetically modified animal models it is known which proteins or drugs can significantly help in healing those conditions.

[0003] However, in many disease contexts, improving human health and prolonging patient lifespan remains a considerable challenge, for the reasons explained below.

[0004] Firstly, therapeutic agents, such as drugs, mRNAs and proteins, are unstable in the extracellular environment. For this reason, specific measures are required to protect them and prolong their half-life, for example, by encapsulation in liposomes or infection with AAV vectors. Moreover, once entered into cells, the therapeutic agents show different half-lives, depending on their nature. For instance, simple molecules with pharmacological action are more stable than proteins, which in turn are more stable than nucleic acids.

[0005] Secondly, therapeutic agents, once administered, must overcome various barriers in the body, such as the cell membrane, hepatic retention or the blood-brain barrier. Therefore, the therapeutic agents must be associated with efficient transport systems, such as liposomes, cellpenetrating peptides or AAV vectors.

[0006] Thirdly, the number of therapeutic particles that can be injected in a single dose is much lower than the number of cells that are present in the body, even assuming an even distribution of the drug that is established, for instance, after an intravenous injection. Moreover, the distribution of therapeutic agents is not uniform, as most of the drug is retained by the liver or cannot reach the cells located deep inside the tissues. It should also be considered that each therapeutic agent has a specific halflife. In this connection, even if a therapeutic agent succeeds in reaching a cell, its action will not last longer than a relatively short time window.

[0007] For the above-mentioned reasons, it is unlikely that therapeutic agents can be delivered to all the cells of a human adult by a limited number of doses, even if a smaller, specific cell population is selected to be targeted by a sophisticated system comprising, for instance, lipid nanoparticles. It is therefore often necessary to administer repeated doses of therapeutic agents to the patient. This entails high costs and possible side effects, such as toxicity or adverse immune reactions over time.

[0008] It is therefore desirable to find new techniques that allow an active principle or a therapeutic agent to be released into a subject more effectively than what is allowed by the conventional therapeutic techniques.

[0009] US 10, 781, 447 B2 describes non-propagating, genetically modified eukaryotic microalgae that are capable of expressing at least one biologically active heterologousRNAi molecule. These microalgae used to orally deliver the RNAi molecule to a target organism, typically an aquatic or terrestrial animal, in its intact and functional form. Specifically, the RNAi molecule is an siRNA for gene silencing within the cells of the target organism.

[0010] US2020 / 063152A1 describes edible vaccines comprising transgenic microalgae expressing at least one exogenous antigen or at least one intermediate organism comprising transgenic microalgae. The antigen-expressing microalgae are used for orally administering the antigen to a target organism maintaining the functional form thereof, such that the antigen is able to cause immunogenic responses in a subject consuming the microalgae, whether human or animal. Similarly, EP2887819B1 describes transgenic microalgae expressing biologically active exogenous proteins to be orally administered to target organisms. Such exogenous proteins are capable of exerting beneficial or therapeutic effects on human or animal subjects consuming the microalgae.

[0011] Also similarly, US2003 / 211089A1 describes the oral administration to a host animal of a biologically active protein contained in an algal cell. The algal cell is prepared by transforming the same using an expression vector comprising a nucleotide sequence codifying for the biologically active protein, expressed under the functional control of a promoter. The biologically active protein is an antigenic epitope and, once the alga has been orally administered, it causes an immune response in the host animal.

[0012] US2010 / 323001A1 describes methods for delivering nucleic acids, including dsRNA, to mammalian target cells in-vivo by intercellular transfer. The dsRNA is delivered to or expressed in a first cell other than the target cell that receives the dsRNA. The first cell can then be used for cell culture production of RNA-containing exo-vesicles, or forautologous or heterologous transplantation into a receiving mammalian organism, so as to provide any nucleic acid that can be delivered by the transfected cell to a distal target cell. Nucleic acids can be delivered to distal organs and tissues by intradermal or subcutaneous administration and by transfection of skin cells, including fibroblasts, with at least one type of nucleic acid in-vivo.

[0013] US 2024 / 173265 A1 relates to a composition containing extracellular vesicles from microalgae (MEVs) that are exogenously loaded with bioactive cargo for therapeutic, industrial, diagnostic and cosmetic uses, and, in particular, for obtaining vaccines, performing anticancer therapies, diagnostics, and other similar uses.

[0014] Molino Joao et al, in " Comparison of secretory signal peptides for heterologous protein expression in micro algae: Expanding the secretion portfolio for Chlamidomonas reinhardtii", PLOS ONE, Vol. 13, no. 2, 6 / 12 / 2018 deal with signal peptides for heterologous protein secretion in Chlamydomonas reinhardtii and relate to the secretion of recombinant proteins into the extracellular environment by microalgae as a known process.

[0015] WO 2025 / 184534 Al discloses non-enveloped capsid delivery systems comprising a viral capsid polypeptide and a heterologous cargo, as well as methods for manufacturing and using such delivery systems.Summary of the invention

[0016] It is therefore an object of the present invention to provide a symbiotic therapeutic technique that ensures a predetermined concentration level of an active principle to be maintained in a treated subject' s target tissue or organ for a time longer than in the case of any conventional therapeutic technique.

[0017] It is a particular object of the invention to provide such a therapeutic technique that makes it possible to reach a subject' s target tissue or organ without having to overcome the barriers that the subject ' s body naturally opposes to the conventional delivery techniques.

[0018] It is another particular object of the invention to provide such a therapeutic technique that ensures a predetermined concentration level of an active principle to be attained in remote cells of a treated subject' s target tissues and organs more reliably than in the case of any conventional administration technique.

[0019] It is a further object of the invention to provide such a therapeutic technique that makes it possible to achieve the above-mentioned concentration level, durability and uniformity goals in organs and tissues without the need of a large number of administrations of an active principle, in comparison with such conventional techniques as oral, intravenous or direct intradermal administration.

[0020] It is also a particular object of the present invention to provide such a therapeutic technique that is more cost-effective than such techniques as lipid nanoparticles or AAV vectors administration to a patient, which can provide performance levels comparable with those allowed by the invention.

[0021] It is also a particular object of the present invention to provide such a therapeutic technique that is safer for the subject being treated than the aforementioned conventional techniques that can provide performance levels comparable with those allowed by the technique according to the invention.

[0022] It is a further object of the present invention to provide a biotechnological system that can be modulated and customised, as required, for delivering the desired peptideand / or protein therapeutic agent to a specific tissue and / or in a specific way.

[0023] According to different aspects of the invention, the above-mentioned objects are achieved by a genetically modified eukaryotic microalga, by an injectable composition for subcutaneous injections, containing said microalga, and by a kit for preparing such a composition, as defined by independent claims 1, 11 and 17, respectively.

[0024] The invention is based, in a plurality of aspects thereof as described below, on a subcutaneously or intradermally injectable genetically modified eukaryotic microalga comprising an expression cassette codifying a biologically active protein, wherein the expression cassette comprises a secretion coding module configured to be translated into a secretion module fused to the biologically active protein, the secretion module configured to mediate the secretion of the biologically active protein outside of the cell wall of the genetically modified eukaryotic microalga, in other words the expression cassette comprises a secretion signal sequence for secreting the therapeutic agent, such that the biologically active protein can be secreted outside of the genetically modified eukaryotic microalga and can be passively diffused in a subject into which the genetically modified eukaryotic microalga is injected.

[0025] By "biologically active protein" it is meant herein, in the broadest sense, a peptide or a polypeptide, i. e. a sequence of amino acids that are mutually linked by peptide bonds, which is capable of acting as an "active principle" or "therapeutic agent", these expressions indicating peptides or polypeptides or complex proteins having the function of hormones and / or enzymes and / or transcription factors and / or protein co-factors that are configured to mediate or otherwise influence a biologicalprocess. The expressions "biologically active protein", "biologically active polypeptide" and "biologically active peptide" are used interchangeably in this specifiction, alone or in combination, as well as the expressions "protein", "polypeptide" and "polypeptide" when referred to an "active principle" or a "therapeutic agent" or the like. These peptides, polypeptides or complex proteins can be either endogenous or exogenous.

[0026] By "subject" a human being or an animal is meant, requiring, for instance, a given local or systemic therapeutic treatment.

[0027] According to one aspect of the invention, the microalga is selected from the group comprised of:- eukaryotic microalgae naturally without flagella, i. e.selected from the group comprised of:- Chlorella spp.;- Scenedesmus spp.;- Micractinium spp.;- Monoraphidium spp.;- Coelastrum spp.;- Pediastrum spp.;- Nannochloropsis spp.;- Trebouxia spp.;- Botryococcus braunii; and- eukaryotic microalgae that are naturally provided with flagella, but have been genetically modified to have no flagella or to have inactive flagella by inactivation of a gene selected from the group comprised of: bld1 (IFT52); bld2 (centrin / CEP164-like); bldlO (CEP135- like); IFT43; IFT121; IFT122; IFT139; IFT140; IFT144; IFT172; IFT88; IFT81; IFT80; IFT74; IFT70; IFT57; IFT54; IFT46; IFT38; IFT27; IFT25; IFT22; IFT20; fla10 (Kinesin-2 motor subunit); fla8 (Kinesin-2); fla3 (FAP163; KAP subunit of kinesin-2).Similarily, the microalga can be selected among the eukaryotic microalgae naturally without cilia, or it can be an eukaryotic microalga naturally provided with cilia, but genetically modified to have no cilia or to have inactive cilia, by inactivation of specific genes.

[0028] In this way, microalgae according to the invention, once they have been subcutaneously or intradermally injected to a subject, cannot migrate in the subject' s body and remain within the subcutaneous or intradermal injection site and continue to release there the therapeutic agent. In the eukaryotic microalgae are photosynthetic eukaryotic microalgae, they can also continue to receive the light passing through the subject ' s skin.

[0029] According to another aspect of the invention, a subcutaneously or intradermally injectable composition comprises the genetically modified eukaryotic microalga defined above, as well as a biocompatible resuspension liquid in which the genetically modified eukaryotic microalga is resuspended.

[0030] According to a further aspect of the invention, a kit for preparing a composition that can be subcutaneously or intradermally injected into a subject comprises the above-mentioned genetically modified eukaryotic microalga, a microalga maintenance medium or support, on which the genetically modified eukaryotic microalga is arranged, and also comprises a biocompatible resuspension liquid for the genetically modified eukaryotic microalga.

[0031] For instance, the biocompatible resuspension liquid of the composition and of the kit for preparing the composition can be an aqueous solution of one or more biocompatible salts that are suitable to maintain physiologically compatible osmolarity and / or pH conditions, such as NaCl, phosphate buffers, TRIS and the like, as known to a person skilled in the art.

[0032] For the reasons explained above, the protein has the essential feature of being able to cross the cell wall of the genetically modified eukaryotic microalgae. This way, once injected, the protein can leave the genetically modified eukaryotic microalgae and can be released into and passively be diffused through the surrounding subcutaneous and / or intradermal environment and then, possibly, also into the subject ' s bloodstream, typically, in order to perform a therapeutic action.

[0033] Accordingly, the genetically-modified eukaryotic microalga is configured to perform the rôle of a releasing agent of a therapeutic agent that has the form of a biologically active protein. The invention provides therefore a symbiotic therapeutic technique, i. e., a therapeutic technique based on the combination of a protein or polypeptide or peptide as a therapeutic agent and a eukaryotic microalga that is genetically modified to produce and secrete the protein or polypeptide or peptide. This therapeutic technique involves a subcutaneous and / or intradermal injection of the genetically modified eukaryotic microalga, so that the genetically modified eukaryotic microalga can continuously secrete specified amounts of the therapeutic agent within the subject over time, in particular, in order to maximise the therapeutic effect and to avoid pile-up of possible toxic levels of the therapeutic agent in the subject.

[0034] Due to the continuous secretion of the therapeutic agent in situ by the genetically modified eukaryotic microalgae, the symbiotic therapeutic technique of the invention allows a persistence of the therapeutic agent in the treated subject' s blood and / or tissues and / or organs that shows to last longer than in the case of any other known administration techniques.

[0035] Moreover, by the symbiotic therapeutic technique of the invention, the active principle persists at constant levels in the subject' s blood for longer times than in the case of other administration systems, as the technique exploits a basal continuous secretion by the microalgae, which remains effective for a prolonged period of time. Actually, higher levels of the therapeutic agent have been observed than in untreated control organisms, both in the bloodstream and in the main organs of a plurality of subjects treated by this symbiotic therapy, after a short time, e. g. 15 days.

[0036] Another significant advantage of the symbiotic therapeutic technique of the invention is that the same does not require multiple administration, i. e., it allows a "one-shot therapy", based on a single injection, as the injected microalgae remain effective at their injection or target site.

[0037] The symbiotic therapeutic technique of the invention also allows a higher safety level with respect to other viral or LNP systems, as it is based on the natural symbiosis of the microalgae with the host organism. In this regard, no signs of inflammation have been observed in the subjects treated by this symbiotic therapy, even over prolonged treatment times of several weeks.

[0038] Moreover, the symbiotic therapeutic technique of the invention can be performed by a very simple preparation process, which contributes to lower the related costs with respect to the prior art therapeutic agent delivery systems.

[0039] Advantageously, the expression cassette of the genetically modified eukaryotic microalgae can include an additional coding module selected from the group comprised of:- a homing module comprising an amino acid sequence codifying for a homing-peptide to deliver a polypeptideor polypeptide portion to a predetermined tissue and / or a predetermined cell population of the subject into which the genetically modified eukaryotic microalga is injected;- a penetrating module comprising an amino acid sequence codifying for a Cell Penetrating Peptide to trigger a polypeptide or polypeptide portion internalization into a cell of the subject across the cell membrane or by induced endocytosis.

[0040] By introducing the homing module and / or the penetrating module into the expression cassette of the microalga, besides the secretion module, the genetically modified microalga, in addition to be configured to act as a delivery agent for a therapeutic agent, is also configured to allow further advantages of the therapeutic technique of the invention, i. e., a higher cell internalization efficiency and / or a higher action specificity, respectively, than other peptide and / or protein therapeutic agent delivery systems, such as LNPs or exosomes.

[0041] The term "microalga" as a delivery agent includes any species of microalgae that is capable of producing a polypeptide or polypeptide portion. Such microalgae can include red, brown, and green microalgae.

[0042] For instance, the genetically modified eukaryotic microalgae can be at least one selected from the group comprised of: Chlorella sorokiniana, Picochlorum sp HM1, Botryococcus braunii, Chlamydomonas reinhardtii, Tetraselmis suecica, Dunaliella salina, Nannochloropsis spp., Chlorella vulgaris, Haematococcus pluvialis, Scenedesmus spp. Preferably, the microalga is Chlamydomonas reinhardtii.

[0043] Obviously, the genetically modified eukaryotic microalga in the composition and in the kit for preparing the composition is similarly selected from the eukaryoticmicroalgae naturally without flagella, in turn selected from the group comprised of Chlorella spp., Scenedesmus spp., Micractinium spp., Monoraphidium spp., Coelastrum spp., Pediastrum spp., Nannochloropsis spp., Trebouxia spp., Botryococcus braunii or from the eukaryotic microalgae that are naturally provided with flagella, but have been genetically modified to have no flagella or flagella made inactive by inactivation of a gene selected from the group comprised of bldl (IFT52), bld2 (centrin / CEP164-like), bld10 (CEP135-like), IFT43, IFT121, IFT122, IFT139, IFT140, IFT144, IFT172, IFT88, IFT81, IFT80, IFT74, IFT70, IFT57, IFT54, IFT46, IFT38, IFT27, IFT25, IFT22, IFT20, fla10 (Kinesin-2 motor subunit), fla8 (Kinesin-2), fla3 (FAP163, KAP subunit of kinesin-2).

[0044] In this way, the genetically-modified eukaryotic microalgae, once injected, can remain in a subcutaneous or intradermal position of the treated subject, in the neighbourhoud to the injection site, at least for the intended duration of the treatment. This makes the symbiotic therapeutic technique of the invention safer, as unfavourable interactions of the genetically modified eukaryotic microalgae with districts of the subject' s body far from the injection site are prevented, and the genetically modified eukaryotic microalgae can be removed from the injection site, once the treatment has been completed, or for any other reason.

[0045] Preferably, the genetically modified eukaryotic microalga is a photosynthetic mictroalga. In this way, the genetically modified eukaryotic microalga does not require any specific source of nourishment arranged within the host ' s body, and is essentially self-sustaining for the entire time of application of the symbiotic therapeutic technique by the light permeating through the subject ' s skin. Such a feature is evidently advantageous in the caseof a genetically modified eukaryotic microalga configured to remain immobilised at an injection site in a part of the subject' s body that is normally exposed to light.

[0046] In particular, the biologically active protein is codified by a plasmid comprising the expression cassette, inserted into the genetically modified eukaryotic microalga by transfection, which is preferably carried out using Agrobacterium Tumefaciens as a transfection vector.

[0047] As an alternative, the biologically active protein can be codified by an exogenous DNA sequence inserted into the genetically modified eukaryotic microalgae as an expression cassette by a technique selected from the group comprised of e.g. CRISPR / CAS, TALENs, electroporation, "Gene gun", viral vectors.

[0048] For example, the secretion coding module of the therapeutic agent comprises an amino acid sequence selected from the group comprised of:SEQ NO. 31, BiPl signal peptide: MAQWKAAVLLLALACASY - SEQ NO. 32, Gametolysin signal peptide:MSLATRRFGAAAALLVAACVLCTAPAWA- SEQ NO. 33, ARS1 Signal peptide:MHARKMGALAVLAVACLAAVASVAHA SEQ NO. 34, IBP1 Signal peptide:MPSSSMKLFAALLIACMAQTSMA- SEQ NO. 35, Glycoside Hydrolase-like protein signal peptide: MRRAIALGVGLALLGLLLPGSLA- SEQ NO. 36, sulfotransferase family Signal peptide MPSTPVAAALRALLCASLLGSLHIARA- SEQ NO. 377, SADlp Signal peptid:MTLRLAQLALATLGVLLLVLAPMPALS- SEQ NO. 38, Prolyl-4-hydroxylase signal peptide:MARRLLLALALAAVLGLAHA- SEQ NO. 39, Mitogen-activated kinase 7 protein signal peptide: MRGIIAAYTSATLLALLLVTWLTHSSA.

[0049] For example, the amino acid sequence codifying for a homing-peptide of the addressing module is selected from the group comprised of:- SEQ NO. 40, human brain homing peptide: CLSSRLDAC;- SEQ NO. 41, human kidney homing peptide: CLPVASC;- SEQ NO. 42, 43, human lung homing peptide:CGFERVRQCPERC, CGFELETC;SEQ NO. 44, human skin homing peptide: CVALCREACGEGC; SEQ NO. 45, human pancreas homing peptide: SWCEPGWCR; SEQ NO. 46, human intestine homing peptide: YSGKWGW; SEQ NO. 47, human uterus homing peptide: GLSGGRS;SEQ NO. 48, human adipose tissue homing peptide:CKCCRAKDC;SEQ NO. 49, human muscle homing peptide: ASSLNIA;SEQ NO. 50, human prostate homing peptide: SMSIARL; SEQ NO. 51-55, human heart homing peptide: CRPPR, CKRAVR, CPKTRRVPC, CRSTRANPC, CARPAR;SEQ NO. 56-59, human tumour tissue homing peptide:CDCRGDCFC, CRGDC, CRGDKGPDC, CNGRCVSGCAGRC.

[0050] For example, the amino acid sequence codifying for a Cell Penetrating Peptide of the penetrating module is selected from the group comprised of:SEQ NO. 60, 61 TAT: GRKKRRQRRRPPQ, RKKRRQRRR;SEQ NO. 62, cTAT: CYGRKKRRQRRRC;SEQ NO. 63, HA- TAT: GDIMGEWGNEIFGAIAGFLGYGRKKRRQRRR; - n-times Arginine repetition (3<n<30): R (n), in particular n=5, RRRRR, SEQ NO. 64;SEQ NO. 65, cR8: CRRRRRRRRC;- n-times PPR repetition (3<n<10): PPR (n), in particular n=5, PPRPPRPPRPPRPPR, SEQ NO. 66;- n-times PRR repetition (3<n<10): PRR (n), in particular n=5, PRRPRRPRRPRRPRR, SEQ NO. 67;SEQ NO. 68-70, MPG: GALFLGFLGAAGSTMGAWSQPKSKRKV,AF LGWLGAWGTMGWSP KKKRK, GALFLAFLAAASLMGLWSQPKKKRKV;SEQ NO. 71, Xentry: LCLRPVG;SEQ NO. 72, BPrPp: MVKSKIGSWILVLFVAMWSDVGLCKKRPKP;SEQ NO. 73, Pep-1: KETWWETWWTEWSQPKKKRKV;SEQ NO. 74, Pep-2: KETWFETWFTEWSQPKKKRKV;SEQ NO. 75, Penetratin: RQIKIWFQNRRMKWKK;SEQ NO. 76, CADY: GLWRALWRLLRSLWRLLWRA;SEQ NO. 77, KALA: WEAKLAKALAKALAKHLAKALAKALKACEA;SEQ NO. 78, Rath: TPWWRLWTKWHHKRRDLPRKPE;SEQ NO. 79, DPV1047: VKRGLKLRHVRPRVTRMDV;SEQ NO. 80, pVEC: LLIILRRRIRKQAHAHSK;SEQ NO. 81, ARF: MVRRFLVTLRIRRACGPPRVRV;SEQ NO. 82, MAP: KLALKLALKALKAALKLA;SEQ NO. 83, Trasportan: GWTLNSAGYLLGKINLKALAALAKKIL; SEQ NO. 84, p28: LSTAADMQGVVTDGMASGLDKDYLKPDD;SEQ NO. 85, VT5: DPKGDPKGVTVTVTVTVTGKGDPKPD;SEQ NO. 86, Bac 7: RRIRPRPPRLPRPRPRPLPFPRPG;SEQ NO. 87, C105Y: CSIPPEVKFNKPFVYLI;SEQ NO. 88, PFVYLI: PFVYLI;SEQ NO. 89, Pep-7: SDLWEMMMVSLACQY.Advantageously, the expression cassette of the genetically modified eukaryotic microalga can also include a protection module comprising an amino acid sequence codifying for a cap module, which can comprise, for instance, a 5- to 30-times repetition of the agcccc sequence such as SEQ NO. 29: agccccagccccagccccagccccagccccagccccagccccagccccagccccagcc cc.In this way, the therapeutic agent is provided with a fused module, i. e. a module obtained by the fusion of two polypeptide chains, at its end, which protects its structure from degradation, thus further prolonging the residence time at a constant level of the therapeutic agent in the subject.Brief description of the pictures

[0051] The invention will be illustrated below with a description of some embodiments, made by way of example and not by way of limitation, with reference to the appended drawings, wherein- Fig. 1A is a schematic representation of the expression cassette of the genetically modified eukaryotic microalga according to the invention;- Figs. 1B–1I schematically show possible modifications of the expression modules of the expression cassette of Fig. 1A;- Fig. 2 is a conceptual diagram of the symbiotic therapy system according to the invention;- Figs. 3A and 3B show the integration of the expression cassette at the level of the nuclear genome of the microalga according to the invention;- Figs. 4A-4C show the production and secretion efficiency of the therapeutic agent by the microalga according to the invention;- Figs. 5A and 5B are diagrams comprising curves representative of the stability over time of the therapeutic agent secreted by the microalga according to the invention;- Fig. 6 includes images showing the absence of immune induction due to the interaction between the microalga according to the invention and immune cells of the treated subject;- Figs. 7A and 7B are diagrams showing blood concentration levels over time of a therapeutic agent secreted by the microalgae of the invention in a treated subject, according to a theoretical secretion model;- Figs. 8A-8C contain images and diagrams show increased basal levels of a therapeutic agent in the blood of a subject treated with the symbiotic therapy according tothe invention;- Fig. 9 is a diagram showing increased basal levels of a therapeutic agent in different organs of a subject treated with the symbiotic therapy according to the invention;- Fig. 10 contains an image and a diagram showing the absence of an inflammatory response in subjects treated with the symbiotic therapy according to the invention.Description of some exemplary embodiments of the invention

[0052] An example of symbiotic therapy for prolonged delivery to a subject of a therapeutic agent consisting of a protein or, more in general, a polypeptide or a peptide that is capable of acting as a hormone and / or as an enzyme and / or as a transcription factor and / or as a protein cofactor that mediates or otherwise influences a given biological process is described below.

[0053] Referring to Fig. 2, the symbiotic therapy of the invention uses a genetically modified eukaryotic microalga 51 as a means to administer a therapeutic agent, i. e., to produce and secrete said protein or polypeptide into a subject 53' s body. According to the invention, genetically modified eukaryotic microalgae 51 are arranged at an intradermal or subcutaneous injection site, briefly s cutaneous injection site 52 of subject 53. This can be achieved by any conventional technique, for instance, by dispersing the microalgae into an injectable resuspension liquid, not shown, and by subsequently injecting the composition obtained this way, having the form of a dispersion, into the subject by means of a conventional liquid injection device 54. For instance, the injected resuspension liquid can be an aqueous solution of one or more biocompatible salts that are suitable to maintain physiologically compatible osmolarity and / or pH conditions,such as NaCl, phosphate buffers, TRIS and the like, which are known to a person skilled in the art. As an alternative, the microalgae can be injected into the subject' s body by tattooing.

[0054] In order to obtain the microalgae 51 to be used in the treatment according to the invention, preferably starting eukaryotic microalgae are used which naturally lack flagella or cilia or which have preliminarily genetically modified to lose the flagella or cilia, or to inhibit the movement of the flagella or of the cilia. In both cases, the eukaryotic microalgae that are used are substantially unable to move autonomously in a fluid medium. The preliminary genetic modification can be carried out according to conventional procedures known to a person skilled in the related art, for instance, through the bldl- / - mutation, which results in the absence of terminal flagella.

[0055] The starting microalgae are preferably selected from the list below, which includes both microalgae naturally lacking flagella and microalgae naturally provided with flagella. The latter must be modified in advance as indicated above, in order to be immobilised, which feature is maintained once the genetically modified eukaryotic microalgae have been injected into subject 53. The list of the starting microalgae is as follows: Chlorella sorokiniana; Picochlorum sp HM1; Botryococcus braunii; Chlamydomonas reinhardtii; Tetraselmis suecica; Dunaliella salina; Nannochloropsis spp.; Chlorella vulgaris; Haematococcus pluvialis; Scenedesmus spp. Furtehr microalgae of known types, not included in the list, can however be used, provided that they can be modified or further modified to behave as a release agent, as described below.

[0056] In order to obtain release agent 51 from the starting eukaryotic microalgae, the latter is modified by integrating an agent codifying for a specific biologicallyactive polypeptide in its own genome. Even this genetical modification can be performed by a conventional technique known to a person skilled in the art.

[0057] In a first approach, not exemplified in this description, the coding agent of the biologically active protein is an exogenous DNA sequence that is inserted into the microalgae 51 as an expression cassette 10, shown in Fig. 1A, in particular, by a conventional technique such as CRISPR / CAS; TALENs; electroporation; "Gene gun"; viral vectors, and the like.

[0058] According to an alternative technique, exemplified in this description, the coding agent is a plasmid that comprises expression cassette 10 and that is inserted into the starting microalgae by transfection. In this example, transfection of a plasmid assisted by Agrobacterium Tumefaciens is described.

[0059] Still with reference to Fig. 1A, in this specific case, SYMBIO 2.0 plasmid 20 has been functionalised by insertion of modules 1-13 to improve the therapeutic agent releasing capacity after injection of genetically modified eukaryotic microalgae 51 into subject 53.

[0060] Figs. 1B–1I schematically show possible alternatives for the expression module of expression cassette of Fig. 1A.

[0061] As shown in Figs. 1B-1I, according to the invention, expression cassette 10 always includes a secretion coding module 5 comprising a secretion signal sequence of the therapeutic agent that allows the release of the latter, in other words, that is capable of inducing secretion of a polypeptide or polypeptide portion produced by genetically modified eukaryotic microalgae 51 outside genetically modified eukaryotic microalgae 51.

[0062] For example, the secretion signal amino acid sequence of secretion coding module 5 can be selected from the group comprised of:SEQ NO. 31, BiPl signal peptide: MAQWKAAVLLLALACASY - SEQ NO. 32, Gametolysin signal peptide:MSLATRRFGAAAALLVAACVLCTAPAWA- SEQ NO. 33, ARS1 Signal peptide:MHARKMGALAVLAVACLAAVASVAHA SEQ NO. 34, IBP1 Signal peptide:MPSSSMKLFAALLIACMAQTSMA- SEQ NO. 35, Glycoside Hydrolase-like protein signal peptide: MRRAIALGVGLALLGLLLPGSLA- SEQ NO. 36, sulfotransferase family Signal peptide MPSTPVAAALRALLCASLLGSLHIARA- SEQ NO. 377, SADlp Signal peptid:MTLRLAQLALATLGVLLLVLAPMPALS- SEQ NO. 38, Prolyl-4-hydroxylase signal peptide:MARRLLLALALAAVLGLAHA- SEQ NO. 39, Mitogen-activated kinase 7 protein signal peptide: MRGIIAAYTSATLLALLLVTWLTHSSA.

[0063] Advantageously, as shown in Figs. 1C, 1F, 1H and 1I, expression cassette 10 can further comprise a coding homing module 6 comprising an amino acid sequence codifying for "homing-peptide" (HP), which is capable of conveying a polypeptide or polypeptide portion secreted by genetically modified eukaryotic microalgae 51 to a specific tissue and / or to a specific cell population of subject 53.

[0064] For example, the amino acid sequence for homing peptide can be selected from the group comprised of:- SEQ NO. 40, human brain homing peptide: CLSSRLDAC;- SEQ NO. 41, human kidney homing peptide: CLPVASC;- SEQ NO. 42, 43, human lung homing peptide:CGFERVRQCPERC, CGFELETC;SEQ NO. 44, human skin homing peptide: CVALCREACGEGC;- SEQ NO. 45, human pancreas homing peptide: SWCEPGWCR; - SEQ NO. 46, human intestine homing peptide: YSGKWGW; - SEQ NO. 47, human uterus homing peptide: GLSGGRS;- SEQ NO. 48, human adipose tissue homing peptide:CKCCRAKDC;- SEQ NO. 49, human muscle homing peptide: ASSLNIA;- SEQ NO. 50, human prostate homing peptide: SMSIARL; - SEQ NO. 51-55, human heart homing peptide: CRPPR, CKRAVR, CPKTRRVPC, CRSTRANPC, CARPAR;- SEQ NO. 56-59, human tumour tissue homing peptide:CDCRGDCFC, CRGDC, CRGDKGPDC, CNGRCVSGCAGRC.

[0065] Advantageously, as shown in Figs. 1E, 1F, 1G and 1I, expression cassette 10 can further include an internalization coding module 7 comprising an amino acid sequence codifying for " Cell Penetrating Peptide" (CPP) that is capable of inducing internalization of the polypeptide or polypeptide portion secreted by genetically modified eukaryotic microalgae 51 within a cell of subject 53 through the cell membrane or by induced endocytosis.

[0066] For example, the amino acid sequence for cell penetrating peptide can be selected from the group comprised of:SEQ NO. 60, 61 TAT: GRKKRRQRRRPPQ, RKKRRQRRR;SEQ NO. 62, cTAT: CYGRKKRRQRRRC;SEQ NO. 63, HA- TAT: GDIMGEWGNEIFGAIAGFLGYGRKKRRQRRR; - n-times Arginine repetition (3<n<30): R (n), in particular n=5, RRRRR, SEQ NO. 64;SEQ NO. 65, cR8: CRRRRRRRRC;- n-times PPR repetition (3<n<10): PPR (n), in particular n=5, PPRPPRPPRPPRPPR, SEQ NO. 66;- n-times PRR repetition (3<n<10): PRR (n), in particular n=5, PRRPRRPRRPRRPRR, SEQ NO. 67;SEQ NO. 68-70, MPG: GALFLGFLGAAGSTMGAWSQPKSKRKV,AF LGWLGAWGTMGWSP KKKRK, GALFLAFLAAASLMGLWSQPKKKRKV;SEQ NO. 71, Xentry: LCLRPVG;SEQ NO. 72, BPrPp: MVKSKIGSWILVLFVAMWSDVGLCKKRPKP;SEQ NO. 73, Pep-1: KETWWETWWTEWSQPKKKRKV;SEQ NO. 74, Pep-2: KETWFETWFTEWSQPKKKRKV;SEQ NO. 75, Penetratin: RQIKIWFQNRRMKWKK;SEQ NO. 76, CADY: GLWRALWRLLRSLWRLLWRA;SEQ NO. 77, KALA: WEAKLAKALAKALAKHLAKALAKALKACEA;SEQ NO. 78, Rath: TPWWRLWTKWHHKRRDLPRKPE;SEQ NO. 79, DPV1047: VKRGLKLRHVRPRVTRMDV;SEQ NO. 80, pVEC: LLIILRRRIRKQAHAHSK;SEQ NO. 81, ARF: MVRRFLVTLRIRRACGPPRVRV;SEQ NO. 82, MAP: KLALKLALKALKAALKLA;SEQ NO. 83, Trasportan: GWTLNSAGYLLGKINLKALAALAKKIL; SEQ NO. 84, p28: LSTAADMQGVVTDGMASGLDKDYLKPDD;SEQ NO. 85, VT5: DPKGDPKGVTVTVTVTVTGKGDPKPD;SEQ NO. 86, Bac 7: RRIRPRPPRLPRPRPRPLPFPRPG;SEQ NO. 87, C105Y: CSIPPEVKFNKPFVYLI;SEQ NO. 88, PFVYLI: PFVYLI;SEQ NO. 89, Pep-7: SDLWEMMMVSLACQY.

[0067] Advantageously, as shown in Figs. 1C, ID, IE and II, expression cassette 10 can further comprise a protection module 9 comprising an amino acid sequence codifying for " Protection cap" (CAP) that is capable of preventing degradation of the biologically active polypeptide within the body of subject 53.

[0068] For example, the amino acid sequence for Protection cap can be a five- to thirty-times repetition of the sequence "agcccc", e.g., it can be SEQ NO. 29: agccccagccccagccccagccccagccccagccccagccccagccccagccccagcccc.

[0069] Referring again to Fig. 1A, the other modules 1-4 and 8-12 of plasmid 20 are now briefly described.- Modules 1 and 13 comprise left-side and right-side border repeat sequences 1 and 13 (Ti LB border and Ti RB border). In the specific case:- SEQ NO. 1 = tggcaggatatattgtggtgtaaac;- SEQ NO. 12 = tgacaggatatattggcgggtaaac.Modules 2 and 3 include any functioning promoter or promoter combination when inserted into a microalga system, capable of inducing the production of a polypeptide or polypeptide portion.For example, modules 2 and 3 can each include sequences selected, independently of each other, from the group comprised of:SEQ NO. 2, HSP70A promoter: cttgacatgattggtgcgtatgtttgtatgaagctacaggactgatttgg cgggctatgagggcgggggaagctctggaagggccgcgatggggcgcgcg gcgtccagaaggcgccatacggcccgctggcggcacccatccggtataaa agcccgcgaccccgaacggtgacctccactttcagcgacaaacgagcact tatacatacgcgactattctgccgctatacataaccactcaSEQ NO. 3 RBCS2 Promoter + 5 ' UTR Promoter + RBCS2 Intron 1: ccgggcgcgccagaaggagcgcagccaaaccaggatgatgtttgatgggg tatttgagcacttgcaacccttatccggaagccccctggcccacaaaggc taggcgccaatgcaagcagttcgcatgcagcccctggagcggtgccctcc tgataaaccggccagggggcctatgttctttacttttttacaagagaagt cactcaacatcttaaaatggccaggtgagtcgacgagcaagcccggcgga tcaggcagcgtgcttgcagatttgacttgcaacgcccgcattgtgtcgac gaaggcttttggctcctctgtcgctgtctcaagcagcatctaaccctgcg tcgccgtttccatttgcagga- SEQ NO. 4, CaMV 35S promoter (short):tgagacttttcaacaaagggtaatatccggaaacctcctcggattccatt gcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggc tcctacaaatgccatcattgcgataaaggaaaggccatcgttgaagatgc ctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcg tggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgt gatatctccactgacgtaagggatgacgcacaatcccactatccttcgca agacccttcctctatataaggaagttcatttcatttggagagaaca SEQ NO. 5, CAMV 35S Promoter (long):agattagccttttcaatttcagaaagaatgctaacccacagatggttaga gaggcttacgcagcaggtctcatcaagacgatctacccgagcaataatct ccaggaaatcaaataccttcccaagaaggttaaagatgcagtcaaaagat tcaggactaactgcatcaagaacacagagaaagatatatttctcaagatc agaagtactattccagtatggacgattcaaggcttgcttcacaaaccaag gcaagtaatagagattggagtctctaaaaaggtagttcccactgaatcaa aggccatggagtcaaagattcaaatagaggacctaacagaactcgccgta aagactggcgaacagttcatacagagtctcttacgactcaatgacaagaa gaaaatcttcgtcaacatggtggagcacgacacacttgtctactccaaaa atatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaa caaagggtaatatccggaaacctcctcggattccattgcccagctatctg tcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgcc atcattgcgataaaggaaaggccatcgttgaagatgcctctgccgacagt ggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaaga cgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactg acgtaagggatgacgcacaatcccactatccttcgcaagacccttcctct atataaggaagttcatttcatttggagagaacacgSEQ NO. 6, CAMV 35S promoter (enhanced): gccaacatggtggagcacgacactctcgtctactccaagaatatcaaaga tacagtctcagaagaccaaagggctattgagacttttcaacaaagggtaa tatcgggaaacctcctcggattccattgcccagctatctgtcacttcatc aaaaggacagtagaaaaggaaggtggcacctacaaatgccatcattgcga taaaggaaaggctatcgttcaagatgcctctgccgacagtggtcccaaag atggacccccacccacgaggagcatcgtggaaaaagaagacgttccaacc acgtcttcaaagcaagtggattgatgtgaacatggtggagcacgacactc tcgtctactccaagaatatcaaagatacagtctcagaagaccaaagggct attgagacttttcaacaaagggtaatatcgggaaacctcctcggattcca ttgcccagctatctgtcacttcatcaaaaggacagtagaaaaggaaggtg gcacctacaaatgccatcattgcgataaaggaaaggctatcgttcaagat gcctctgccgacagtggtcccaaagatggacccccacccacgaggagcat cgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgat gtgatatctccactgacgtaagggatgacgcacaatcccactatccttcg caagacccttcctctatataaggaagttcatttcatttggagaggacacg ctgaSEQ NO. 7, Nos Promoter: gatcatgagcggagaattaagggagtcacgttatgacccccgccgatgac gcgggacaagccgttttacgtttggaactgacagaaccgcaacgttgaag gagccactcagccgcgggtttctggagtttaatgagctaagcacatacgt cagaaaccattattgcgcgttcaaaagtcgcctaaggtcactatcagcta gcaaatatttcttgtcaaaaatgctccactgacgttccataaattcccct cggtatccaattagagtctcatattcactctcaatccaaataatctgca SEQ NO. 8, ZmUbil Promoter ctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcat tgcatgtctaagttataaaaaattaccacatattttttttgtcacacttg tttgaagtgcagtttatctatctttatacatatatttaaactttactcta cgaataatataatctatagtactacaataatatcagtgttttagagaatc atataaatgaacagttagacatggtctaaaggacaattgagtattttgac aacaggactctacagttttatctttttagtgtgcatgtgttctccttttt ttttgcaaatagcttcacctatataatacttcatccattttattagtaca tccatttagggtttagggttaatggtttttatagactaatttttttagta catctattttattctattttagcctctaaattaagaaaactaaaactcta ttttagtttttttatttaataatttagatataaaatagaataaaataaag tgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaa acatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcga cgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaa gcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcga gagttccgctccaccgttggacttgctccgctgtcggcatccagaaattg cgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcc tctcacggcaccggcagctacgggggattcctttcccaccgctccttcgc tttcccttcctcgcccgccgtaataaatagacaccccctccacaccctct ttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcc cccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctc cccccccccccctctctaccttctctagatcggcgttccggtccatggtt agggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttg tgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtca gacacgttctgattgctaacttgccagtgtttctctttggggaatcctgg gatggctctagccgttccgcagacgggatcgatttcatgattttttttgt ttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttg tgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctg tttcaaactacctggtggatttattaattttggatctgtatgtgtgtgcc atacatattcatagttacgaattgaagatgatggatggaaatatcgatct aggataggtatacatgttgatgcgggttttactgatgcatatacagagat gctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttc attcgttctagatcggagtagaatactgtttcaaactacctggtgtattt attaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagt ttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgg gttttactgatgcatatacatgatggcatatgcagcatctattcatatgc tctaaccttgagtacctatctattataataaacaagtatgttttataatt attttgatcttgatatacttggatgatggcatatgcagcagctatatgtg gatttttttagccctgccttcatacgctatttatttgcttggtactgttt cttttgtcgatgctcaccctgttgtttggtgttacttctgcag Module 4 comprises Kozac sequence SEQ ID. 9: gccacc. Module 8 acts as a cutting site for restriction enzymes within plasmid 20.For example, module 8 can comprise a sequence selected from the group comprised of:- SEQ NO. 25, BspEI: tccggaSEQ NO. 26, Spel: actagtSEQ NO. 27, BstEII: ggtgaccSEQ NO. 28, Aatll: gacgtc.Module 10 comprises one or more stop codons or termination codons, for example it can comprise the taatag sequence, SEQ NO. 30.Module 11 comprises a sequence working as 3 ' UTR in an mRNA codifying for a polypeptide or fragment thereof within microalga 51.For example, such a sequence working as 3 ' UTR can be selected from the group comprised of:SEQ NO. 21, PSAD 3 ' UTR: gcgttctggcagcagctggaccgcctgtaccatggagaagagctttactt gccgggatggccgatttcgctgattgatacgggatcggagctcggaggctttcgcgctaggggctaggcgaagggcagtggtgaccagggtcggtgtggg gtcggcccacggtcaattagccacaggaggatcagggggaggtaggcacg tcgacttggtttgcgaccccgcagttttggcggacgtgctgttgtagatg ttagcgtgtgcgtgagccagtggccaacgtgccacacccattgagaagac caaccaacttactggcaatatctgccaatgccatactgcatgtaatggcc aggccatSEQ NO. 22, HSP70A 3 ' UTR: tcggcttcgccccagactgaggagtgcgggaggcgccggcgggtttgacg gctggcgccgtggactgggtgtgtgggtgcgccggttgggcggctgtggc gcggcctagggcccggacgtgggccgggcgctgtattgatgtgtgggaac ggcagacgccgctgtgcgttgtgtgtgaatacgtacctatatgcccggcg gcgctgtagcactgatgctgtgtttcgcgcgtgtcttgtgctccttgtgt ttaacaacctggttggattgggacacccgcacggtctacatactcagagc aggactgagctgattggtcggtcggccggcgattgattttgccaacatgc gctgaagtgcagcgttgagttcggactacgtgggatttgtggttgttagc tagataggccccgggctgctatctgttgcgtagcggcccgtgggagcgaa cgcgactgaggctgtggctgcacgcatccgggctgttgaatggctagggt cgtgcgcggaaggccgttgctgagccatgccaagtagagtaggatgacga tgatgttataagaaaccgagatggcacctagctccaactgagttgggtgc ggccttaggggagaggcgtgcaggcaggtgcaagttccaaagcatgagag tggtgagtgagaggcgggagtggtggatggaagtgcatggagggggtctc gacagaactcaacggcccgtcacggacaattcgaagggcggagccgggat cagagtcaacacggacgctgccccacattgggtggtgtgtaaaagcagaa gattSEQ NO. 23, RBCS2 3 ' UTR: tccgtgtaaatggaggcgctcgttgatctgagccttgccccctgacgaac ggcggtggatggaagatactgctctcaagtgctgaagcggtagcttagct ccccgtttcgtgctgatcagtctttttcaacacgtaaaaagcggaggagt tttgcaattttgttggttgtaacgatcctccgttgattttggcctctttc tccatgggcgggctgggcgtatttgaagcgSEQ NO. 24, NITI 3 ' UTR: atcgctagagattgtggccacggttggatcatgcgaccagcaggattgga tccgaatccaggcatctgggcggatgccagggcaggggcaggggcagcgg tcgtgggagcgtgtgtgagccggataagggctggcacaggccacggccgcagcggccttttgtgcagtttgacgacggaagtgtgcgtctgtgtggttgt gtgtgagagctaggcaggaccgcagggtcagacagagtgcgcccggcttg gctgcggcgcactgggacgcgttggcagtttaaaacctgcgctgagggga caaacgagtttggcaacagtaggcagttaaaagatagaatgtgtaggtca gtttcccagtgggcaaatgagttgtgcaagcctgcaaacggcaggaagca tggcaaggatattactgattgactgcagcaggggatagcagtagtggcac agcagagtgcccgagagagtgtgtgcacgtgctagttttggagtcaggca ccgccgttgagatgatgtattgatgacgcagcatttcatgtgacataggg aggctttccatgcggttattatattatgtcaggaaagtgcctgacagcgt ttagcgcgtggggagaagtacgaccctccgtgggctgaacccacgttagg gcgtgggttggtcaggagggtgggtgccatgcatgaagcagagggccggg gcgttagcccgttttggtgagagcttgtttgatgcaatgcgatacataat cataagggggttttggcgcgtgagagtgcgacctgtgtatggcagcacgc catcggtgccaaccggcgaaggtgcaggaggtgcaggtaaagctacatat agacaccccctgccgcacagttgtaaattaagcaagcg.Module 12 comprises a nucleotide sequence capable of inducing transcription termination of the mRNA codifying for a polypeptide or polypeptide portion in microalga 51.For example, such a transcription terminator sequence can be selected from the group comprised of:- SEQ NO. 10, Nos terminator:gatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgc cggtcttgcgatgattatcatataatttctgttgaattacgttaagcatg taataattaacatgtaatgcatgacgttatttatgagatgggtttttatg attagagtcccgcaattatacatttaatacgcgatagaaaacaaaatata gcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactag atcg- SEQ NO. 11, Ocs terminator:ctgctttaatgagatatgcgagaagcctatgatcgcatgatatttgcttt caattctgttgtgcacgttgtaaaaaacctgagcatgtgtagctcagatc cttaccgccggtttcggttcattctaatgaatatatcacccgttactatc gtatttttatgaataatattctccgttcaatttactgattgtaccctact acttatatgtacaatattaaaatgaaaacaatatattgtgctgaataggtttatagcgacatctatgatagagcgccacaataacaaacaattgcgtttt attattacaaatccaattttaaaaaaagcggcagaaccggtcaaacctaa aagactgattacataaatcttattcaaatttcaaaagtgccccaggggct agtatctacgacacaccgagcggcgaactaataacgctcactgaagggaa ctccggttccccgccggcgcgcatgggtgagattccttgaagttgagtat tggccgtccgctctaccgaaagttacgggcaccattcaacccggtccagc acggcggccgggtaaccgacttgctgccccgagaattatgcagcattttt ttggtgtatgtgggccccaaatgaagtgcaggtcaaaccttgacagtgac gacaaatcgttgggcgggtccagggcgaattttgcgacaacatgtcgagg ctcagca.

[0070] In vitro tests have been carried out to evaluate some important aspects of the symbiotic therapy according to the invention. The results are shown in Figs. 3A to 6 and described hereinafter.

[0071] In the specific example described above, once the delivery agent, i. e., eukaryotic microalgae 51 had been transfected with the peptide and / or protein therapeutic agent by Agrobacterium tumefaciens, the delivery agent has shown stable integration in its own genome of portion 20 of SYMBIO 2.0 plasmid codifying peptide and / or protein therapeutic agent.

[0072] In other specific examples, the test has been repeated for three other peptide and / or protein therapeutic agents, namely:- Sirt6, referred to as SEQ NO. 18:agcgtgaactacgccgccggcctgagcccctacgccgacaagggcaagtgcggc ctgcccgagatcttcgacccccccgaggagctggagcgcaaggtgtgggagctg gcccgcctggtgtggcagagcagcagcgtggtgttccacaccggcgccggcatc agcaccgccagcggcatccccgacttccgcggcccccacggcgtgtggaccatg gaggagcgcggcctggcccccaagttcgacaccaccttcgagagcgcccgcccc acccagacccacatggccctggtgcagctggagcgcgtgggcctgctgcgcttc ctggtgagccagaacgtggacggcctgcacgtgcgcagcggcttcccccgcgac aagctggccgagctgcacggcaacatgttcgtggaggagtgcgccaagtgcaag acccagtacgtgcgcgacaccgtggtgggcaccatgggcctgaaggccaccggccgcctgtgcaccgtggccaaggcccgcggcctgcgcgcctgccgcggcgagctg cgcgacaccatcctggactgggaggacagcctgcccgaccgcgacctggccctg gccgacgaggccagccgcaacgccgacct gagcat caccctgggcaccagcctg cagatccgccccagcggcaacctgcccctggccaccaagcgccgcggcggccgc ctggtgatcgtgaacctgcagcccaccaagcacgaccgccacgccgacctgcgc atccacggctacgtggacgaggtgatgacccgcctgatgaagcacctgggcctg gagatccccgcctgggacggcccccgcgtgctggagcgcgccctgccccccctg ccccgcccccccacccccaagctggagcccaaggaggagagccccacccgcatc aacggcagcatccccgccggccccaagcaggagccctgcgcccagcacaacggc agcgagcccgccagccccaagcgcgagcgccccaccagccccgccccccaccgc ccccccaagcgcgtgaaggccaaggccgtgcccagc,Nrf2, referred to as SEQ NO. 19: cagcccgcccagcacatccagagcgagaccagcggcagcgccaactacagccag gtggcccacatccccaagagcgacgccctgtacttcgacgactgcatgcagctg ctggcccagaccttccccttcgtggacgacaacgaggtgagcagcgccaccttc cagagcctggtgcccgacatccccggccacatcgagagccccgtgttcatcgcc accaaccaggcccagagccccgagaccagcgtggcccaggtggcccccgtggac ctggacggcatgcagcaggacatcgagcaggtgtgggaggagctgctgagcatc cccgagctgcagtgcctgaacatcgagaacgacaagctggtgaagcagaacggc cccaagacccccgtgcacagcagcggcgacatggtgcagcccctgagccccagc cagggccagagcacccacgtgcacgacgcccagtgcgagaacacccccgagaag gagctgcccgtgagccccggccaccgcaagacccccttcaccaaggacaagcac agcagccgcctggaggcccacctgacccgcgacgagctgcgcgccaaggccctg cacatccccttccccgtggagaagatcatcaacctgcccgtggtggacttcaac gagatgatgagcaaggagcagttcaacgaggcccagctggccctgatccgcgac atccgccgccgcggcaagaacaaggtggccgcccagaactgccgcaagcgcaag ctggagaacatcgtggagctggagcaggacctggaccacctgaaggacgagaag gagaagctgctgaaggagaagggcgagaacgacaagagcctgcacctgctgaag aagcagctgagcaccctgt acctggaggtgttcagcatgctgcgcgacgaggac ggcaagccctacagccccagcgagtacagcctgcagcagacccgcgacggcaac gtgttcctggtgcccaagagcaagaagcccgacgtgaagaagaac, or human follistatin, referred to as SEQ NO. 20: gccagcagcctgaacatcgccggcaactgctggctgcgccaggccaagaacggc cgctgccaggtgctgtacaagaccgagctgagcaaggaggagtgctgcagcaccggccgcctgagcaccagctggaccgaggaggacgtgaacgacaacaccctgttc aagtggatgatcttcaacggcggcgcccccaactgcatcccctgcaaggagacc tgcgagaacgtggactgcggccccggcaagaagtgccgcatgaacaagaagaac aagccccgctgcgtgtgcgcccccgactgcagcaacatcacctggaagggcccc gtgtgcggcctggacggcaagacctaccgcaacgagtgcgccctgctgaaggcc cgctgcaaggagcagcccgagctggaggtgcagtaccagggccgctgcaagaag acctgccgcgacgtgttctgccccggcagcagcacctgcgtggtggaccagacc aacaacgcctactgcgtgacctgcaaccgcatctgccccgagcccgccagcagc gagcagtacctgtgcggcaacgacggcgtgacctacagcagcgcctgccacctg cgcaaggccacctgcctgctgggccgcagcatcggcctggcctacgagggcaag tgcatcaaggccaagagctgcgaggacatccagtgcaccggcggcaagaagtgc ctgtgggacttcaaggtgggccgcggccgctgcagcctgtgcgacgagctgtgc cccgacagcaagagcgacgagcccgtgtgcgccagcgacaacgccacctacgcc agcgagtgcgccatgaaggaggccgcctgcagcagcggcgtgctgctggaggtg aagcacagcggcagctgcaacagcatcagcgaggacaccgaggaggaggaggag gacgaggaccaggactacagcttccccatcagcagcatcctggagtgg.

[0073] Even in these cases, the delivery agent has shown stable integration in its own genome of portion 20 of the SYMBIO 2.0 plasmid codifying for each of the three above-mentioned therapeutic agents. The effective and stable integration of expression cassette 10 into the nuclear genome of the alga is shown in Figs. 3A and 3B, referring to PCR assays and subsequent electrophoresis on 2% agarose gels.

[0074] In particular, Fig. 3A refers to PCR products amplified with primers binding the integrating portion 20 of the plasmid:- nptll Forward primer, designated as SEQ NO. 13:cctgatgctcttcgtccaga- nptll Reverse primer, referred to as SEQ NO. 14:tattcggctatgactgggca,to assess the presence of the expression cassette in the genome of the microalgae, while Fig. 3B refers to PCR products amplified with primers binding the non-integratingportion of the plasmid:- Ori Forward primer, referred to as SEQ NO. 15:atacctgtccgcctttctcc- Ori Reverse primer, designated as SEQ NO. 16:caagaactctgtagcaccgc,to check for residual presence of the non-integrated plasmid in microalga 51.

[0075] In vitro tests were also performed to assess the effectiveness of microalgae 51 in producing and secreting the therapeutic agent. Figs. 4a-4c provide evidence of the successful secretion of the peptide and / or protein therapeutic agent by the microalgae that remains viable in vitro.

[0076] More in detail, Fig. 4a is a fluorescence microscope image of a microalga having a fluorescent therapeutic agent contained within its own secretory vesicles, namely:- GFP, referred to as SEQ NO. 17:gtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg gacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgat gccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgccc gtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagc cgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaa ggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacc cgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaag ggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaac tacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaag gtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgac cactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaac cactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgat cacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggac gagctgtacaag.

[0077] Fig. 4b refers to a spot assay stained with Bradford reagent at various known concentrations of BSA (Bovine Serum Albumin), and of the supernatant filtered after 1 day of culture of the microalgae transformed with SYMBIO 2.0, still bearing SEQ NO. 17 as the therapeutic agent, while Fig. 4c is a fluorescence stereo-microscope image of colonies of microalgae, transformed with SYMBIO 2.0 still bearing SEQ NO. 17 as the therapeutic agent, showing the secretion of the fluorescent therapeutic agent into the solid culture medium, TAP agar.

[0078] A high in-vitro stability of the secreted therapeutic agent was also assessed, as shown in Figs. 5A and 5B, which were obtained by simulating the half-life of the therapeutic agent in the human bloodstream using fetal bovine serum (FBS). More in detail, the two diagrams in Figs. 5A and 5B are scatter plots showing how the stability of the therapeutic agent secreted by the microalgae transformed with SYMBIO 2.0 bearing SEQ NO. 17 as the therapeutic agent varies over time in the presence of different concentrations of FBS (Fig. 5A) and at different initial concentrations of therapeutic agent (Fig. 5B), respectively.

[0079] Experimental tests were also carried out to assess whether the presence of microalgae 51 can cause an inflammatory phenotype in vitro. To this purpose, the microalgae transformed with SYMBIO 2.0 bearing SEQ NO. 17 as a therapeutic agent were arranged and maintained for 3 days in co-culture with mixed populations of immune cells, periodically performing an aggregation test, i. e. testing whether clusters of immune cells had formed as an inflammatory phenotype. More in detail, the microalgae transformed with SYMBIO 2.0 bearing SEQ NO. 17 as a therapeutic agent was co-cultured in the wells of a 96-well plate with a population of mixed immune cells at differentratios. As a positive control, immune cells without the delivery agent were stimulated by LPS. As a negative control, immune cells were placed in the absence of the microalgae. No inflammatory responses were detected in any of the in vitro co-culture conditions, unlike in the positive control described above, as shown in Fig. 6, where microscopic magnifications of the co-culture condition and of the negative control are shown bottom right in the figure.

[0080] Besides the in-vitro tests described above, an in-vivo experimentation of the symbiotic therapy of the invention was performed. Figs. 8-10 relates to this in-vivo experimentation. Before the in-vivo tests, the blood concentration over time of the therapeutic agent secreted by the microalgae was investigated theoretically by a suitable secretion mathematical model. Figs. 7A and 7B refer to this simulation. The simulation allows to forecast that the therapeutic agent can be evenly distributed to all the cells of a target organ or tissue of a subject by the symbiotic therapy of the invention. This is due to the continuous and constant therapeutic agent release over time by the microalgae, which remain alive. Moreover, according to the above simulations, the secreted therapeutic agent entering the bloodstream is not able to attain harmful concentrations, as the blood concentration of the therapeutic agent is limited, as a result of a dynamic balance between the amount of the secreted therapeutic agent, the half-life of the therapeutic agent and the amount of the therapeutic agent that is internalised by the cells.

[0081] More in detail, Fig. 7A shows that the simulated plasma concentration of the therapeutic agent can stabilise at a constant value within a predetermined time, ranging between 5 and 15 minutes, regardless of the initial concentration of the therapeutic agent. Instead, Fig. 7Bshows how, the simulated plasma concentration of the therapeutic agent can stabilise at a constant value in a predetermined time varying between 3 and 4 hours, regardless of the initial concentration of the microalgae.

[0082] Microalgae obtained by transforming systems consisting of microalgae transformed with SYMBIO 2.0 and having SEQ NO. 18 and SEQ NO. 20 as therapeutic agents were injected subcutaneously into male and female Danio rerio specimens, fishes also known as zebrafishes, in order to assess the effectiveness of the symbiotic therapeutic technique in vertebrate organisms and possibly draw conclusions about the possibility to treat humans as well.15 days after the treatment, the injected specimens (n=6) were compared with non-injected control specimens (n=4) in connection to such relevant features as the presence of therapeutic agent in the blood and in various tissues, and as the presence of inflammation as a result of the subcutaneous injection of the microalgae.

[0083] Concerning the presence of the therapeutic agent in the blood, only 15 days after the treatment, an increase by between 60% and 100% of the therapeutic agent plasma concentration was observed in the specimens treated with the symbiotic therapeutic technique of the invention, in comparison with the untreated control specimens. This can be seen in Figs. 8B and 8C, respectively, in which Dotblots on PVDF membrane are shown indicating the plasma levels of the secreted peptide and / or protein therapeutic agents, codified by SEQ NO. 18 and SEQ NO. 20, respectively, and marked with specific antibodies interacting with these peptide and / or protein therapeutic agents. These levels were normalised against the total protein levels present in each sample marked on PVDF membrane using Bradford reagent, Fig.8A. The quantifications of this analysis are shown as barplots on the right side of the diagrams of Figs. 8D and8E, respectively between specimens treated with symbiotic therapy and control specimens. Statistical significance was calculated using t-tests (*p<0.15, **p<0.05, ***p<0.01).

[0084] Concerning the presence of the therapeutic agent in the different tissues, an increase by between 2 and 2.5 times was observed with respect to normal physiological levels, with an internalisation rate of the therapeutic agent in the cells of between 60% and 90%, which is much higher than the corresponding values reported in the literature for prior technique systems. This is shown in Fig. 9, which reports a quantification of therapeutic agent levels in different organs of the specimens treated with the symbiotic therapy of the invention (n=6) and OF untreated SPECIMENS (n=4). Statistical significance was calculated using t-test (*p<0.15, **p<0.05, ***p<0.01).

[0085] Concerning the presence of inflammation by subcutaneous injection of microalgae according to the symbiotic therapeutic technique of the invention, it was observed not only the absence of inflammation in the specimens treated with symbiotic therapy in comparison with the control specimens, but also a significant decrease was observed in the levels of the pro-inflammatory marker IL1-beta in the blood of the treated specimens, in comparison with the untreated specimens. This can be seen in Fig. 10, showing a PVDF membrane dot-blot revealing the plasma levels of the pro-inflammatory cytokine ILl-beta in specimens treated with the symbiotic therapy of the invention and in control specimens. These levels were also normalised for the total protein levels present in each PVDF membrane-marked sample by means of Bradford's reagent. The quantifications of this analysis re shown as barplots in Fig. 10. Statistical significance was calculated using t-test (*p<0.15, **p<0.05, ***p<0.01)

[0086] The results of the experimental tests described above show that the symbiotic therapeutic technique of the invention, which is based on the subcutaneous injection of microalgae genetically modified to act as releasing agents, allows intra-cellular diffusion levels of the therapeutic agents into various organs from 2 to 2.5 times higher than the normal physiological levels, with an internalisation rate of the therapeutic agent into the cells of between 60% and 90%. These values are much higher than the corresponding values, reported in the literature, concerning previous administration systems, and indicate that continuous secretion of the peptide and / or protein therapeutic agent over time can potentially reach every cell in the body, without causing inflammation. The specimens treated with this symbiotic therapy show a significant decrease in inflammation levels, measured as plasma ILl-beta levels, compared to untreated control specimens. Moreover, the specimens that received the symbiotic therapy of the invention were all alive and viable 15 days after the treatment, and showed no visible signs of stress, indicating a safe symbiosis between the microalgae and the treated subject.EXAMPLES OF POSSIBLE APPLICATIONSExample 1: Treatment of muscular atrophy

[0087] Muscle atrophy is a loss or reduction of muscle mass and strength, typically caused by inactivity, ageing, injury or disease. It occurs when muscles are not used regularly, leading to a decrease in the size of muscle fibres. Common causes include prolonged immobility, for example, after an injury or surgery, neurological conditions, malnutrition or chronic diseases such as cancer or rheumatoid arthritis. Muscular atrophy also affects astronauts due to prolonged weightlessness in space.

[0088] Today, several treatments are available to treat muscle atrophy, many of which involve an appropriate combination of exercise and a high-protein diet. However, these treatments usually lead to limited improvement of the pathologic condition and are not always feasible for elderly or injured people. Molecular therapies are also known to treat muscular atrophy using myostatin inhibitors, growth hormone or gene therapies (e. g. mRNA encapsulated in lipid nanoparticles) to stimulate muscle growth. These therapies have been successful in genetically modified animal models and in vitro, but allow only a temporary improvement in the quality of life when transposed to human patients.

[0089] As anticipated, a common problem of the conventional above-mentioned therapeutic agent delivery systems is that they do not allow all target cells to be reached, and only work for a short period of time, requiring multiple doses that can be harmful and very costly for the patient.

[0090] The symbiotic therapy system based on the subcutaneous or intradermal injection of the microalgae according to the invention can substantially improve the condition of patients suffering from muscle atrophy. Indeed, the microalgae can be transformed with a plasmid such as SYMBIO 2.0 bearing the gene codifying for human follistatin, SEQ NO. 20, above, so as to provide a constant basal stimulus for muscle growth and strengthening, potentially curing such a condition.Example 2: Treatment of type 1 diabetes

[0091] Type 1 diabetes is a chronic autoimmune disease characterised by the destruction of the beta cells of the pancreas, which are responsible for insulin production. Insulin is an essential hormone for glucose metabolism, and its deficiency leads to high blood sugar levels(hyperglycaemia) and long-term complications affecting the cardiovascular system, kidneys, eyes and nerves. The precise cause of type 1 diabetes is not fully understood, but it is believed that genetic and environmental factors, such as viral infections, trigger an autoimmune response against beta cells. The disease usually develops during childhood or adolescence, but can also occur in adulthood.

[0092] Type 1 diabetes is commonly treated by administering exogenous insulin with multiple daily injections or continuous infusion via pumps. Despite some technical improvements, such as continuous blood glucose monitoring systems and automated pumps, the natural glycaemic control cannot be fully restored. Current research is focusing on cell therapy, which involves transplanting pancreatic islets or stem cells differentiated into beta cells, or immunotherapy to modulate the autoimmune response, as well as more advanced insulin delivery systems. However, many of these therapies are still in the experimental phase and encounter difficulties such as immune rejection, limited duration of benefits and high costs.

[0093] The symbiotic therapy system based on subcutaneous or intradermal injection of the microalgae according to the invention can improve the condition of patients with Type 1 diabetes. In fact, the microalgae can be transformed with a plasmid such as SYMBIO 2.0 bearing the gene codifying for human insulin, so as to ensure a constant release of the hormone and compensate for the lack of pancreatic beta cells.Example no. 3: Biohacking-Delivery of functional enzymes

[0094] Alcohol degradation relies mainly on the activity of two enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). The deficiency of these enzymes is often due to genetic variants and leads to pile-up ofacetaldehyde, a toxic by-product of alcohol metabolism. This can cause symptoms such as facial flushing, tachycardia, nausea, headache and increased sensitivity to the toxic effects of alcohol.

[0095] Research is ongoing to develop targeted therapeutic approaches, such as specific inhibitors to reduce acetaldehyde accumulation or gene therapies to restore the enzyme function of the above-mentioned enzymes. Another experimental approach involves the use of molecules that mimic the action of the deficient enzymes, promoting faster and safer degradation of alcohol. However, these treatments are still in the early stages of development and face challenges related to safety, effectiveness and administration.

[0096] The symbiotic therapy system based on subcutaneous or intradermal injection of the microalgae according to the invention can substantially improve a person ' s ability to dispose of alcohol. In fact, the microalgae can be transformed with a plasmid such as SYMBIO 2.0 bearing genes codifying for human ALDH and ADH enzymes, allowing a constant release thereof, and a subsequent internalisation by cells, in particular, liver cells. This would allow people to drink safely while mitigating the side effects of alcohol and drunkenness.Example 4: Anti-ageing therapy

[0097] Ageing is a complex systemic syndrome characterised by a progressive functional decline of organs and systems, caused by the accumulation of cellular and molecular damage over time. This process is influenced by genetic, environmental and lifestyle factors, and entails a reduction in the regenerative capacity of tissues, alterations in the immune system (immunosenescence), loss of muscle mass (sarcopenia), metabolic dysfunction and anincreased risk of chronic diseases such as diabetes, cardiovascular diseases, cancer and neurodegenerative diseases. At the cellular level, ageing is associated with processes such as telomere shortening, accumulation of reactive oxygen species (ROS), mitochondrial dysfunction and persistent systemic inflammation, known as "inflammaging".

[0098] Although ageing is inevitable, modern therapeutic strategies aim to slow the biological processes causing it, in order to improve quality of life and prolong healthspan. These approaches include lifestyle interventions, such as a balanced diet and regular exercise, and innovative pharmacological treatments, including senolytics (drugs that eliminate senescent cells), calorie restriction mimetics and molecules that modulate autophagy or mitochondrial function. Moreover, gene therapies and stem cell-based approaches are emerging as potential solutions to regenerate damaged tissue and restore organ function. However, scientific, economic and ethical obstacles are slowing down the transition of these therapies from clinical research to medical practice.

[0099] The symbiotic therapy system based on the subcutaneous or intradermal injection of the microalgae according to the invention can substantially improve the condition of elderly patients. Indeed, the delivery agent can be transformed with a plasmid such as SYMBIO 2.0 bearing genes codifying for key proteins such as the above-mentioned Nrf2 (SEQ NO. 19), or Sirt6 (SEQ NO. 18), which are able to counteract ageing by a constant release of those proteins, which are subsequently internalised by the cells in a systemic manner. This way, the peptides / proteins can act in a targeted and safe manner in the implementation of their cellular function, e. g. ROS clearance, without the need ofexpensive drugs that, on the contrary, are often stopped in the liver or in the intestine.

[0100] The foregoing description is capable of showing the invention from a conceptual point of view so that others, using the known technique, will be able to modify and / or adapt in various applications such embodiments without further research and without departing from the inventive concept, and, therefore, it is understood that such adaptations and modifications will be considered as equivalent to the embodiments described. The means and materials for realising the various functions can be of various kinds without departing from the scope of the invention. It is understood that the expressions or terminology used are purely descriptive and, therefore, not limiting.

Claims

CLAIMS1. A genetically modified eukaryotic microalga (51) comprising an expression cassette (10) codifying a biologically active protein, wherein said genetically modified eukaryotic microalga is selected from the group comprised of:- a eukaryotic microalga naturally without flagella, selected from the group comprised of:- Chlorella spp.;- Scenedesmus spp.;- Micractinium spp.;- Monoraphidium spp.;- Coelastrum spp.;- Pediastrum spp.;- Nannochloropsis spp.;- Trebouxia spp.;- Botryococcus braunii; and- a eukaryotic microalga naturally provided with flagella but genetically modified to have no flagella or to have flagella made inactive by inactivation of a gene selected from the group comprised of: bld1 (IFT52); bld2 (centrin / CEP164- like); bld10 (CEP135-like); IFT43; IFT121; IFT122; IFT139; IFT140; IFT144; IFT172; IFT88; IFT81; IFT80; IFT74; IFT70; IFT57; IFT54; IFT46; IFT38; IFT27; IFT25; IFT22; IFT20; fla10 (Kinesin-2 motor subunit); fla8 (Kinesin-2); fla3 (FAP163; KAP subunit of kinesin-2),such that said genetically modified eukaryotic microalga (51) can be injected and remain within a subject' s (53) subcutaneous or intradermal injection site (52)and wherein said expression cassette (10) comprises: - a secretion coding module (5) configured to be translated into a secretion module fused to saidbiologically active protein, said secretion module configured to mediate the secretion of said biologically active protein outside of a cell wall of said genetically modified microalgae (51), such that said biologically active protein can be secreted outside of said genetically modified eukaryotic microalga (51), and can be passively diffused in said subject (53) into which said genetically modified eukaryotic microalga (51) is injected.

2. The genetically modified eukaryotic microalga (51) according to claim 1, wherein said expression cassette (10) comprises at least one further coding module selected from the group comprised of:- a homing module (6) comprising an amino acid sequence codifying for a homing-peptide to deliver a polypeptide or polypeptide portion to a predetermined tissue and / or to a predetermined cell population of said subject (53);- a penetrating module (7) comprising an amino acid sequence codifying for a Cell Penetrating Peptide to trigger a polypeptide or polypeptide portion internalisation into a cell of said subject (53) through the cell membrane or by induced endocytosis; - a protection module (9) comprising an amino acid sequence codifying for a cap module with a protective function.

3. The genetically modified eukaryotic microalga (51) according to claim 1, wherein said genetically modified eukaryotic microalga is selected from the group comprised of:Chlorella sorokiniana;Picochlorum sp HM1;Botryococcus braunii;- Chlamydomonas reinhardtii;- Tetraselmis suecica;- Dunaliella salina;- Nannochloropsis spp.;- Chlorella vulgaris;- Haematococcus pluvialis;- Scenedesmus spp.

4. The genetically modified eukaryotic microalga (51) according to claim 1, wherein said genetically modified eukaryotic microalga is a photosynthetic microalga.

5. The genetically modified eukaryotic microalga (51) according to claim 1, wherein said biologically active protein is codified by an expression cassette inserted into a system selected from the group comprised of: - a plasmid comprising said expression cassette (10), inserted into said microalga by transfection by a predetermined transfection vector;- an exogenous DNA sequence inserted into said microalga (10) by a technique selected from the group comprised of CRISPR / CAS; TALENs; electroporation; 'Gene gun'; viral vectors.

6. The genetically modified eukaryotic microalga (51) according to claim 5, wherein said transfection vector is Agrobacterium Tumefaciens.

7. The genetically modified eukaryotic microalga (51) according to claim 1, wherein said secretion coding module (5) of said therapeutic agent comprises an amino acid sequence selected from the group comprised of: - SEQ NO. 31, BiPl signal peptide:MAQ WKAAVL LLALACASY- SEQ NO. 32, Gametolysin signal peptide:MSLATRRFGAAAALLVAACVLCTAPAWA- SEQ NO. 33, ARS1 signal peptide:MHARKMGALAVLAVACLAAVASVAHASEQ NO. 34, IBP1 signal peptide:MPSSSMKLFAALLIACMAQTSMA SEQ NO. 35, Glycoside Hydrolase-like protein signal peptide: MRRAIALGVGLALLGLLLPGSLASEQ NO. 36, sulfotransferase family signal peptide: MPSTPVAAALRALLCASLLGSLHIARASEQ NO. 37, SADlp signal peptide:MTLRLAQLALATLGVLLLVLAPMPALS SEQ NO. 38, prolyl-4-hydroxilase signal peptide: MARRLLLALALAAVLGLAHA SEQ NO. 39, Mitogen-activated kinase 7 protein signal peptide: MRGIIAAYTSATLLALLLVTWLTHSSA.

8. The genetically modified eukaryotic microalga (51) according to claim 2, wherein said amino acid sequence codifying for a homing-peptide is selected from the group comprised of:- SEQ NO. 40, human brain homing peptide: CLSSRLDAC; - SEQ NO. 41, human kidney homing peptide: CLPVASC; - SEQ NO. 42, 43, human lung homing peptide:CGFERVRQCPERC, CGFELETC;- SEQ NO. 44, human skin homing peptide:CVALCREACGEGC;- SEQ NO. 45, human pancreas homing peptide:SWCEPGWCR;- SEQ NO. 46, human intestine homing peptide:YSGKWGW;- SEQ NO. 47, human uterus homing peptide: GLSGGRS; - SEQ NO. 48, human adipose tissue homing peptide:CKCCRAKDC;- SEQ NO. 49, human muscle homing peptide: ASSLNIA; - SEQ NO. 50, human prostate homing peptide:SMSIARL;- SEQ NO. 51-55, human heart homing peptide: CRPPR,CKRAVR, CPKTRRVPC, CRSTRANPC, CARPAR;SEQ NO. 56-59, human tumoral tissue homing peptide: CDCRGDCFC, CRGDC, CRGDKGPDC, CNGRCVSGCAGRC.

9. The genetically modified eukaryotic microalga (51) according to claim 2, wherein said amino acid sequence codifying for a Cell Penetrating Peptide is selected from the group comprised of:SEQ NO. 60, 61 TAT: GRKKRRQRRRPPQ, RKKRRQRRR; SEQ NO. 62, cTAT: CYGRKKRRQRRRC;SEQ NO. 63, HA- TAT:GDIMGEWGNEIFGAIAGFLGYGRKKRRQRRR;- n-times Arginine repetition (3<n<30): R (n), in particular n=5, RRRRR, SEQ NO. 64;SEQ NO. 65, cR8: CRRRRRRRRC;- n-times PPR repetition (3<n<10): PPR (n), in particular n=5, PPRPPRPPRPPRPPR, SEQ NO. 66; - n-times PRR repetition (3<n<10): PRR (n), in particular n=5, PRRPRRPRRPRRPRR, SEQ NO. 67; SEQ NO. 68-70, MPG: GALFLGFLGAAGSTMGAWSQPKSKRKV, AFLGWLGAWGTMGWSPKKKRK,GALFLAFLAAASLMGLWSQPKKKRKV;SEQ NO. 71, Xentry: LCLRPVG;SEQ NO. 72, BPrPp: MVKSKIGSWILVLFVAMWSDVGLCKKRPKP; SEQ NO. 73, Pep-1: KETWWETWWTEWSQPKKKRKV;SEQ NO. 74, Pep-2: KETWFETWFTEWSQPKKKRKV;SEQ NO. 75, Penetratin: RQIKIWFQNRRMKWKK;SEQ NO. 76, CADY: GLWRALWRLLRSLWRLLWRA;SEQ NO. 77, KALA: WEAKLAKALAKALAKHLAKALAKALKACEA; SEQ NO. 78, Rath: TPWWRLWTKWHHKRRDLPRKPE;SEQ NO. 79, DPV1047: VKRGLKLRHVRPRVTRMDV;SEQ NO. 80, pVEC: LLIILRRRIRKQAHAHSK;SEQ NO. 81, ARF: MVRRFLVTLRIRRACGPPRVRV;SEQ NO. 82, MAP: KLALKLALKALKAALKLA;SEQ NO. 83, Trasportan:GWTLNSAGYLLGKINLKALAALAKKIL;SEQ NO. 84, p28: LSTAADMQGVVTDGMASGLDKDYLKPDD;SEQ NO. 85, VT5: DPKGDPKGVTVTVTVTVTGKGDPKPD; SEQ NO. 86, Bac 7: RRIRPRPPRLPRPRPRPLPFPRPG; SEQ NO. 87, C105Y: CSIPPEVKFNKPFVYLI;SEQ NO. 88, PFVYLI: PFVYLI;SEQ NO. 89, Pep-7: SDLWEMMMVSLACQY.

10. The genetically modified eukaryotic microalga (51) according to claim 2, wherein said amino acid sequence codifying for the protection module comprises a 5 to 30-times repetition of the agcccc sequence, in particular of SEQ NO. 29: agccccagccccagccccagccccagccccagccccagccccagccccagcccc agcccc.

11. A subcutaneously or intradermally injectable composition comprising:- a genetically modified eukaryotic microalga (51) comprising an expression cassette (10) codifying a biologically active protein;- a biocompatible resuspension liquid in which said genetically modified eukaryotic microalga (51) is resuspended,wherein said expression cassette (10) comprises a secretion coding module (5) configured to be translated into a secretion module fused to said biologically active protein, said secretion module configured to mediate the secretion of said biologically active protein outside of a cell wall of said genetically modified eukaryotic microalga (51), such that said biologically active protein can be secreted outside of said genetically modified eukaryotic microalga (51), and can be passively diffused in said subject (53).

12. The composition according to claim 11, wherein said expression cassette (10) comprises at least one module selected from the group comprised of:- a homing module (6), comprising an amino acid sequence codifying for a homing-peptide to deliver a polypeptide or polypeptide portion to a predetermined tissue and / or a predetermined cell population of said subject (53);- a penetrating module (7) comprising an amino acid sequence codifying for a Cell Penetrating Peptide to trigger a polypeptide or a polypeptide portion internalization into a cell of said subject (53) through the cell membrane or by induced endocytosis - a protection cap module (9) comprising an amino acid sequence codifying for a cap module with a protective function.

13. The composition according to claim 11, wherein said genetically modified eukaryotic microalga (51) is selected from the group comprised of:- eukaryotic microalgae naturally without flagella selected from the group comprised of:- Chlorella spp.;- Scenedesmus spp.;- Micractinium spp.;- Monoraphidium spp.;- Coelastrum spp.;- Pediastrum spp.;- Nannochloropsis spp.;- Trebouxia spp.;- Botryococcus braunii; and- eukaryotic microalgae naturally provided with flagella but genetically modified to have no flagella or to have flagella made inactive by inactivation of a gene selected from the groupcomprised of: bld1 (IFT52); bld2 (centrin / CEP164- like); bld10 (CEP135-like); IFT43; IFT121; IFT122; IFT139; IFT140; IFT144; IFT172; IFT88; IFT81; IFT80; IFT74; IFT70; IFT57; IFT54; IFT46; IFT38; IFT27; IFT25; IFT22; IFT20; fla10 (Kinesin-2 motor subunit); fla8 (Kinesin-2); fla3 (FAP163; KAP subunit of kinesin-2),such that said genetically modified eukaryotic microalga (51), once injected, remains within the subcutaneous or intradermal injection site (52) of said subject (53).

14. The composition according to claim 11, wherein said genetically modified eukaryotic microalga (51) is selected from the group comprised of:- Chlorella sorokiniana;- Picochlorum sp HM1;- Botryococcus braunii;- Chlamydomonas reinhardtii;- Tetraselmis suecica;- Dunaliella salina;- Nannochloropsis spp.;- Chlorella vulgaris;- Haematococcus pluvialis;- Scenedesmus spp.

15. The composition according to claim 11, wherein said biologically active protein is codified by an expression cassette inserted into a system selected from the group comprised of:- a plasmid comprising said expression cassette (10), inserted into said microalga by transfection by a predetermined transfection vector;- an exogenous DNA sequence inserted into said microalga by a technique selected from the group comprised of CRISPR / CAS; TALENs; electroporation; "Gene gun"; viral vectors.

16. The composition according to claim 15, wherein said transfection vector is Agrobacterium Tumefaciens.

17. A kit for preparing a composition to be subcutaneously or intradermally injected into a subject (53), said kit comprising- a maintenance medium for said microalga;- a genetically modified eukaryotic microalga (51) comprising an expression cassette (10) codifying a biologically active protein, said genetically modified eukaryotic microalga (51) being arranged [grown?] on said maintenance medium;- a biocompatible resuspension liquid for said genetically modified eukaryotic microalga (51), wherein said expression cassette (10) comprises a secretion coding module (5) configured to be translated into a secretion module fused to said biologically active protein, said secretion module configured to mediate the secretion of said biologically active protein outside of a cell wall of said genetically modified eukaryotic microalga (51), such that said biologically active protein can be secreted outside of said genetically modified eukaryotic microalga (51), and can be passively diffused in said subject (53).

18. The kit according to claim 17, wherein said expression cassette (10) further comprises at least one module selected from the group comprised of:- a homing module (6) comprising an amino acid sequence codifying for a homing-peptide to deliver a polypeptide or polypeptide portion to a predetermined tissue and / or a predetermined cell population of said subject (53);- a penetrating module (7) comprising an amino acid sequence codifying for a Cell Penetrating Peptide to trigger a polypeptide or a polypeptide portioninternalization into a cell of said subject (53) through the cell membrane or by induced endocytosis; - a protection module (9) comprising an amino acid sequence codifying for a cap module with a protective function.

19. The kit according to claim 17, wherein said genetically modified eukaryotic microalga (51) is selected from the group comprised of:- eukaryotic microalgae naturally without flagella selected from the group comprised of:- Chlorella spp.;- Scenedesmus spp.;- Micractinium spp.;- Monoraphidium spp.;- Coelastrum spp.;- Pediastrum spp.;- Nannochloropsis spp.;- Trebouxia spp.;- Botryococcus braunii; and- eukaryotic microalgae naturally provided with flagella but genetically modified to have no flagella or to have flagella made inactive by inactivation of a gene selected from the group comprised of: bld1 (IFT52); bld2 (centrin / CEP164- like); bld10 (CEP135-like); IFT43; IFT121; IFT122; IFT139; IFT140; IFT144; IFT172; IFT88; IFT81; IFT80; IFT74; IFT70; IFT57; IFT54; IFT46; IFT38; IFT27; IFT25; IFT22; IFT20; fla10 (Kinesin-2 motor subunit); fla8 (Kinesin-2); fla3 (FAP163; KAP subunit of kinesin-2),,such that said genetically modified eukaryotic microalga (51), once injected, remains within the subcutaneous or intradermal injection site (52) of said subject (53),20. The kit according to claim 17, wherein said genetically modified eukaryotic microalga (51) is selected from the group comprised of:- Chlorella sorokiniana;- Picochlorum sp HM1;- Botryococcus braunii;- Chlamydomonas reinhardtii;- Tetraselmis suecica;- Dunaliella salina;- Nannochloropsis spp.;- Chlorella vulgaris;- Haematococcus pluvialis;- Scenedesmus spp.

21. The kit according to claim 17, wherein said biologically active protein is codified by a medium selected from the group comprised of:- a plasmid comprising said expression cassette (10), inserted into said microalga by transfection by a predetermined transfection vector;- an exogenous DNA sequence inserted into said microalga as an expression cassette (10) by a technique selected from the group comprised of CRISPR / CAS; TALENs; electroporation; "Gene gun"; viral vectors.

22. The kit according to claim 21, wherein said transfection vector is Agrobacterium Tumefaciens.

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