WO2024018037A1 - Microalgae expressing glp-2 and uses thereof - Google Patents

Abstract

The present invention relates to a microalgae product capable of expressing glucagon-like peptide (GLP), preferably GLP-2, and its use in the pharmaceutical or nutraceutical field in the treatment and prevention of disorders affecting the gastrointestinal system, including in particular acute and chronic inflammatory diseases.

Description

MICRO ALGAE EXPRESSING GLP-2 AND USES THEREOF

Field of Invention

The present invention relates to microalgae capable of expressing glucagon-like peptide (GLP), preferably GLP-2 or analogs of GLP-2, and uses thereof in pharmaceuticals or as a dietary supplement in the treatment and prevention of disorders affecting the gastrointestinal system, including in particular acute and chronic inflammatory diseases.

Background of the invention

A bioactive compound is any compound present in foods, animals, or plants that has an effect on the body that consumes it. In that direction, bioactive ingredients are those that, when inserted in foods, provide some effect of improvement to health. Bioactive ingredients or active ingredients, often referred to as functional ingredients, are compounds extracted from a source food, such as fruits, cereals, vegetables, and food processing residues, which preserve their characteristics even after extraction. In the same context, the term nutraceutical product or "functional food" defines all those foods, fresh or processed, which, through their normal consumption within the normal diet, by containing bioactive compounds, offer potential improvement in human health by reducing the occurrence of certain diseases. These functional foods aim to promote and support the well-being of the human body, and their development is extremely valuable from the perspective not only of treatment, for patients with gastointestinal (GI) disorders, but also of complementary and alternative prevention and containment of these diseases.

Usually functional foods contain, within them, specific natural or additional minerals, vitamins, fatty acids, dietary fiber and/or biologically active substances such as, for example, phytochemicals, antioxidants, probiotics and, also, proteins.

In the scientific landscape, there are several studies that support how certain categories of functional foods, containing probiotics, prebiotics, specific plant extracts, act positively on the digestive system, driving the food industry toward the production and development of innovative foods, capable of containing beneficial living substances using, increasingly predominantly, state-of-the-art genetic technologies.

It is necessary to emphasize that in the context of GI disorders, complementary strategies turn out to be essential not only for the reduction of the onset of such disorders, but also, and more importantly, for the management of physiological consequences related to an inflammatory state that adversely affect a normal lifestyle. Moreover, such disorders greatly impact the cost of public health, as very often, a correct diagnosis is not obtainable in a short time.

GI diseases and, in particular, chronic inflammatory bowel diseases (IBDs) such as inflammatory bowel syndrome, Crohn's disease, and ulcerative colitis are characterized by a chronic inflammatory state and, in recent years, their incidence, and prevalence has increased considerably in synergy with their severity. In addition, triggers for such intestinal dysfunctions that can result in severe intestinal failure include all conditions for which there is a change in the intestinal microbiota (drug use, alcohol, presence of malignant neoplasms, etc.).

For example, although there is a genetic predisposition for the occurrence of IBDs, the development of these diseases appears to be multifactorial, encompassing lifestyle, diet, and endogenous factors, including the composition of the gut microbiota. High intakes of mono/di-saccharides, and total fat have been shown to significantly increase the risk of developing IBDs, while a diet rich in vegetables, fruits and/or dietary fiber, protects against the onset of Crohn's disease and ulcerative colitis; these are also associated with probiotics and prebiotics which modulate the gut microflora and reduce the likelihood of IBD progression. Certain dietary patterns, rather than individual foods or nutrients, in fact, may be critical to susceptibility toward these diseases. Irritable bowel syndrome (IBS) is considered a chronic, common and little-known disease condition that is treated pharmacologically with different active ingredients, dietary factors and psychotherapeutic forms without, however, in lasting success.

Among the main drugs approved by regulatory agencies and used in the treatment of IBD at different stages are those that can regulate major inflammatory cytokines, immunomodulatory drugs, anti- TNF agents, antibiotics, corticosteroids and others

(https://link.springer.eom/article/10.1007/s00210-019-01698-z). Unfortunately, however, a large percentage of patients with IBD are in the age group of over 60 years and have comorbidities such as cardiovascular disease and/or diabetes that make the treatment of GI disorders more complex and difficult. In addition, some patients are found to be unresponsive to anti-IBD therapies. All these co-factors, therefore, make it necessary to develop new therapeutic strategies in the treatment of GI failure.

It is now known in therapeutic protocols that glucagon-like peptide (GLP) possesses numerous functions in the human body involving the reduction of blood glucose levels, control of body weight, inhibition of gastric emptying, reduction of food ingestion, increased proliferation of intestinal villi, and enhancement of intestinal growth and nutrient absorption; therefore, GLP peptides and dipeptidyl peptidase IV (DPP-IV) inhibitors have recently attracted the attention of researchers for the treatment of IBD. Several animal models, in fact, have shown how GLP administration improves the clinical course of colitis. In particular, the use of GLP-2 in the treatment of IBDs and IBSs appears to be a new therapeutic option for intestinal failure. In several experimental models, GLP-2 reversed the atrophy of the intestinal mucosa induced by parenteral nutrition and accelerated the process of endogenous adaptation in rats that had a large part of the small intestine severed. GLP-2 also significantly attenuated intestinal damage, and weight loss, in rat models with indomethacin-induced enteritis. GLP-2 also exerted reparative and cytoprotective actions in rodents in which intestinal inflammation was induced by chemotherapy treatments. Human clinical results, at the same time, have fully elucidated the beneficial effects of GLP-2 in patients with intestinal mucosal damage, confirming its ability to significantly increase energy uptake, reduce water loss and nitrogen absorption in treated subjects compared with controls. Body weight and lean body mass, moreover, increased, as opposed to fat mass, which, on the other hand, decreased; the time required for gastric emptying was also increased by 50%. Finally, histological analysis of intestinal tissue revealed how the depth and height of intestinal villi also increased following GLP-2 administration (Julie Lovshin, Daniel J. Drucker, in Encyclopedia of Endocrine Diseases, 2004).

GLP-2 is a peptide hormone composed of 33 amino acids released by intestinal L-endocrine cells upon nutrient ingestion and exerts its trophic action on the epithelium of the small and large intestine through stimulation of cell proliferation and inhibition of apoptosis. GLP-2 also upregulates intestinal glucose transporter activity and reduces gastric emptying and gastric acid secretion. GLP- 2 activity is regulated, in part, through renal clearance and cleavage by dipeptidyl peptidase IV aminopeptidase. The actions of GLP-2, in addition, are transduced by the glucagon-like peptide-2 receptor (GLP-2R), which represents a member of the G-protein-coupled receptor superfamily. GLP- 2R is involved in the increased adenylate cyclase activity.

Recently, several approaches have been developed to improve the half-life of circulating GLP-2, such as (i) structural modifications that affect the cleavage sites of degrading enzymes (DPP-IV and other proteases), (ii) fusion with proteins or lipids that allow better bindingto albumin or other circulating proteins, (iii) combining the sequence or formulation of GLP-2 with lipids/phospholipids or excipients that preserve the primary structure of the peptide hormone.

In all of these approaches, regardless of the modifications or formulation applied to improve its bioavailability and/or half-life, GLP-2 is obtained primarily by chemical synthesis or biologically by recombinant DNA (cDNA) technology, with several purification steps downstream in the process. Based on all these considerations, it is evident that there is a need to make available systems capable of expressing a bioavailable, low-cost form of GLP-2 for use in the treatment and prevention of GI disorders that is easily obtainable and suitable for preferably oral administration. Microalgae are single-celled plant organisms belonging to both the prokaryotic and eukaryotic kingdoms. Microalgae produce energy through the photosynthetic process, removing carbon dioxide from the atmosphere and releasing oxygen at the end of the process. Their special position in phylogenetic evolution makes them model organisms, as they share characteristics common to both bacteria and higher plants. The unicellular nature of microalgae means that they must respond to environmental stresses quickly and efficiently (to ensure their survival), maintaining the genome in a highly reactive euchromatic state. Number of studies, concerning microalgae, derive from the large production of secondary metabolites that are perfectly placed in the nutraceutical and pharmaceutical fields, these being potent antioxidants, essential amino acids and polyunsaturated fatty acids (co3 and co6). Microalgae comprise a wide range of mainly aquatic, unicellular, and eukaryotic organisms (including green algae, diatoms, and brown algae) that engage in photosynthetic activity, and contain cytoplasmic organelles such as mitochondria and chloroplasts. The chromatin structure of microalgae is distinct from other eukaryotic organisms; in fact, this is heavily colored highlighting a more compact nucleosomal structure and a close association of DNA with histone protein components. These differences, which are more present in green microalgae, indicate a differential mechanism in the regulation of gene expression at the histone chromatin level. In addition, structural chromatin differences in microalgae may be explanatory regarding stable nuclear transgene expression that is, in fact, difficult and transient due to chromatin-mediated gene silencing itself (H. Cerutti, A.M. I, N.W. Gillham, J.E. Boynton, Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas, The Plant Cell9:925-945 (1997)). Several anti-apoptotic gene constructs derived from mammalian cells combined with fluorescent reporter genes were introduced within model algae to evaluate their expression. The results showed that expression was low and no fluorescence gene expression could be detected, confirming the difficulty in the expression of transgenes within the microalgal genome.

Microalgae are considered an important source of healthy nutrients for human needs and are important in that biomass and biofuels can also be derived from their cultivation. Genetic engineering and stable expression (maintained over multiple generations) of different transgenes would open new horizons and greatly enhance the value and opportunity of cultivating microalgae for purposes of improving healthy well-being. However, as described earlier, it has been difficult to achieve a stable and sufficiently high level of gene expression, so a new methodology to help overcome this obstacle is extremely useful. Such an approach must consider gene silencing related to the unique and resistant histone presence of microalgae, including green microalgae.

The present invention reports a totally innovative approach regarding the use of specific microalgal strains and specific growth and reproduction techniques to express and produce GLP-2 and/or GLP- 2 peptide analogs as an endogenous metabolite to be used, for example in the form of freeze-dried biomass or humid biomass absorbed over dried functionalized food flour, as a bio-active material for animal and human use.

Summary of the invention

It is therefore an object of the invention a nucleic acid molecule comprising or consisting of: a. a coding polynucleotide sequence having at least 85% homology to SEQ ID No. 28 or which matches perfectly or is complementary to SEQ ID No. 28 or having at least 85% homology to any of SEQ ID No. 53-64 or which matches perfectly or is complementary to any of SEQ ID No. 53-64; and b. at least one linker polynucleotide sequence with at least 85% homology to any of SEQ ID No 1-21; wherein the linker polynucleotide sequence is linked to the 5 'end and/or the 3' end of the coding polynucleotide sequence.

Preferably said nucleic acid molecule comprises or consists of: c. a coding polynucleotide sequence having at least 85% homology to SEQ ID No. 28 or which matches perfectly or is complementary to SEQ ID No. 28 or which matches perfectly or is complementary to SEQ ID No. 28 or having at least 85% homology to any of SEQ ID No. 53-64 or which matches perfectly or is complementary to any of SEQ ID No. 53-64; and d. a linker polynucleotide sequence with at least 85% homology to any of SEQ ID No 1- 21, linked to the 5' end of the coding polynucleotide sequence; and e. a linker polynucleotide sequence with at least 85% homology to any of SEQ ID No 1- 21, linked to the 3' end of the coding polynucleotide sequence.

Still preferably said nucleic acid molecule has at least 85% homology to any of SEQ ID No 29-42 or has at least 85% homology to any of SEQ ID No 65-76. It is a further object of the invention an host cell comprising said nucleic acid molecule, wherein said host cell is an algal cell; preferably said host cell belongs to the class of Chlorophyceae and/or to the class of Trebouxiophyceae; still preferably said host cell to the species Haematococcus spp. and/or Chlorella spp.; even more preferably said host cell belongs to Haematococcus pluvialis and/or Chlorella vulgaris.

In a further embodiment the invention provides said host cell for use in the treatment and/or prevention of intestinal dysbiosis, chronic inflammatory bowel disease (IBD), for the treatment of irritable bowel disorders, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, to strengthen and improve the intestinal microbiota in human or veterinary animals; preferably said host cell according is for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders.

It is a further object of the invention a GLP2 expressing algal biomass comprising at least one host cell as defined above and/or a lysate and/or an extract of said host cell.

Preferably said biomass comprises the GLP2 peptide of SEQ ID No. 43 and/or GLP2 peptide analogs comprising SEQ ID No 43 or comprises the GLP2 peptides of SEQ ID No. 44-52 and/or analogs comprising SEQ ID No. 44-52.

It is a further object of the invention a method for obtaining said GLP2 expressing algal biomass comprising the following steps: a. induce thermal stress in a culture of microalgae by heating at a temperature between 35° and 50° C for a time between 300 and 600 seconds; b. expose the microalgae culture to UV rays (UV-A, UV-B and UV-C) for one or more time intervals between 5 and 15 min; c. inoculate the culture treated in step b in fresh liquid medium; d. suspend the microalgae culture from step c) in a solution composed of 16-50% (v / v) of 0.35 M mannitol and 0.2-0.6% of a mixture of lytic enzymes comprising at least one of the following enzymes: cellulase, cellulase CP, hemicellulose, chitinase, 0-D- glucanase, macerozyme, helicase, driselasi, lytic enzyme L, pectinase, protease, xylanase, cutinase, P-D-glucuronidanase, cellobiohydrolase, mixtures of them; e. incubate the microalgae culture treated in step d) at a temperature between 30 - 40° C for 4-8 h; f. combine the culture incubated from step e) in 10 ml of culture medium, with 0.1 - 0.5% of a solution comprising the isolated nucleic acid as defined in the previous paragraphs, in the presence of 10-35 % polyethylene glycol (PEG-X).

In the method of the invention the microalgae preferably belong to the Chlorophyceae class and or to the Trebouxiophyceae, more preferably belong to the Haematococcus spp. and/or Chlorella spp., even more preferably belong to the Hematococcus pluvialis species and/or Chlorella vulgaris.

It is a further object of the invention the GLP-2 expressing algal biomass as defined above or obtainable by the method above for use in the treatment and/or prevention of intestinal dysbiosis, chronic inflammatory bowel disease (IBD), for the treatment of disorders deriving from irritable bowel, for the containment of the progression of osteoporosis, for the improvement of disorders deriving from bone resorption, to strengthen and improve the intestinal microbiota in human or veterinary animals; preferably said GLP-2 expressing algal biomass is for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders.

It is a further object of the invention a pharmaceutical composition comprising the host cell of the invention or the GLP-2 expressing algal biomass of the invention and at least one pharmacologically acceptable excipient.

Preferably said pharmaceutical composition is for use in the treatment and/or prevention of intestinal dysbiosis and inflammatory bowel disease (IBD), for the treatment of disorders deriving from irritable bowel, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, to strengthen and improve the intestinal microbiota in human or veterinary animals; preferably for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders.

It is a further object of the invention a supplement or food product or drinking product comprising the host cell of the invention or the GLP-2 expressing algal biomass of the invention.

It is a further object of the invention the non-therapeutic use of the GLP-2 expressing algal biomass of the above supplement or food product or product to drink in the nutraceutical sector or as a basic ingredient in preparations of supplements and/or as an agent for the prevention and/or treatment of intestinal dysbiosis, chronic inflammatory bowel diseases (IBD), for the treatment of disorders deriving from irritable bowel, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, to strengthen and improve the intestinal microbiota, inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders.

The invention will be illustrated with reference to the following figures .

Figure 1. Effect of natural products with pharmacological properties, extracted from engineered algae, in a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. (A) Effect of tested compounds on body weight in the TNBS-induced colitis mouse model. Body weight was monitored daily until sacrifice. Weight change over time is expressed as a percentage of initial body weight. Lines indicate the mean ± standard error of each experimental group; (B) Effect of derivatives extracted from artificial seaweed on colonic length in TNBS-induced colitis mice. At the end of the study (day +3 from TNBS infusion), the two spots were excised and the length was determined with a slide caliper. Data shown represent mean + ES of 8 mice/group. ***P<0.0001 vs TNBS (test-t); (C) Effect of artificial seaweed extracted derivatives on health quality index (Health score, as defined in the results description section) in mice with TNBS-induced colitis; (D) Effect of derivatives extracted from engineered algae on DAI index (Disease Activity Index, as defined in the results description section) in mice with TNBS-induced colitis; (E) Effect of derivatives extracted from engineered algae on macroscopically visible colonic damage in mice with TNBS-induced colitis; (F) All mice in the different groups in the study were treated with TNBS. Mortality changed from the control group ( 37.5% mortality) if the mice ate seaweed withGLP2 ( 12.5% mortality) or did only Mesalazine ( 25% ) or ate seaweed not expressing GLP2 ( 50%); (G) Analysis of Body Weight and food intake of BTBR mice involved in the study. Mice treated with both lOOuL and 200 uL contrarily to the control group take less food (Right Panel) and consequently do not gain weight (Left Panel) as a consequence of socializing and playing more; (H): BTBR Mice and Behavioral Tests: Marble burying (Left Panel ) and Self grooming (Right Panel) . The treatment albeit reduced in time to 3 weeks of observation with GLP2 alga specifically and significantly changed the OCD type behavior of BTBR mice (socializing attitude) while leaving the self grooming attitude unchanged.

Figure 2. Cell viability reported as a function of the tested dose of algae, expressed as the absorbance value of formazan crystals that is directly proportional to cell viability. (A) 24 hours; (B) 48 hours; (C) Cell viability expressed as % compared to control (untreated cells = 0%) The results show a positive effect on cell viability at 24 and 48 hours after treatment with the GLP-2 expressing alga compared to the control alga. On HepG2, the effect is visible only at 24h.

Figure 3. Pharmacokinetic study in C57BL/6 male mice (n=6) with single dose treatment p.o. administration of 0.100 ml per mouse of humid biomass microalgae expressing GLP-2 peptides. The sampling time was following: the predosing and 15 min, Ih and 6h after dosing.

Figure 4. Schematic representation showing the method of the invention according to one embodiment.

Detailed description of the invention

The invention is based on the development of a novel strategy for the biosynthesis of active compounds by unicellular microalgae. In particular, within the present invention specific microalgal genotypes have been selected for the production of bioactive molecules under controlled conditions, which are available and suitable for the preparation of nutraceutical and/or pharmaceutical products. Chlorophyceae are one of the classes of green microalgae, distinguished mainly on the basis of ultrastructural morphology. Usually their coloration is caused by the predominance of photosynthetic pigments such as chlorophyll a and chlorophyll b. The chloroplast can have a discoidal, plate-shaped, reticulate, cup-shaped, spiral or ribbon-like conformation depending on the species to which it belongs. Most species belonging to this class, have one or more storage compartments called pyrenoids, located within the chloroplast. Pyrenoids, in turn, contend with various proteins including starch. Some species of green microalgae can store nutrient sources in the form of oily droplets. They usually have a cell wall consisting of an inner layer of cellulose and an outer layer of pectose. Trebouxiophyceae is a further class of green microalgae suitable for the purposes of the invention, in particular Chlorella spp.

The present invention is related to the production of exogenous proteins within a host cell, represented by a microalgal cell belonging to the class Chlorophyceae and/or Trebouxiophyceae. In some embodiments, the host cell is used as a biofactory for protein production. In some embodiments of the invention, the recombinant Chlorophyceae host cell is Haematococcus spp. In some embodiments of the invention, the recombinant Trebouxiophyceae host cell is Chlorella spp. In some embodiments of the invention the host cell is Haematococcus pluvialis and/or Chlorella vulgaris.

It therefore forms an object of the invention an isolated nucleic acid molecule comprising or consisting of: a. a polynucleotide coding sequence having at least about 50 percent homology, preferably at least about 60 percent, preferably at least about 70 percent, preferably at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 98 percent to SEQ ID No. 28 or that appears perfectly or is substantially complementary to SEQ ID No. 28, or its fragments or functional derivatives; b. at least one linker polynucleotide sequence with at least about 50 percent homology, preferably at least about 60 percent, preferably at least about 70 percent, preferably at least about 85 percent , at least about 90 percent, at least about 95 percent, at least about 98 percent to any one of SEQ ID No 1-21; wherein the linker polynucleotide sequence is operatively bound to the 5' and/or 3' end of the coding sequence.

As used herein, the term “homology” refers to sequences characterized by similarity at the nucleotide level or amino acid level as discussed herein.

A nucleic acid molecule that is complementary to the nucleotide sequence of SEQ ID NO 28 is one that is sufficiently complementary to the nucleotide sequence of SEQ ID NO 28 that it can hydrogen bond with few or no mismatches to the nucleotide sequence shown in SEQ ID NO 28 thereby forming a stable duplex.

As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.

Preferably the linker polynucleotide sequences with at least about 50 percent homology, preferably at least about 60 percent, preferably at least about 70 percent, preferably at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 98 percent to any one of SEQ ID No 1-21 are two and are bound at the 5' and 3' ends, respectively, of the coding polynucleotide sequence. Preferably the isolated nucleic acid molecule is of sequence corresponding to SEQ IDs No 29-42, or functional fragments and derivatives.

It is a further object of the invention a host cell comprising the isolated nucleic acid molecule as defined above, wherein said host cell is an algal cell, preferably said host cell belongs to the class Chlorophyceae and/or Trebouxiophyceae, preferably said host cell belongs to the species Haematococcus spp. and/or Chlorella spp..

The invention also relates to the host cell for use in the treatment and/or prevention of intestinal dysbiosis, chronic inflammatory bowel disease (IBD), for the treatment of disorders resulting from irritable bowel syndrome, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, for strengthening and improving the intestinal microbiota, preferably for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disordersand autism spectrum disorders.

In a further embodiment the invention is directed to an algal biomass comprising at least one host cell as defined above and/or a lysate and/or extract and/or secretion of said host cell and comprising expressed GLP2 (SEQ ID No 43) and GLP2 peptide analogs .

As used herein, a GLP2 peptide analog is a peptide comprising the GLP2 sequence HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID No. 43) elongated at the N or C terminal with additional aminoacids corresponding to or encoded by the specific linkers of the invention. Said GLP2 analog is a functional analog as it maintains the same biological properties of the GLP2 peptide of SEQ ID No 43.

Further forming an object of the invention is a method for obtaining an algal biomass expressing GLP2 and/or GLP2 peptide analogs comprising the following steps: a. Induce heat stress in a culture of microalgae belonging to the class Chlorophyceae by heating at a temperature between 35 and 50°C for between 300 and 600 seconds; b. Expose the microalgae culture to UV light (UV-A, UV-B and UV-C) for one or more time intervals between 5 and 15 min; c. Inoculate the culture treated in step b into fresh liquid medium to restore normal biophysiological conditions; d. Suspend the microalgae culture pretreated according to steps (a), (b) and (c) in a macerating solution consisting of 16-50% (v/v) 0.35 M mannitol and 0.2-0.6% lytic enzyme mixture comprising at least one of the following enzymes: cellulase, cellulase CP, hemicellulase, chitinase, 0-D-glucanase, macerozyme, helicase, driselase, lytic enzyme L, pectinase, protease, xylanase, cutinase, P-D-glucuronidanase, cellobiohydrolase, or mixtures thereof; e. Incubate the microalgae culture treated in step (d) at a temperature of 30 to 40°C for 4 to 8 h; f. combine the culture incubated from step (e) in 10 ml of culture medium, with 0.1 to 0.5 % of a solution comprising the nucleic acid isolated according to any of claims 1 or 2, in the presence of 10 to 35 % polyethylene glycol (PEG-X).

In a preferred form of realization microalgae belong to the classes Chlorophyceae, Trebouxiophyceae, preferably microalgae belong to the species Haematococcus spp., Chlorella spp. Further forming an object of the invention is an algal biomass expressing GLP-2 and /or GLP-2 peptide analogs obtainable by the method defined above.

The present invention also relates to a pharmaceutical composition comprising the invention's host cell or biomass and at least one pharmacologically acceptable excipient.

Preferably said pharmaceutical composition is for use in the treatment and/or prevention of intestinal dysbiosis, chronic inflammatory bowel disease (IBD), for the treatment of disorders resulting from irritable bowel syndrome, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, for strengthening and improving the intestinal microbiota, preferably for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis, and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders.

Further forming an object of the invention is a food supplement or drinking product comprising the host cell or biomass as defined above.

It is an object of the invention to make nontherapeutic use of the host cell, biomass or suppiementor food or drink product, as defined above, in the nutraceutical field or as a basic ingredient in supplement or drug preparations and/or as an agent for the prevention and/or treatment of chronic inflammatory bowel disease (IBD), for the treatment of disorders arising from irritable bowel syndrome, for the containment of the progression of osteoporosis, for the amelioration of disorders arising from bone resorption, for strengthening and improving the intestinal microbiota, preferably of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis. In addition, the products of the invention find use in treatment, prevention and as supplements in situations of intestinal dysbiosis preferably due to eating disorders and autism spectrum disorders.

For this innovative process, effective but not limiting growth conditions for the host cell include (i) efficient growth media, (ii) bioreactor temperature, (iii) pH, and (iv) oxygenation. Efficient growth medium is defined as any culture medium in which a microalgal cell, such as the Chlorophyceae cell, is usually grown. Such medium typically includes an aqueous phase containing assimilable sources of carbon, nitrogen and phosphate, as well as mineral salts, metals and other appropriate nutrients such as, for example, vitamins. Non-limiting examples of suitable media and growth conditions are described in the "Examples" section. Cells of the present invention can be cultured in conventional fermentation photobioreactors, shaking flasks, test tubes, microtiter plates, and Petri dishes. Culture can be carried out at appropriate temperature, pH and oxygen content for the recombinant cell.

According to the present invention, the term "transformation" identifies any methodology by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted within microbial cells. Within microbial cells, the term "transformation" is used primarilyto describe a genetic, heritable change caused by the acquisition of exogenous nucleic acids by the microorganism, and is essentially synonymous with the term "transfection." Several methodologies suitable for the introduction of exogenous nucleic acid molecules inside algal hostcells are known, such as (i) shotgun with gold particles, (ii) electroporation, (iii) micro injection, (iv) lipofection, (v) adsorption, (vi) infection, and (vii) protoplastic fusion.

A biomass according to the present invention is a composition comprising transformed algal cells and/or extracts and/or lysates or other derivatives. More specifically, the biomass of the invention is obtained after lysis of the cell culture and comprises, inter aha, the GLP2 peptide and the GLP2 peptide analogs.

In the present invention, a methodology for transforming a competent algal host cell has been developed that involves two main steps: a) pretreating the host cell with an enzyme, and b) introducing an exogenous nucleic acid molecule into the host cell. The method of the invention is well explained in Figure 4. According to the present invention, the enzyme can have cellulase, protease, P-glucoronase and various combinations of these activities.

The expression system of the invention, which allows the GLP-2 protein to be expressed within the algal cell, includes regulatory control elements that are active in microalgal cells. Many of these elements, including several promoters, are active in different species; therefore, the novel regulatory sequences, described as aspects of the invention, can be used not only in the algal cells described here, but also in cells belonging to different species characterized by the same evolutionary mechanisms . The design and construction of theexpression systems covered by the invention use standard biomolecular technologies known to persons skilled in the art. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd edition.

In an embodiment of the invention, the expression system or expression vector comprises a polynucleotide sequence encoding for the GLP-2 protein associated with any promoter sequence, or 5' linker, and possibly a terminator sequence, or 3' linker. The 5' linker, the GLP-2 coding sequence and the 3' linker are operatively linked so that they are functional within the host cell. The expression system may also include additional regulatory sequences that are functional within the genome of the host cell. Inducible or constitutively active sequences can be used. Suitable control elements, also include any regulatory element associated with the expression of the nucleicacid molecules described here.

The present invention is also directed to the algal host cell comprising the expression system described above and/or to a biomass obtained from or comprising said algal cell.

The expression system of the invention preferably comprises at least one of the nucleic acid molecules isolated in the present invention and described herein. In addition, all genetic elements of the expression system are sequences associated with previously isolated nucleic acid molecules. In some embodiment protocols, the nucleic acid sequence encoding for the GLP-2 protein, or coding sequence, is stably integrated into the genome of the host cell, while in others, said coding sequence is operatively linked to a promoter linker sequence and/or a terminator linker sequence, both of which are functional in the host cell. The linker sequences to which the coding sequence is operatively linked include, but are not limited to, the novel nucleic acid sequences described inthe present invention. In some embodiments, moreover, the coding sequence is optimized for the codon belonging specifically to the host cell of Haematococcus spp. and/or Chlorella spp. so as to maximize translation efficiency.

The recombinant algal cell that is the subject of the invention is capable of expressing therapeutic proteins. A "therapeutic protein," as used herein, includes proteins useful for the treatment or prevention of diseases, pathological conditions, and various disorders in both animals and humans. The terms "prevention" and "treatment" refer to both therapeutic treatment and prophylactic or preventive measures in which the objective is to prevent or slow (reduce) an undesirable pathophysiological condition, disease, or disorder, or to achieve beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, butare not limited to, the alleviation of symptoms or signs associated with a pathological condition or disorder of normal physiology; reducing the severity of a condition, disease, or disorder; stabilization of a condition, disease, or disorder (or better, situations in which the condition, disease, or disorder is stable and does not worsen over time) delay in the onset or progression of the condition, disease, or disorder; improvement of the condition, disease, or disorder; remission (total or partial and detectable or undetectable) of the condition, disease, or disorder; or enhancement or improvement of a condition, disease, or disorder. Treatment includes eliciting a clinically meaningful response without excessive side effects and also prolonging survival over expected survival if treatment is not received. In the present invention, the therapeutic protein is glucagon-like peptide 2 (GLP-2) and analogs thereof. Protein produced or expressed by the algae cell according to the invention can be produced on a commercial scale. Commercial scale includes protein production from a microorganism grown in an aerated bioreactor (biofermentor) of size > 100 L, > 1,000 L, > 10,000 L, or > 100,000 L. In some forms of implementation, commercial-scale production is performed in an aerated biofermentor with agitation. The protein produced by the algae cell can also accumulate within the cell or can be secreted by the cell, for example, into the culture medium as a soluble protein. The protein produced can be recovered from the cell, the culture medium, or the fermentation medium in which the cell itself is grown. The same biomass expressing the GLP-2 protein and GLP-2 analog, can be used directly for the purposes of the invention, which include therapeutic and preventive uses and nontherapeutic uses, for example, in the preparation of a nutraceutical product or active ingredient as for pharmaceutical use thereafter.

In addition, the present invention is directed to a method for producing a recombinant protein; the methodology also includes culture conditions for the microalgal cells of the invention such that a polynucleotide sequence coding for a protein can be expressed.

Proteins produced by the method elaborated in this invention can also be purified using a variety of standard protein purification techniques such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reversed phase chromatography, chromatographyusing concanavallin A, chromatofocusing, and differential solubilization. In some embodiments, proteins produced by the method described in the present invention are isolated in "substantially pure" form. In this context, "substantially pure" refers to a purity that allows effective use of the protein as a commercial product and/or ingredient to be used in combination with others. In some embodiments, the recombinant protein accumulates within the cell and is recovered from the cell; in some embodiments, the host cell of the method belongs to the Chlorophyceae class, while in other embodiments, the host cell is from Haematococcus spp. In some embodiments, the host cell belongs to the Trebouxiophyceae class, while in other embodiments is Chlorella spp. In some realizations, the recombinant protein is a therapeutic protein, a food enzyme or an industrial enzyme. In some realizations, the recombinant protein is GLP-2. In some realizations, the recombinant protein is a therapeutic protein that includes a secretion signal sequence, such as the GLP2 analog.

Nucleic acids

Isolated nucleic acid molecules or polynucleotide sequences that constitute the expression systemin the algal cell form the object of the invention. The nucleic acid sequences described here include the 5' and 3' linker sequences and coding sequences, particularly the GLP-2 coding sequence. An isolated nucleic acid molecule can be a DNA molecule, RNA molecule (e.g., mRNA) or derivatives of them (e.g., cDNA). Although the phrase "nucleic acid molecule" refers principally to the physical nucleic acid molecule, and although the phrases "nucleic acid sequence" or "polynucleotide sequence" refer primarily to the sequence of nucleotides present on the nucleic acid molecule, the phrases are used interchangeably, especially in reference to a nucleic acid molecule, polynucleotide sequence, or nucleic acid sequence encoding a protein. In some embodiments, a nucleic acid molecule isolated by the present invention is produced using recombinant DNA technology (such as, for example, cloning and amplification by polymerase chain reaction (PCR)) or by chemical synthesis. Isolated nucleic acid molecules include naturally occurring nucleic acid molecules and their homologs, including, but not limited to, naturally occurring allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or reversed in such a way that these modifications provide the desired effect on the sequence, function, and/or biological activity of the encoded peptide or protein.

A double-stranded DNA present in this invention, includes a single-stranded DNA and its complementary strand, the sequence of which mirrors the sequence of the single-stranded DNA. As such, nucleic acid molecules of the present invention, may be double-stranded or single- stranded and also include those nucleic acid molecules that form stable hybrids under high "stringency" conditions with a sequence of the invention and/or with a sequence complementary to a sequence of the invention. Methods for tracing a complementary sequence are known to experts in the field.

The term "protein" includes single-chain polypeptide molecules as well as multiple polypeptide complexes in which the individual constituent polypeptides are bound through covalent and noncovalent means. The term "polypeptide" includes peptides of two or more amino acids in length, typically having more than 5, 10 or 20 amino acids.

The human GLP-2 peptide is a 33 amino acid peptide, having the following sequence: HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID No. 43).

In the present invention the GLP-2 peptide refers also to peptides of other mammals, including:

The new nucleic acid molecules of the present invention can be used in any genus of microalgae in which they are found to be functional. In some forms of embodiment, the nucleic acid molecules are used in algae belonging to the class Chlorphyceae. In some forms of embodiment, recombinant nucleic acid molecules are used in the species Haematococccus spp. In some forms of embodiment, recombinant nucleic acid molecules are used in the class of Trebouxiophyceae and/or in the species Chlorella spp. As used in this invention, a recombinant microorganism has a genome that has been modified (i.e., mutated or changed) from its natural (i.e., naturally occurring or wild-type) form using recombinant technology. A recombinant microorganism according to the present invention, may include a microorganism in which nucleic acid molecules have been inserted, deleted, or modified (i.e., mutated, e.g., by nucleotide insertion, deletion, substitution, and/or inversion) such that the modifications provide the desired effect within the microorganism.

Linker promoters and terminals

The present invention is directed to the 5' and 3' linker sequences. The 5' or promoter linker is a DNA sequence that directs transcription of a coding region associated with it. The 3' or terminal linker, on the other hand, is the gene sequence that marks the end of transcription of genomic DNA.

The linker of the invention (promoter or terminal) is any of the following sequences: CGGGGCAACTCAAGAAATTC (SEQ ID No 1) GTCTGGCCGAGGTCTGGTTCCTGTGCC (SEQ ID No 2)

ACTGCACATCGCTGCAGTCT (SEQ ID No 3) CGCGTCGGGGCCTGCCTAAG (SEQ ID No 4) TTACCTGCCACACAAGCCTG (SEQ ID No 5) CGTGCTACTGGGGTCTGGCAG (SEQ ID No 6) CACATGCCATCCGAGTCGTC (SEQ ID No 7) CACAACCATACTGGCGAAGT (SEQ ID No 8) ATGGCCACGC (SEQ ID No 9) CTCTACCCAC (SEQ ID No 10)

CCGGACTGCCATAGCACAGCTAGACGA (SEQ ID No 11) GTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 12) ACTGACTGCCATAGCACAGCTAGACGA (SEQ ID No 13) ATTTGCTGCATGACTGGATCAATGCGACGA (SEQ ID No 14) GTCTGGCCTGACGTATGATCGATGCCATAAATGC (SEQ ID No 15) ATGCCCTGATCCCAATGATGGACGA (SEQ ID No 16) GTCTGGCCGAAACTGATTTGGCCATGAC (SEQ ID No 17) GAGCGTGCTGAAATGCATGCGACGA (SEQ ID No 18) GTCTGGCCCCCGGGTATAGTAGCTGAC (SEQ ID No 19) CCCGGGTATAGTAGCTGACTGCGACGA (SEQ ID No 20) GTCTGGGAGCGTGCTGAAATGCATG (SEQ ID No 21)

It therefore forms an object of the invention to have an isolated nucleic acid molecule comprising a polynucleotide sequence that is at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 1 - SEQ ID NO:21, in which the polynucleotidesequence functions as a promoter and/or terminal linker at least in algae belonging to the class Chlorophyceae. The invention also relates to an isolated nucleic acid molecule comprising a polynucleotide sequence that hybridizes with any of the SEQ ID NO:1 - SEQ ID NO:21 sequences or that hybridizes with a polynucleotide sequence that is at least 95 percent identical to any of the SEQ ID NO: 1 - SEQ ID NO:21 sequences.

The isolated nucleic acid molecule may include a polynucleotide sequence that is completely complementary to any of the SEQ ID NO: 1 - SEQ ID NO:21 sequences or to a polynucleotide sequence that is at least 95% identical to any of the SEQID NO:1 - SEQ ID NO:21 sequences.

MATERIALS AND METHODS

Cell cultures

Microalgae, belonging to the genus Chlorophyceae, were grown in the culture medium shown in Table 1, under illumination with an intensity of 120 mmol photons m s^-2-1 in an alternating cycle 25 of 16 h of light and 8 h of dark, at a temperature of 25 °C. The cultures were agitated by mechanical shaker (g24 environmental incubator shaker, American Laboratory Trading) at 70 rpm throughoutthe growth time. Table 1 : composition of the growth medium

The percentages shown in the table may vary depending on the species, genotype and initial concentration of microalgae cultures.

Polynucleotide design

Through bioinformatics research, some microalgae-specific genes related to the photosynthesis pathway were analyzed. The following sequences were derived from specific algal sequences:

CGGGGCAACTCAAGAAATTC (SEQ ID No 1)

GTCTGGCCGAGGTCTGGTTCCTGTGCC (SEQ ID No 2)

ACTGCACATCGCTGCAGTCT (SEQ ID No 3)

CGCGTCGGGGCCTGCCTAAG (SEQ ID No 4)

TTACCTGCCACACAAGCCTG (SEQ ID No 5)

CGTGCTACTGGGGTCTGGCAG (SEQ ID No 6)

CACATGCCATCCGAGTCGTC (SEQ ID No 7)

CACAACCATACTGGCGAAGT (SEQ ID No 8)

ATGGCCACGC (SEQ ID No 9)

CTCTACCCAC (SEQ ID No 10)

CCGGACTGCCATAGCACAGCTAGACGA (SEQ ID No 11)

GTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 12)

ACTGACTGCCATAGCACAGCTAGACGA (SEQ ID No 13)

ATTTGCTGCATGACTGGATCAATGCGACGA (SEQ ID No 14)

GTCTGGCCTGACGTATGATCGATGCCATAAATGC (SEQ ID No 15)

ATGCCCTGATCCCAATGATGGACGA (SEQ ID No 16)

GTCTGGCCGAAACTGATTTGGCCATGAC (SEQ ID No 17) GAGCGTGCTGAAATGCATGCGACGA (SEQ ID No 18)

GTCTGGCCCCCGGGTATAGTAGCTGAC (SEQ ID No 19)

CCCGGGTATAGTAGCTGACTGCGACGA (SEQ ID No 20)

GTCTGGGAGCGTGCTGAAATGCATG (SEQ ID No 21)

The sequence of the human GLP-2 gene is as follows:

CATGCTGATGGTTCTTTCTCTGATGAGATGAACACCATTCTTGATAATCTTGCCGCCAG

GGACTTTATAAACTGGTTGATTCAGACCAAAATCACTGAC (SEQ ID No. 28)

The following oligonucleotides comprising the human GLP2 gene sequence and at least one promoter linker, preferably a promoter linker and a terminal linker (underlined in the list below), derived from the specific algal sequences and previously described, were then synthesized:

CGGGGCAACTCAAGAAATTCCATGCTGATGGTTCTTTCTCTGATGAGATGAACAC CATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAA ATCACTGACGTCTGGCCGAGGTCTGGTTCCTGTGCC (SEQ ID No 29)

ACTGCACATCGCTGCAGTCTCATGCTGATGGTTCTTTCTCTGATGAGATGAACAC CATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAA ATCACTGACCGCGTCGGGGCCTGCCTAAG (SEQ ID No 30)

TTACCTGCCACACAAGCCTGCATGCTGATGGTTCTTTCTCTGATGAGATGAACAC CATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAA ATCACTGAC (SEQ ID No 31)

CGTGCTACTGGGGTCTGGCAGCATGCTGATGGTTCTTTCTCTGATGAGATGAACA CCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAA AATCACTGAC (SEQ ID No 32)

CACATGCCATCCGAGTCGTCCATGCTGATGGTTCTTTCTCTGATGAGATGAACAC CATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAA ATCACTGAC (SEQ ID No 33)

CACAACCATACTGGCGAAGTCATGCTGATGGTTCTTTCTCTGATGAGATGAACAC CATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAA ATCACTGAC (SEQ ID No 34)

ATGGCCACGCCATGCTGATGGTTCTTTCTCTGATGAGATGAACACCATTCTTGAT AATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGACCAAAATCACTGACC TCTACCCAC (SEQ ID No 35)

CCGGACTGCCATAGCACAGCTAGACGACATGCTGATGGTTCTTTCTCTGATGAGA TGAACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCA GACCAAAATCACTGACGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 36) ACTGACTGCCATAGCACAGCTAGACGACATGCTGATGGTTCTTTCTCTGATGAGA TGAACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCA GACCAAAATCACTGACGTCTGGCCGAGGTCTGGTTCCTGCCTAG (SEQ ID No 37)

ATTTGCTGCATGACTGGATCAATGCGACGACATGCTGATGGTTCTTTCTCTGATG AGATGAACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGAT TCAGACCAAAATCACTGACGTCTGGCCTGACGTATGATCGATGCCATAAATGC

(SEQ ID No 38)

ATGCCCTGATCCCAATGATGGACGACATGCTGATGGTTCTTTCTCTGATGAGATG

AACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGA

CCAAAATCACTGACGTCTGGCCGAAACTGATTTGGCCATGAC (SEQ ID No 39)

GAGCGTGCTGAAATGCATGCGACGACATGCTGATGGTTCTTTCTCTGATGAGATG AACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCAGA CCAAAATCACTGACGTCTGGCCCCCGGGTATAGTAGCTGAC (SEQ ID No 40)

CCCGGGTATAGTAGCTGACTGCGACGACATGCTGATGGTTCTTTCTCTGATGAGA TGAACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCA GACCAAAATCACTGACGTCTGGGAGCGTGCTGAAATGCATG (SEQ ID No 41)

ACTGACTGCCATAGCACAGCTAGACGACATGCTGATGGTTCTTTCTCTGATGAGA TGAACACCATTCTTGATAATCTTGCCGCCAGGGACTTTATAAACTGGTTGATTCA GACCAAAATCACTGACAAAAAAAAAAAAAAAAAAGTCTGGCCGAGGTCTGGTTC

CTGCCTAG (SEQ ID No 42)

The sequences of the GLP-2 gene of other mammals are as follows:

The following oligonucleotides comprising the GLP2 gene sequence from other species and at least one promoter linker, preferably a promoter linker and a terminal linker (underlined in the list below), derived from the specific algal sequences and previously described, were also synthesized by using the same method of the invention:

Treatment of microalgae

The microalgae culture was resuspended at a ratio of 1 :5 to 1 :25, depending on cell concentrations, in a macerating solution which constitutes a lytic mixture. The macerating solution contained 16- 50% (v/v) 0.35 M mannitol and 0.2-0.6% lytic enzyme mixture (Table 2). The lytic mixtures were then incubated at 30-37°C for 4-8 h. The composition of the lytic mixture, temperature and incubation time changed according to the microalgae species.

Table 2: Lytic enzymes

The microalgae solutions after treatment with the lytic mixture were combined, in a final volume of 10 ml of culture medium, with 0.1 to 0.5 % v/v of polynucleotide diluted 2, 5, 10, 20, 25, or 50 times (initial concentration 100 pM), depending on the nucleotide length and the resultingmolecular weight of each polynucleotide (Table 3)

Table 3: Polynucleotide dilutions and percentages of lithium mixture used

10-35 % w/V Polyethylene glycol (PEG-X) chemical melting agent was added to the culture

(Table 4), calculated in consideration of the biomass obtained from the previous step.

Subsequently, the following steps were taken: i) The mixture was centrifuged at 1000 to 7000 rpm for 2 to 15 minutes; ii) The supernatant was removed and the pellet was washed with 2-15 ml of culture medium; iii)The mixture was centrifuged at 1000 to 7000 rpm for 2 to 15 minutes.

Steps i, ii and iii are repeated 2 to 5 times. When finished, the pellet is resuspended in 1 to 50 ml of standard culture medium. Centrifugation speed and duration vary according to the concentrations of PEG used (Table 4)

Table 4: PEG concentrations (% w/v) and processing parameters.

DNA extraction and amplification reaction by PCR

After several subcultures, PCR on DNA was conducted using Phire Plant Direct PCR Master Mix

(Thermo Fisher). The following primers were used in the reaction:

Pri GLP regl - Forward AGACATGCTGATGGTTCTTT (SEQ ID No 22) Pri GLP reg2 - Forward TCTCTGATGAGATGAACACC (SEQ ID No 23) Pri GLP reg4 - Forward GATTTCCCAGAAGAGGTCG (SEQ ID No 24) Pri GLP regl - Reverse AACATTTCAAACATCCCACG (SEQ ID No 25) Pri GLP reg2 - Reverse GCAGGTGATGTTGTGAAGAT (SEQ ID No 26)

Pri GLP reg4 - Reverse CGGCAAGATTATCAAGAATGG (SEQ ID No 27)

The amplification protocol is as suggested in the Phire Plant Direct PCR Master Mix brochure. (Thermo Scientific™ - Cat. Number: Fl 60S - https : //www. thermofisher , com/ order/ catalog/product/F 160S )

To extract metabolites contained within culture, this was sonicated for 15 min (1/2 W converterset to 100 % . Modulated pulse. UH-500B probe - Probe 600 ml - 50% pulse duty cycle). Then the sonicated culture was centrifuged at 4500 rpm for 10 min in order to separate cell debris from the supernatant. Depending on the different applications (in vivo or in vitro), the supernatant and/or biomass were subjected to lyophilization.

Quantification of GLP-2 (pg/g) in microalgae biomass by Mass Spectometry analysis

Mass Spec analysis was used to determine the concentration of the GLP-2 and GLP-2 analogs in dried biomass by lyophilization.

The humid microalgae biomass expressing GLP-2 was collected and freeze-dried to obtain a green powder and 100 mg of this material used to extracted the desired peptide hormone.

Methodology: - Sample weighing

- Extracted with 20 mL of methanol

- Doing three freeze/thaw cycles with sonication lasting 30 min each

- Leave to stand over night

- The next day centrifuge and 1 mL of supernatant dried by N2

- Added 1 mL of Ammonium bicarbonate 50 mM to the dried sample.

- Sonicated for 20 min

- Centrifuge and supernatant dried in Savant

- Resuspended in 200 pL of HCOOH 0,1%

- Centrifuge and supernatant in vial for LC-MS/MS “MRM” analysis

Ipl of supernatant were analysed by using a AB-sciex 5500 QTRAP® system with a HPLC chromatography system Exion LC™. The mobile phase was generated by mixing eluent A (0.1 % Eormic Acid in water) and eluent B (0.1 % Eormic Acid in acetonitrile) and the flow rate was 0.200 mL/min. Chromatographic gradient was from 20% to 90% in 4 min, hold for 2 min, then return to 20 % in 1 min. Tandem mass spectrometry was performed using Turbo VTM ion source operated in positive ion mode, and the multiple reaction monitoring (MRM) mode was used for the selected analytes.

The extracted mass chromatogram peaks were integrated using Skyline software for data processing.

Pharmakokinetic Studies

A pharmacokinetic study was conducted in C57BL/6 Male mice (n=6) by single dose treatment by p.o. administration of 0.100 ml per mouse of humid biomass microalgae expressing GLP-2 peptides. The sampling time was following: the predosing and 15 min , Ih and 6h after dosing. To preserve the peptide stability, blood samples was collected in presence of a protease inhibitor cocktail to be analyzed by LC-MS and by highly specific and very sensitive (picomols) ELISA sandwich to determine the PK parameters (results are shown in Pig. 3).

RESULTS

In vitro studies

Quantification of GLP-2 (pg/ml) in microalgae biomass by ELISA assay

To determine the amount of GLP-2 expressed by algal biomass, a human glucagon-like peptide 2 (GLP2) ELISA kit (0.156-10 ng/mL) was used. 100 mg of dry algal biomass, obtained by freeze- drying the crude product, was dissolved in 10 mL of water containing 50% acetonitrile and 0.1% trifluoroacetic acid. The extraction step performed is reported in the materials and methods, in fact, the solution was sonicated for 15 min and then, by centrifugation, the algal biomass was separatedfrom the supernatant. The supernatant was lyophilized and 100 pl was resuspended in diluent buffer (provided by the kit).

The analysis protocol used includes the following steps:

1. Prepare all reagents, samples and standards.

2. Add 100 pl of sample to each well. Incubate 2 hours at 37°C

3. Aspirate and add 100 pl of prepared detection reagent A. Incubate 1 hour at 37°C

4. Vacuum and wash 3 times

5. Add 100 pl of prepared detection reagent B. Incubate 1 hour at 37°C

6. Vacuum and wash 5 times

7. Add 90 pl of substrate solution. Incubate 15-25 min at 37°C

8. Add 50 pl of blocking solution.

9. Read the absorbance immediately at 450 nm.

Interpolating the different measurements made, with the calibration line derived from the assay of the GLP-2 standard provided by the ELISA kit, the amount of peptide present in the sample is in the range of 150 to 2000 pg/ml.

Cytotoxicity Assays

Biocompatibility experiment conducted through Mi l assay of the extract derived from control cells of Haematococcus spp. and GLP-2

5x10³ IEC-6 cells were seeded and allowed to grow overnight. CTRL and GLP-2 cells of Haematococcus spp were sonicated and filtered to separate the precipitated phase from the liquid phase. Different concentrations of algal extracts were tested (starting from 25% v/v. Serial 1:2 dilutions were made until the final concentration of 0.39 % v/v was reached). MTT assay was conducted after 24 and 48 hours to check cell viability. The protocol used involves the following steps:

1. Remove the treatment medium: For adherent cells, carefully aspirate the medium.

2. Add 50 pl of serum-free medium and 50 pl of MTT reagent to each well.

3. Background control wells were prepared: 50 pL of MTT reagent + 50 pL of cell culture medium (without cells)

4. Incubate the plate at 37°C for 3 hours.

5. After incubation, add 150 pl of MTT solvent to each well.

6. Wrap the plate in aluminum foil and shake on an orbital shaker for 15 minutes.

7. Read the absorbance at OD = 590 nm.

Data analysis and statistics were performed using GraphPad Prism. Haematococcus pluvialis was used in the experiment.

In vivo studies

Animal Efficay Studies

Two different efficacy studies were conducted in animal models (mouse) with the aim of verifying non toxicity in vivo and measuring the efficacy of GLP2 produced in alga . The first study was conducted on a mouse model of inflammatory bowel disease (TNBS mouse model) and a second study conducted on Autism Spectrum Disorder Animal Model (BTBR T+tf/J mouse mode - Gut- BRAIN axis report )

Animal Model (TNBS)

Inflammatory bowel disease (IBD) is a multifactorial disease involving immunological, environmental and genetic factors. Although there are no animal models that effectively mimic human inflammatory bowel disease, experimental models allow to analyze the mechanisms of chronic intestinal inflammation. IBD can be induced in mice by a 2,4,6-trinitrobenzene sulfonic acid (TNBS)- ethanol enema that elicits an immune response and colitis. In order to evaluate the efficacy of GLP-2 contained in the biomass of our microalgae on a mouse model of IBD, it was decided to use the TNBS-ethanol-induced colitis model. Based on the scientific publication by Morris et al. (Morris G.P., Beck P.L., Herridge M.S., Depew W.T., Szewczuk M.R., Wallace J.L.Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology. 1989;96(3):795-803. [PMID: 2914642]), ethanol and TNBS (trinitrobenzenesulfonic acid) at a dose of 100 mg/kg are co-administered intrarectally to rats. Ethanol is used as a means to effectively destroy the intestinal barrier and allow TNBS to interact with proteins in colon tissue.

After colitis induction, mice develop several manifestations of acute colitis. These include soft stool formation and occult or even bloody diarrhea. Intracolonic administration of TNBS/ethanol,in mice induces severe disease characterized by bloody diarrhea and dramatic body weight loss during the first week. In detail, colitis was induced by rectal administration of 2 mg/100 ml TNBS (Sigma- Aldrich, St. Louis, MO, USA) in 45% ethanol (Merck, Darmstadt, Germany) using a vinylcatheter placed 3.5 cm proximal to the anus. During the procedure, mice were anesthetized using Rumpun/Zoletil. After instillation of the catheter, the animals were kept upright for 30 sec. Control mice underwent identical procedures but were instilled with 45% ethanol dissolved in phosphate- buffered saline (PBS). Mice were monitored daily for survival, body weight, rectal bleeding and stool consistency. All animals were sacrificed on day 5 of the experiment for cervical dislocation.

Immunocompetent mice were kept fasting for 24 h before colitis induction. At day 0, mice were anesthetized by inhalation of isoflurane, 2 mg of TNBS in 100 pL of 50% ethanol solution were administered slowly into the colon through a medical-grade catheter (3.5 F) inserted gently about 4 cm into the anus.

Food and water were administered ad libitum after TNBS instillation. Mice were divided into the following experimental groups (8 mice/group):

1) TNBS 2 mg/topo ir + PBS 1 mL/topo os

2) TNBS 2 mg/topo ir + Mesalazine 1 mL/topo (100 mg/Kg) os

3) TNBS 2 mg/topo ir + Algae CTRL (300pL) os

4) TNBS 2 mg/topo ir + Algae CTRL (lOOpL) os

5) TNBS 2 mg/topo ir + Algae GLP2 (300pL) os

6) TNBS 2 mg/topo ir + Algae GLP2 (lOOpL) os

(os: oral administration; ir: intra-rectal administration; GLP2 algae are GLP2-expressing algae according to the invention; CTRL algae are control algae)

Mesalazine, an anti-inflammatory agent used in the treatment of inflammatory bowel disease (positive disease control) was administered orally by gastric gavage from three hours before the TNBS infusion and for the next three days (once daily in the morning, 4 total doses). Algae were administered orally by gavage from seven days before TNBS infusion and for the next three days (once daily in the morning, 10 total doses). Mice were sacrificed by CO asphyxiation2 in the afternoon 3 days after intrarectal administration of TNBS.

The following parameters were recorded during the study: Clinical signs.

Daily body weight (for measuring weight loss).

The day of sacrifice:

Blood samples were collected and frozen from 3 mice of groups 1, 3, 5 and 6. the two points were explanted, measured in length with a slide caliper, immediately frozen inliquid nitrogen, and stored at -80°.

The following parameters were recorded ex vivo:

- length of the colon.

Dosages PBS: 1 mL/mouse TNBS: 100 pL in 50% ethanol/mouse solution. Mesalazine: 1 mL/mouse GLP2 and CTRL algae: 300pL and lOOpL/mouse (two doses for both types of algae)

Frequency of administration TNBS: only once at time 0 to induce colitis. Mesalazine: daily from three hours before TNBS infusion until the mice are sacrificed. GLP2 and CTRL algae: daily from seven days before TNBS infusion until mice are sacrificed.

Results

Impact on weight loss

To study the potential therapeutic action of derivatives extracted from artificial algae and the potential adverse effects due to administration of test compounds in a TNBS model of colitis, changes in body weight over time were monitored because weight loss in mice represents one of the parameters of disease severity.

As expected, mice that were instilled with TNBS (disease control) developed severe disease characterized by a body weight loss of about 14% from their initial body weight.

Prior to TNBS infusion, no changes in body weight and/or adverse effects were observed during prophylactic oral treatment with seaweed.

Among the two doses of GLP2 tested, a 100 pL dose showed a protective effect against TNBS- induced colitis, comparable to the positive control (Mesalazine-treated group), attenuating clinical symptoms and weight loss (Figure 1A).

Both groups treated with CTRL algae, in which no effect was expected, showed marked weight loss as did the TNBS control disease group.

Impact on colon length

To assess the impact of artificial algae derivative treatments on the inflammatory response in the mouse model with colitis, at the end of the study, the length of the explanted colon was measured with a slide caliper. This parameter makes it possible to estimate the level of inflammation in the colon, since colitis could cause edema of the large intestine and a decrease in the length of the entire colon.

Figure 1(B) shows that, macroscopically, the colon is shorter in the groups of mice treated with TNBS and both doses of CTL alga, with a reduction of about 70% compared with the positive control group (Mesalazine).

These results are very encouraging; in fact, mice treated with GLP2 alga at both doses used, but especially at the 100-pL dose, showed markedly improved macroscopic signs, with a significant reduction in inflammatory activity and restoration of colonic length at the level of Mesalazine- treated group.

Impact on clinical manifestation scores

To assess the putative ability of the compounds under investigation to repair colon inflammation and damage by reducing its clinical manifestations in this model, indices of DAI, colon damage, and health-related quality of life in mice were monitored and evaluated over time.

The DAI score is a marker of disease activity and was determined based on the methods of Murano et al. (Murano M. et al. (2000): Therapeutic effect of intracolonically administered nuclear factor K B (p65) antisense oligonucleotide on mouse dextran sulphate sodium (DSS)-induced colitis. Clin

Exp Immunol 120, 51-58) and calculated as the sum of the weight loss score, diarrheal score, and hematochezia score.

The health quality of mice (health score) is a marker extrapolated from the human endpoint scoring system, according to which, based on predetermined physiological or behavioral signs, a score can be assigned to the health status of the animal enrolled in the experimental study.

Finally, the colon damage index scores colon features in TNBS-treated mice such as hyperemia, thickening and ulceration.

As shown in Figures 1(C), 1(D) and 1(E), the severity of colitis symptoms tended to worsen especially in CTL-treated mice, possibly due to a toxic effect with higher scores than mice in the TNBS-treated (disease control) group alone.

In contrast, an improvement in clinical manifestations of colitis, including phenotypic and behavioral characters, reduction in weight loss and stool consistency, was observed in both groupsadministered with GLP2 alga especially at the lowest dose, which was able to bring these signs ofdisease back almost to the levels of the positive control group.

In addition, as shown in Figure 1(F) and Table 5, the mice that received GLP2 algae had a lower mortality rate (and thus higher survival) than both the control and Mesalazine groups.

Table 5 : Mortality rate

Animal Model for Autism Spectrum (BTBR T+ Itpr3tf/J (BTBR)

In recent years, scientific research toward the study of metabolic and inflammatory disorders related to central neurological disorders has expanded greatly with strong evidence on thecorrelation of gut integrity and behavioral issues.

Autism (ASD) is a neuro-psychiatric disorder characterized primarily by substantial impairments in social interaction, difficulties in communication, and stereotyped and repetitive behaviors (9).

Several studies have reported the presence of altered gut microbiota and eating disorders in autism spectrum disorders in both humans and animal models (10, 11), hypothesizing a possible involvement of dysbiosis in worsening symptoms (11). In addition, a range of epidemiological evidence has shown a relationship between exposure to a fatty diet by pregnant women and the risk of developing neurological disorders in their offspring including autism (12).

Animal models currently used by the scientific community as a model of the autism spectrum include mice of the BTBR T+ Itpr3tf/J strain (BTBR; Meyza,K.Z., Blanchard, D.C., The BTBR mouse model of idiopathic autism. Current view on mechanisms. Neurosci. Biobehav. Rev. (2017)). In fact, presenting the 3 key symptoms (problems with socialization, communication, and repetitive behaviors), he summarizes the main biochemical, neuropathological, and behavioral features found in human pathology (13, 14). In addition, recent studies have shown that a nutritional approach can induce metabolic and behavioral changes in these mice. For example, when subjected to fatty diet from the time of weaning, such strain shows even more antisocial behavior and cognitive rigidity. This strain therefore constitutes an important investigative tool for the study of metabolic disorders related to autism (15-17).

Examining oxidative metabolism, among the abnormalities in energy metabolism, a key role is played by mitochondria. Several studies in both patients and BTBR animal models have shown that mitochondrial dysfunction is a very common condition in autism (18, 19). However, it is stillunclear whether this dysfunction is a cause or effect of ASD.

Mitochondria, in addition to being the main cellular energy powerhouse (contributing to the production of about 90 percent of the energy used by our body), are known to synthesize key molecules during inflammatory and oxidative processes, serving as the main source of free radicals. Therefore, it is not surprising that mitochondrial dysfunction is associated with inflammation and other metabolic disorders in which cellular oxidative damage is caused by reactive oxygen species (ROS) production that exceeds physiological antioxidant activity (20). So, mitochondrial dysfunction can be both the cause and the consequence of inflammatory processes and cause metabolic adaptations that could be protective or become progressively harmful (21). The trial was conducted with 6 genetically modified male BTBR T+ Itpr3tf/J (BTBR) strain miceat the age of 3 months. The animals were fed a standard laboratory diet (15.88 kJ/g) for 3 weeks and were divided into two groups: the first group (control) received daily intragastric administration of vehicle (water), the second group (treated) received daily intragastric administration of Haematococcus spp- GLP2 extract of lOOpl/day for 2 weeks and 200pl/dayfor an additional 7 days (Haematococcus pluvialis was used for this experiment). Body weight and food intake were monitored daily. In addition, behavioraltests were performed by means of the "Marble burying test" and "self grooming." These tests consist of assessing repetitive/perversive behaviors such as number of marbles or enrichment material buried in the Marble burying Test or time spent cleaning and rubbing the body in self grooming. In self-grooming, animals are placed one per cage, and after 5 minutes of settling in, the time the animal spends in cleaning/scrubbing the various body parts is calculated for 10 minutes. In the marble burying test, on the other hand, mice are placed individually in a cage whose floor has been covered with a layer of sawdust. After an initial adaptation period, 20 marbles of 1cm diameter were placed on the sawdust at a regular distance from each other. Their behavior and the number of marbles hidden by the animals were evaluated. A marble was considered hidden ifit was two-thirds covered by the sawdust.

The administration of GLP2 algae caused no adverse effects to the animals, which showed no signsof distress with either dose. In addition, the results obtained showed that no changes in major metabolic parameters were observed following the 3 weeks of treatment regarding food intake and body weight gain between the two experimental groups (Fig. G). Following behavioral investigations aimed at assessing the repetitive/perseverative behaviors characteristic of the BTBR phenotype, in the Marble burying Test, the alga-GLP2 -treated mice showed more disinterested behavior in burying the marbles (thus very positive effect), while regarding self grooming the differences between the two groups for the short period of treatment showed no statistically significant differences (Fig. H).

Discussion

The conceptual idea behind the invention is based on the use of microalgae as natural and green bioreactors, capable of producing and transporting molecules of interest. GLP-2, as described above, plays a key role in intestinal well-being, and microalgae represent optimal carriers. The platform aims to design edible microalgae to make naturally produced GLP-2 to be used directly as a carrier for gut delivery. The innovation of this technology lies in the combination of an already useful product for humanhealth (microalgae high in antioxidants) with a molecule of pharmaceutical interest (GLP-2 and/or GLP-2 peptide analogs).

The methodology used, exploits the possible ability of microalgae to incorporate exogenous DNA. Forthis purpose, a polynucleotide containing the GLP-2 coding sequence was synthesized and two linkers were joined at the terminal end, coding for a structural gene of the microalgae, to enable targeted insertion. No physical or biological vector was used. A novel methodology was applied and special conditions were optimized that allowed the microalgae to incorporate the synthetic polynucleotide. Once the GLP-2 was inserted, the microalgae were first selected on solid medium and at a later stage were inoculated into the specific liquid culture medium and allowed to grow following standard growth protocols.

At the end of the culture cycle (12-15 days), the biomass was harvested and processed to obtain the extract in accordance with what was described in the experimental part.

The TNBS-induced colitis model has proven very useful in understanding the pathophysiology of intestinal inflammation and is a powerful tool for evaluating interventions to prevent or amelioratethe disease.

The in vivo study carried out in the present invention aims to evaluate the protective and antiinflammatory effect of derivatives extracted from engineered algae in an experimental mouse model of TNBS-induced colonic inflammation. The hypothesis, based on suggestions from the literature, is that they might have a marked effect of reconstructing the intestinal epithelium, thus reducing the process of dysbiosis resulting in anti-inflammatory activity and improvement of the intestinal microbiota. It also might be able to prevent oxidative damage by scavenging free radicals.

Overall, the experimental data obtained in the invention confirmed the high protective and antiinflammatory effects with improvement in clinical manifestations of colitis obtained by administering the GLP2 alga of the invention at both doses used, but especially at the lowest dose (lOOul) .

The in vivo model correlating with autism spectrum also confirmed that the tested product is not toxic in mice (that although genetically modified for the BTBR gene have a normal life span) and provided a clear evidence that the improvement of the epithelium quality results in less inflammation and, consequently, better quality of the microbiota. Through the gut-brais axis these effects have a positive impact on autistic behavior.

Bibliography

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Claims

CLAIMS . A nucleic acid molecule comprising or consisting of: a. a coding polynucleotide sequence having at least 85% homology to SEQ ID No. 28 or is complementary to SEQ ID No. 28 or having at least 85% homology to any of SEQ ID No. 53-64 or is complementary to any of SEQ ID No. 53-64; and b. at least one linker polynucleotide sequence with at least 85% homology to any of SEQ ID No 1-21; wherein the linker polynucleotide sequence is linked to the 5 'end and/or the 3' end of the coding polynucleotide sequence of a).

2. The nucleic acid molecule according to claim 1 comprising or consisting of: a. a coding polynucleotide sequence having at least 85% homology to SEQ ID No. 28 or is complementary to SEQ ID No. 28 or is complementary to SEQ ID No. 28 or having at least 85% homology to any of SEQ ID No. 53-64 or is complementary to any of SEQ ID No. 53-64; and b. a linker polynucleotide sequence with at least 85% homology to any of SEQ ID No 1- 21, linked to the 5' end of the coding polynucleotide sequence of a); and c. a linker polynucleotide sequence with at least 85% homology to any of SEQ ID No 1- 21, linked to the 3' end of the coding polynucleotide sequence of a).

3. The nucleic acid molecule according to any one of claims 1 or 2 having at least 85% homology to any of SEQ ID No 29-42 or having at least 85% homology to any of SEQ ID No 65-76.

4. A host cell comprising the nucleic acid molecule according to any one of claims 1 to 3, where said host cell is an algal cell.

5. The host cell according to claim 4 wherein said host cell belongs to the class of Chlorophyceae and/or wherein said host cell belongs to the class of Trebouxiophyceae.

6. The host cell according to claim 5 wherein said host cell belongs to the species Haematococcus spp. and/or Chlorella spp..

7. The host cell according to claim 6 wherein said host cell belongs to Haematococcus pluvialis and/or Chlorella vulgaris.

8. The host cell according to any one of claims 4-7 for use in the treatment and/or prevention of intestinal dysbiosis, chronic inflammatory bowel disease (IBD), for the treatment of irritable bowel disorders, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, to strengthen and improve the intestinal microbiota in human or veterinary animals. The host cell according to claim 8 for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders. A GLP2 expressing algal biomass comprising at least one host cell according to any of claims 4- 7 and/or a lysate and/or an extract of said host cell. The GLP2 expressing algal biomass according to claim 10 wherein said biomass comprises the GLP2 peptide of SEQ ID No. 43 and/or GLP2 peptide analogs comprising SEQ ID No 43 or wherein said biomass comprises the GLP2 peptides of SEQ ID No. 44-52 and/or analogs comprising SEQ ID No. 44-52. A method for obtaining the GLP2 expressing algal biomass according to any of claims 10 and 11 comprising the following steps: a. induce thermal stress in a culture of microalgae by heating at a temperature between 35° and 50° C for a time between 300 and 600 seconds; b. expose the microalgae culture to UV rays (UV-A, UV-B and UV-C) for one or more time intervals between 5 and 15 min; c. inoculate the culture treated in step b in fresh liquid medium; d. suspend the microalgae culture from step c) in a solution composed of 16-50% (v / v) of 0.35 M mannitol and 0.2-0.6% of a mixture of lytic enzymes comprising at least one of the following enzymes: cellulase, cellulase CP, hemicellulose, chitinase, 0-D- glucanase, macerozyme, helicase, driselasi, lytic enzyme L, pectinase, protease, xylanase, cutinase, P-D-glucuronidanase, cellobiohydrolase, mixtures of them; e. incubate the microalgae culture treated in step d) at a temperature between 30 - 40° C for 4-8 h; f. combine the culture incubated from step e) in culture medium with 0.1 - 0.5% of a solution comprising the isolated nucleic acid according to any one of claims 1-3, in the presence of 10-35 % polyethylene glycol (PEG-X).

13. The method according to claim 12 wherein the microalgae belong to the Chlorophyceae class and or to the Trebouxiophyceae.

14. The method according to any of claims 12-13 wherein the microalgae belong to the Haematococcus spp. and/or Chlorella spp..

15. The method according to claim 14 wherein the microalgae belong to the Hematococcus pluvialis species and/or Chlorella vulgaris.

16. The GLP-2 expressing algal biomass according to any of claims 10 and 11 or obtainable by the method according to any one of claims 12 -15 for use in the treatment and/or prevention of intestinal dysbiosis, chronic inflammatory bowel disease (IBD), for the treatment of disorders deriving from irritable bowel, for the containment of the progression of osteoporosis, for the improvement of disorders deriving from bone resorption, to strengthen and improve the intestinal microbiota in human or veterinary animals.

17. The GLP-2 expressing algal biomass according to claim 16 for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders.

18. A pharmaceutical composition comprising the host cell according to any one of claims 4-9 or the biomass according to any one of claims 10-11 or the biomass obtainable from the method according to any one of claims 12 - 14 and at least one pharmacologically acceptable excipient.

19. The pharmaceutical composition according to claim 18 for use in the treatment and/or prevention of intestinal dysbiosis and inflammatory bowel disease (IBD), for the treatment of disorders deriving from irritable bowel, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, to strengthen and improve the intestinal microbiota in human or veterinary animals.

20. The pharmaceutical composition according to claim 19 for use in the treatment and/or prevention of inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders.

21. Supplement or food product or drinking product comprising the host cell according to any one of claims 4 -9 or the biomass according to any one of claims 10-11 or the biomass obtainable from the method according to any one of claims 12 - 14. Non-therapeutic use of the biomass according to any one of claims 10-11 or the biomass obtainable from the method according to any one of claims 12 - 14 or of the supplement or food product or product to drink according to claim 21 in the nutraceutical sector or as a basic ingredient in preparations of supplementsand/or as an agent for the prevention and/or treatment of intestinal dysbiosis, chronic inflammatory bowel diseases (IBD), for the treatment of disorders deriving from irritable bowel, for the containment of the progression of osteoporosis, for the improvement of disorders resulting from bone resorption, to strengthen and improve the intestinal microbiota, inflammatory bowel syndrome, Crohn's disease and ulcerative colitis and/or intestinal dysbiosis due to eating disorders and autism spectrum disorders.