ES2382625A1 - Nanoparticles for the prevention and/or treatment of diseases of the mucus membranes - Google Patents

Abstract

Nanoparticles for the prevention and/or treatment of mucosal diseases. The present invention refers to polymeric nanoparticles which comprise a polynucleotide encoding a MUC5AC protein modified so that it presents the N-terminal end of MUC5AC linked to the domain of dimerization located at the C-terminal end, or a plasmid or a genetic construction that contains it, and its use for the preparation of a medicine for the prevention and/or treatment of a disease that occurs with a mucin deficiency. It also refers to a pharmaceutical composition that comprises said nanoparticles and its use for the preparation of a medicine for the prevention and/or treatment of a disease that occurs with a mucin deficiency.

Description

Nanoparticles for the prevention and / or treatment of mucosal diseases.

The present invention is within the field of biotechnology, and more specifically of biomedicine. Specifically, it refers to nanoparticles comprising a polynucleotide encoding a modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end, or a plasmid or a genetic construct containing it, and its use for the elaboration of a medicine for the prevention and / or treatment of a disease that has a mucin deficiency.

STATE OF THE PREVIOUS TECHNIQUE

The mucous membranes of the organism are one of the most sensitive elements to both inflammatory and infectious conditions. Within these mucous membranes, one of the most important elements are the mucins. These mucins are a heterogeneous group of high molecular weight glycoproteins and are the fundamental element of the body's mucous secretions. Within this group there are described at least 17 different proteins with different characteristics of both expression and functional.

These mucins are especially relevant in the case of the ocular mucosa, where the mucins have high importance in the homeostasis of the mucous tissue, as well as in the lubrication of both corneal and conjunctival epithelia during flickering. In addition, the mucins act at the ocular level as stabilizers of the tear film preventing the desiccation of the epithelium and as a barrier against the entry of both foreign agents and pathogens.

Due to the importance of these mucins, the deficiency in them carries several ocular pathological problems. These problems fundamentally affect a set of structures present in the anterior part of the eye as a whole called "ocular surface" which is considered an established functional unit (Stern et al, 1998. Cornea. 17: 584-589). This ocular surface is a mucous tissue similar to other mucous membranes in the body such as the mucosa of the gastro-intestinal tract, the mucosa of the respiratory tract, the urinary mucosa and the vaginal mucosa.

Examples of diseases that occur with alteration in the regulation of mucin production are, for example, autoimmune diseases that occur with inflammation. In these cases it can lead, depending on the disease, an overproduction or a subproduction of the mucins characteristic of the ocular surface. This variation in the levels of the mucins causes that homeostasis produced by them does not occur and therefore results in serious pathological processes that may have an influence on visual function.

One of the diseases with the highest incidence, in relation to mucins and the ocular surface, is the so-called dry eye syndrome. This disease affects, for example, 10% of the adult population (Viso et al, 2009. Ophthalmic Epidemiol. 16: 15-21) reaching 14.6% in the population over 65 years (Schein et al, 1997. Am J Ophthalmol. 124: 723-728). This disease is strongly related to aging, as well as to sex since it mostly affects women. Thus, the prevalence of this disease can reach 40% in postmenopausal women (Schaumberg et al, 2003. Am J Ophthalmol. 136: 318-326).

Other important diseases, which do not reach the incidence of dry eye syndrome are serious allergic diseases such as atopic keratoconjunctivitis (AKC) or vernal keratoconjunctivitis (VKC), which can reach an incidence of 4.4% and 3, 8%, respectively, of the total ocular allergy patients in Japan (Uchio et al, 2008. Graefe's Arch Clin Exp Ophthalmol. 246: 291-296). In other studies, the incidence reaches 40% in the case of atopic keratoconjunctivitis and 46% in the case of vernal keratoconjunctivitis, of the total ocular allergy patients in Brazil (Belfort et al, 2000. Acta Ophthalmol Scand. 78 : 38-40). In all these diseases alterations in mucin expression levels have been seen (Argüeso et al, 2002. Invest Opththalmol Vis Sci. 43: 1004-1011; Dogru et al, 2005. Curr Eye Res. 30: 897-908). On the other hand, some pathologies of the ocular mucosa, can be produced by the continuous use of contact lenses, which can lead to subclinical inflammation and therefore to the decrease of mucin production (Corrales et al, 2009. Optom Vis Sci. 86: 1051-1058), which is a major health and social impact.

Within the mucins, one of the most clinically relevant is the mucin MUC5AC, which is specific to the goblet cells of the conjunctiva. The production of mucin by these cells is strongly decreased (up to 90% reduction) in various eye diseases such as keratoconjunctivitis sicca, Sjögren's syndrome, pemphigoid, Stevens Johnson's syndrome (Kunert et al, 2001. Invest Ophthalmol Vis Sci. 42: 2483-2489), in atopic patients with corneal ulcers (Dogru et al, 2005. Cornea 24: S18-S23; Dogru et al, 2005. Curr Eye Res. 30: 897-908) You can also be reduced as a result of the development of dry eye symptom secondary to other diseases or eye treatments (Heimann et al, 2001. Graefe's Arch Clin Exp Ophthalmol.

239: 488-495). Similarly, passive exposure to tobacco smoke produces a significant reduction in MUC5AC levels (Rummenie et a, 2008. Cytokine 43: 200-208).

At present there are no treatments that allow the functional recovery of the inflamed ocular surface in a chronic way, but the existing treatments are only useful to alleviate the symptoms of the diseases. The search for a treatment that allows the recovery of the disease is necessary, for which a good approach can be the treatment of the underlying inflammation, allowing a normalization of the ocular surface that finally allows to recover the levels of the components that are altered . Within this recovery of levels could be framed a therapy that allows the functional recovery of mucin levels.

Due to the high absenteeism caused by eye discomfort and the decrease of visual faculties, as well as the psychological damage associated with the loss of vision that these chronic problems produce, and the absence of effective treatments, the development of a therapy would be highly beneficial gene that allows a functional recovery of mucin-producing cells. This would entail a restoration of their levels and therefore a recovery of the visual diseases associated with this defect.

The development of gene therapies has been harmed by the search for very ambitious goals through which it has tried to achieve a systemic response to genetic treatments. However, a more local treatment would allow a simpler development of therapy, and with it better results through an efficient therapeutic response.

One of the biggest problems that gene therapy has faced has been the development of adequate vehicles. The approach to a systemic therapy is complex and difficult, since there are many existing barriers, and of a very different nature within an organism. However, a more local approach reduces barriers and simplifies the search for the vehicle. Even so, the need for adequate vehicles has been the biggest problem in gene therapy (Nathan Blow, 2007. Nature. 450: 1117-1120).

Currently, among the most promising vehicles for the transport of genetic material are nanoparticular systems made from biomaterials. These have great potential, although to date they have not provided the expected results. Therefore, it is necessary to develop new systems capable of harnessing their potential (Friede et al, 2005. Adv Drug Deliv Rev. 57: 325-331).

One of the most used polymers is chitosan, which is a polymer cited as essential in the formation of nanoparticles by ionic crosslinking. Chitosan nanoparticles are being questioned as there are studies that question their usefulness against simple solutions of bioactive molecules with the polymer (Dyer et al, 2002. Pharm Res. 19: 998-1008). In addition, the possible cytotoxicity of chitosan nanoparticles has also been noted (Loretz and Bernkop-Schnürch, 2007. Nanotoxicology. 1: 139-148).

There is therefore a need to develop nanoparticular systems based on biocompatible materials and reagents of low cytotoxicity that provide high control over the physical-chemical properties of the nanoparticles, which can be obtained by simple and effective procedures, and that are capable of effectively associating genetic material and to achieve an adequate biological effect with it.

DESCRIPTION OF THE INVENTION

The present invention relates to nanoparticles useful for the preparation of a medicament that allows the recovery of mucin production. Said nanoparticles are associated with a polynucleotide encoding a modified MUC5AC protein, a genetic construct or a vector containing said polynucleotide. This composition allows the recovery of mucin production, which is why it is useful for the development of a drug for the treatment of mucosal diseases associated with a mucin deficiency.

The present invention demonstrates how the inventors have developed a plasmid that includes a polynucleotide capable of encoding a mucin, which can be associated with nanoparticles. When the nanoparticular system with the associated plasmid comes into contact with human cells, the nanoparticles act as vehicles of the genetic material, without presenting cytotoxic activity, and allow the cells to produce the mucin encoded by the plasmid. In addition, the present invention demonstrates the ability of the composition to recover tear production in mice with induced dry eye, demonstrating the usefulness of said composition for the development of medicaments for the treatment of mucosal diseases that occur with mucin deficiency .

On the other hand it should be noted that the similarity between the mucins, allows a mucin to replace, at least partially, the functionality of others, in case of deficiency of any of them. Therefore, the composition of the present invention would be used for the treatment of mucosal diseases that occur with mucin deficiency.

In the present invention a plasmid has been associated that integrates a sequence encoding the modified MUC5AC protein (whose sequence is SEQ ID NO: 1), which corresponds to the C and N terminal ends of the native MUC5AC protein. This nucleotide sequence (SEQ ID NO: 2) coding for the modified mucin is substantially shorter (7.7 kilobases) than the coding sequence of the complete mucin (16 kilobases), which has the advantage of greater ease of cloning in plasmids, genetic constructs or vectors. This shorter coding sequence encodes the MUC5AC mucin that has fused the C-terminal and N-terminal ends, and that maintains the most important domains of the original protein such as the dimerization domain (present at the C-terminal end ), the polymerization domain, some of the different glycosylation points in the complete protein, and the signal peptide for its correct conduction through the secretory pathway. This protein, therefore, has the N-terminal end of the MUC5AC (amino acids 1-2,448) attached to the dimerization domain present at the C-terminal (amino acids 5,220-5,333), eliminating the central region where highly repeated sequences are found. rich in proline, threonine and serine (PTS). In the present invention it is demonstrated that said modified MUC5AC protein is useful for treating diseases that occur with mucin deficiency such as dry eye syndrome.

Therefore, a first aspect of the invention relates to polymeric nanoparticles, hereafter referred to as nanoparticles of the invention, comprising a polynucleotide encoding a modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end.

The expression "modified MUC5AC protein presenting the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end" refers to a protein presenting the N-terminal end of the MUC5AC that includes both the polymerization domain, as the signal peptide for its correct conduction by the secretory pathway, with the dimerization domain of the C-terminal end of the MUC5AC protein. An example of a modified MUC5AC protein presenting the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end is, for example, but not limited to, the sequence protein SEQ ID NO: 1, which has the amino acids 1-2,448 of the MUC5AC protein (corresponding to the N-terminal end) attached to amino acids 5,220

5,333 (corresponding to the dimerization domain of the C-terminal end). In the same way as with many proteins, such as among homologous proteins in different organisms, a polypeptide could have variations in the amino acid sequence with respect to that described by way of example without a substantial alteration in the protein. This means that this variation would not be determinant in the polypeptide neither for its structure nor for its activity. These variations are generated by substitutions, deletions or additions. Such substitutions occur between amino acids that have similar characteristics such as polarity, size or charge, and include, but are not limited to, substitutions between glutamic acid (Glu) and aspartic acid (Asp), between Lysine (Lys) and Arginine (Arg), between asparagine (Asn) and glutamine (Gln), between serine (Ser) and threonine (Thr), and between the amino acids that make up the alanine (Ala), leucine (Leu), valine (Val) and isoleucine group (Ile). The variations can be artificially generated variations such as by mutagenesis or direct synthesis. These variations do not cause changes in the characteristics or essential properties of the polypeptide, so said variants maintain their same biological activity. On the other hand, additions, or deletions of few amino acids at the ends of the sequences selected to give rise to the protein of the example, would not cause substantial modifications in size or functionality thereof, so they would also be found within the expression "Modified MUC5AC protein presenting the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end." Therefore, in a preferred embodiment of this aspect of the invention, the modified MUC5AC protein having the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end has at least 90%, preferably 95 %, more preferably 98% identity of with respect to SEQ ID NO: 1. In a more preferred embodiment, the modified MUC5AC protein presenting the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end is SEQ ID NO: 1.

On the other hand, a polynucleotide that codes for a modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end would be any polynucleotide that codes for a protein or polypeptide included under the designation " modified MUC5AC protein presenting the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end. ” Due to the degeneracy of the genetic code, in which several nucleotide triplets give rise to the same amino acid, there are several nucleotide sequences that give rise to the same amino acid sequence. An example of a polynucleotide encoding a modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end would be, for example, but not limited to, the sequence polynucleotide SEQ ID NO: 2. The terms "nucleotide sequence", "nucleotide sequence", "nucleic acid", "oligonucleotide" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length that may or may not be chemical or biochemically modified. They therefore refer to any polyiribonucleotide or polydeoxyribonucleotide, both single stranded and double stranded. The polynucleotide of the invention can be found in nature and obtained by isolation by methods known in the state of the art, or obtained artificially by conventional cloning and selection methods, or by sequencing. The polynucleotide, in addition to the coding sequence, can carry other elements such as, but not limited to, introns, non-coding sequences at the 5 'or 3' ends, ribosome binding sites, or stabilizing sequences. These polynucleotides can additionally also include coding sequences for additional amino acids that may be useful, for example, but not limited to increasing the stability of the peptide generated from it or allowing a better purification thereof.

In another preferred embodiment of this aspect of the invention, the polynucleotide has at least 90%, preferably 95%, more preferably 98% identity with respect to SEQ ID NO: 2. In a more preferred embodiment the polynucleotide is SEQ ID NO: 2.

The terms "peptide", "polypeptide", "protein" or "amino acid sequence" are used interchangeably in the present invention, and refer to a polymeric form of amino acids of any length, which may or may not be, chemically or biochemically modified.

The term "identity", as used herein, refers to the proportion of identical nucleotides or amino acids between two nucleotide or amino acid sequences that are compared. Sequence comparison methods are known in the state of the art and include, but are not limited to, the GAG program, including GAP (Devereux et al. 1984, Nucleic Acids Research 12: 287 Genetics Computer Group University of Wisconsin, Madison , (WI); BLAST or BLASTN, and FASTA (Altschul et al. 1999, J. Mol. Biol. 215: 403-410) Additionally, the Smith Waterman algorithm should be used to determine the degree of identity of two sequences.

The polynucleotide encoding the modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end, may be included in gene constructs or vectors. Therefore, a preferred embodiment of the first aspect of the invention relates to nanoparticles comprising a polynucleotide encoding a modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end, where the polynucleotide It is included in a genetic construct or vector. In a more preferred embodiment of the first aspect of the invention, the vector in which the polynucleotide is included is a plasmid.

The genetic construct can include control sequences operatively linked to the polynucleotide. The term "control sequence", as used herein, refers to nucleotide sequences that are necessary to effect the expression of the sequences to which they are linked. The term "control sequences" is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous. Examples of control sequences are, for example, but not limited to, promoters, transcription initiation signals, transcription termination signals, polyadenylation signals or transcriptional activators.

The term "operatively linked", as used herein, refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended manner. A control sequence "operatively linked" to a polynucleotide is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences.

As used herein, the term "promoter" refers to a region of DNA, generally "upstream" or "upstream" of the transcription start point, which is capable of initiating transcription in a cell. This term includes, for example, but not limited to, constitutive promoters, cell or tissue specific promoters or inducible or repressible promoters. Control sequences depend on the origin of the cell in which the nucleic acid is to be expressed. Examples of prokaryotic promoters include, for example, but not limited to, promoters of the trp, recA, lacZ, lacI, tet, gal, trc, or tac genes of E. coli, or the promoter of the a-amylase gene of B. subtilis For the expression of a nucleic acid in a prokaryotic cell, the presence of an upstream ribosomal binding site of the coding sequence is also necessary. Appropriate control sequences for the expression of a polynucleotide in eukaryotic cells are known in the state of the art. Both the polynucleotide of the invention and the genetic construct of the invention comprising the polynucleotide can be introduced, for example, into a cloning vector or an expression vector to allow replication or expression. Preferably, said vector is an appropriate vector for expression of the peptide of the invention.

The term "cloning vector", as used in the present description, refers to a DNA molecule in which another DNA fragment can be integrated, without losing the capacity for self-replication. Examples of expression vectors are, but are not limited to, plasmids, cosmids, DNA phages or artificial yeast chromosomes. The term "expression vector", as used in the present invention, refers to a cloning vector suitable for expressing a nucleic acid that has been cloned therein after being introduced into a host cell. Said nucleic acid is generally operatively linked to control sequences.

The term "expression" refers to the process by which a polypeptide is synthesized from a polynucleotide. It includes transcription of the polynucleotide into a messenger RNA (mRNA) and the translation of said mRNA into a protein or polypeptide. Expression can take place in a host cell, but also by any process of protein expression in vivo.

The nanoparticles can be formed by an ionic interaction mechanism that causes the joint precipitation of the components in the form of nanoclusters as a result of the addition of a cross-linking agent that has an electric charge. The ionic interaction procedure has advantages that do not require the use of chemical elements other than the components of the nanoparticles themselves that can cause toxicity or incompatibility of the system. The use of an ionic crosslinking agent allows the cross-linking of the ionic polymer giving rise to the nanoparticles. This procedure is a fast and reliable, economical and easily scalable process for industrial production.

Therefore, in another preferred embodiment of the first aspect of the invention, the nanoparticles are formed by:

i) at least one polymer with an electric charge, and

ii) an ionic crosslinking agent.

Examples of polymers useful for the formation of nanoparticles are, for example, but not limited to, hyaluronic acid or salts thereof, colominic acid or derivatives, glucomannan or derivatives, chondroitin sulfate, queratane sulfate, dextran sulfate, dermatan sulfate, heparin, carrageenan, gellan, albumin, collagen or gelatin. These polymers also include polymers on which modifications have been made such as enzymatic fragmentation, chemical fragmentation or derivatization by, for example, but not limited to, the anionization or cationization of the polymer prior to the preparation of the particles. These polymers can have a natural, semi-synthetic or synthetic origin. It is interesting that the polymers used are natural or semi-synthetic polymers since this allows to minimize the possible rejection of the treated individuals to the nanoparticles. Therefore, in a more preferred embodiment of this aspect of the invention, the polymer of (i) is a natural or semi-synthetic polymer.

Within the polymers of (i), one of the ones that provides the best results is cationized gelatin. Therefore, in an even more preferred embodiment of this aspect of the invention, the naturally occurring polymer with an electric charge (i) is cationized gelatin.

Examples of ionic crosslinking agents are, for example, but not limited to, phosphates, sulfates, citrates or salts thereof, spermine or salts thereof, or spermidine or salts thereof. These agents have different electrical charges (phosphates, sulfates and citrates anionic charge; spermine and spermidine cationic charge). Among these, one of the best characterized and the best result is tripolyphosphates. Therefore, in another more preferred embodiment of this aspect of the invention, the ionic crosslinking agent (ii) is a tripolyphosphate. In an even more preferred embodiment, the ionic crosslinking agent is sodium tripolyphosphate.

Optionally, the nanoparticles may comprise an additional element, which is an ionic polymer of electric charge opposite to the first polymer of the nanoparticles. The addition of this polymer allows a better ionic gelation, and allows to modify the characteristics of the nanoparticles obtained both in size, as in structural characteristics and surface electric charge. Therefore, in a more preferred embodiment of the first aspect of the invention, the nanoparticles are further formed by an element (iii) which is at least one polymer of charge opposite to the polymer (i).

Examples of polymers may be, for example, but not limited to, hyaluronic acid or salts thereof, colominic acid or derivatives, glucomannan or derivatives, chondroitin sulfate, queratane sulfate, dextran sulfate, dermatan sulfate, heparin, carrageenan, gelane, albumin, collagen or gelatin. In an even more preferred embodiment the polymer (iii) of opposite charge to the polymer (i) is dextran sulfate or chondroitin sulfate.

Due to the formation of the nanoparticles by ionic crosslinking, the polynucleotide, either independently or included in a vector or a gene construct, can be retained inside them by purely physical entrapment, due to the difference in its size and that of the mesh formed in said crosslinking process. On the other hand, due to the ionic character of the polymers used, either in (i) or in (iii), the polynucleotide can also be attached to the nanoparticles as a result of an electrostatic interaction between it and said polymers.

In the present invention, "nanoparticle" is understood as a stable system, with homogeneous reproducible and modulable characteristics, perfectly distinguishable from self-assembled systems, which are formed as a consequence of a controlled process of ionotropic cross-linking of the polymer provided with the same constituent charge mediated by ionic crosslinking agents. The electrostatic interaction that results between the different components of the nanoparticles in the cross-linking process generates characteristic physical entities, which are independent and observable, whose average size is less than 1 μm.

"Average size" in the present invention is understood as the average diameter of the nanoparticles, which can be measured using standard procedures known to a person skilled in the art, such as, but not limited to, the one used in the examples herein. invention. The nanoparticles of the invention have an average size between 1 and 999 nm, preferably between 50 and 600 nm, and more preferably between 100 and 400 nm. This average particle size is influenced by their composition, as well as by the environmental conditions in which they are formed.

The nanoparticles can have different surface electric charges (potential Z) depending on their composition, and the proportions of each of the elements. This charge may have positive, negative or 0 values, preferably, the electrical charge of the nanoparticles is between +50 mV and -50mV, more preferably between +45 and 0mV, and even more preferably between +40 and + 10mV. The measurement of the electric charge can be carried out by methods known in the state of the art, such as that used in the examples of the present invention.

The nanoparticles, once they have formed and present the associated polynucleotide, can undergo a lyophilization process to allow their preservation during storage while maintaining their original characteristics and allowing the handling of smaller volumes. This lyophilization may slightly increase the crosslinking of the particles. Freeze-drying requires the prior addition of molecules that act as cryoprotectants or lioprotectors such as, but not limited to, glycols, or sugars such as glucose, sucrose or trehalose at a concentration of between 1 and 5%. Therefore, in another preferred embodiment, the nanoparticles of the invention are in lyophilized form.

Another aspect of the invention relates to the use of the nanoparticles of the invention for the preparation of a medicament or alternatively to the nanoparticles of the invention for use as a medicament.

As demonstrated in the examples of the present invention, the nanoparticles of the invention have utility in recovering mucin production by body cells. Therefore, another aspect of the invention relates to the use of the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, or alternatively to the nanoparticles. of the invention for use as a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency.

By "mucosal disease with a mucin deficiency" is understood in the present invention all that pathology that is produced by, or associated with a total or partial decrease in the production of mucins in the mucous membranes. These diseases include, for example, but not limited to, diseases that affect the ocular mucosa and / or other mucous membranes of the body, such as limbic insufficiency syndrome, neurotrophic keratitis, herpetic keratitis, alkali burn, burn thermal, radiation burn, Steven-Johnson syndrome, toxic epidermal necrosis, pemphigus, mucous membrane pemphigoid, eye allergies, vernal keratoconjunctivitis, atopic keratoconjunctivitis, sicca keratoconjunctivitis or dry eye syndrome, Infectious, immune and irritative-toxic-drug conjunctivitis, Primary Sjögren's Syndrome

or secondary to other autoimmune diseases (such as scleroderma, mesenchymopathy, lupus, rheumatoid arthritis, myopathies or primary viral cirrhosis), atopy, graft-versus-host disease, salivary glandular hypofunction and xerostomia, and ichthyosis .

In the examples of the present invention it is demonstrated that in cells present in the ocular mucosa, the use of nanoparticles comprising a plasmid carrying a polynucleotide encoding the modified MUC5AC protein presenting the N-terminal end of MUC5AC bound to the domain dimerization located at the Cterminal end, allows the expression of this mucin. Therefore, a preferred embodiment of this aspect of the invention relates to the use of the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, where the mucosa is the ocular mucosa, or alternatively to the nanoparticles for use as a medicine for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, where the mucosa is the ocular mucosa.

In the present invention it is demonstrated that the nanoparticles of the invention allow the recovery of tear production in a disease that is deficient in mucins, and more specifically with a deficiency in the mucin MUC5AC. Therefore, another preferred embodiment relates to the use of the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a mucosal disease that occurs with a mucin deficiency, where the mucin is MUC5AC, or alternatively to the nanoparticles for use as a medicine for the prevention and / or treatment of a mucosal disease that presents with a mucin deficiency, where the mucin is MUC5AC. In a more preferred embodiment of the invention it refers to the use of the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a mucosal disease that is present with a mucin deficiency, where the mucosa is The ocular mucosa and the mucin is MUC5AC, or alternatively to the nanoparticles of the invention for use as a medicament for the prevention and / or treatment of a disease of a mucosa that occurs with a mucin deficiency, where the mucosa is the mucosa eyepiece and the mucin is MUC5AC.

Another preferred embodiment relates to the use of the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency where the disease of a mucosa that has a Mucin deficiency is selected from the list comprising: limbic insufficiency syndrome, neurotrophic keratitis, herpetic keratitis, alkaline burn, thermal burn, radiation burn, Steven-Johnson syndrome, toxic epidermal necrosis, pemphigus, pemphigoid of mucous membranes , eye allergies, vernal keratoconjunctivitis, atopic keratoconjunctivitis, sicca keratoconjunctivitis or dry eye syndrome, infectious, immune and irritative-toxic-drug conjunctivitis, Sjögren's syndrome primary or secondary to other autoimmune diseases (such as scleroderma, mesenquuspathy, lupus disease rheumatoid arthritis, myopathies or cirrus is viral primary), atopy, graft-versus-host disease, salivary glandular hypofunction, xerostomia, or ichthyosis, or alternatively to the nanoparticles of the invention for use as a medicament for the prevention and / or treatment of a mucosal disease that It has a mucin deficiency, where the disease of a mucosa that has a mucin deficiency is selected from the list comprising: limbic insufficiency syndrome, neurotrophic keratitis, herpetic keratitis, alkali burn, thermal burn, radiation burn, Steven-Johnson syndrome, toxic epidermal necrosis, pemphigus, mucous membrane pemphigoid, eye allergies, vernal keratoconjunctivitis, atopic keratoconjunctivitis, keratoconjunctivitis sicca or dry eye syndrome, infectious conjunctivitis, immune and irritative secondary-syndromes, primary toxicity syndrome to other autoimmune diseases (such as scleroderma , mesenchymopathy, lupus, rheumatoid arthritis, myopathies or primary viral cirrhosis), atopy, graft-versus host disease, salivary glandular hypofunction, xerostomia, or ichthyosis. A more preferred embodiment of this aspect of the invention relates to the use of the nanoparticles of the invention for the preparation of a medicament for the prevention and / or treatment of a mucosal disease that occurs with a mucin deficiency where the disease of a mucosa that has a mucin deficiency is dry eye syndrome, or alternatively to the nanoparticles of the invention for use as a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency , where the disease of a mucosa that has a mucin deficiency is dry eye syndrome.

Another aspect of the invention relates to a pharmaceutical composition, hereinafter pharmaceutical composition of the invention, comprising the nanoparticles of the invention. In a preferred embodiment of this aspect of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In another more preferred embodiment of this aspect of the invention, the pharmaceutical composition further comprises another active ingredient. In an even more preferred embodiment of this aspect of the invention, the pharmaceutical composition further comprises another active ingredient and a pharmaceutically acceptable carrier.

As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity, a metabolic effect, an immunological effect or another different effect on the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of the human being or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.

The compositions of the present invention can be formulated for administration to an animal, and more preferably to a mammal, including humans, in a variety of ways known in the state of the art. Thus, they can be, without limitation, in aqueous or non-aqueous solutions, in emulsions or in suspensions. Examples of non-aqueous solutions are, for example, but not limited to, propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, or injectable organic esters, such as ethyl oleate. Examples of aqueous solutions are, for example, but not limited to, water, alcoholic solutions in water, or saline media. Aqueous solutions may be buffered or not, and may have additional active or inactive components. Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, or the like, or nutrients, including glucose, dextrose, vitamins and minerals. Alternatively, the compositions can be prepared for administration in solid form. The compositions may be combined with various inert carriers or excipients, including but not limited to: binders, such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients, such as starch or lactose; dispersing agents, such as alginic acid or corn starch; lubricants, such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or flavoring agents, such as peppermint or methyl salicylate.

The composition of the invention is especially advantageous for constituting a liquid composition or any composition comprising the nanoparticles of the invention and which is in the form of a gel, cream, ointment, or balm for topical use.

Such compositions and / or their formulations can be administered to an animal, including a mammal and, therefore, to the human being, in a variety of ways, including, but not limited to, oral, oral, sublingual, ocular, nasal, pulmonary, otic, intrauterine, parenteral, intraperitoneal, intravenous, intradermal, intrastromal, intraarticular, intrasinovial, intrathecal, intralesional, intraarterial, intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intracapsular, topical, through transdermal patches, rectal, vaginal, by administering a suppository, percutaneous, nasal spray, surgical implant, internal surgical paint, infusion pump or catheter. The nanoparticles of the invention, due to their usefulness in the mucous membranes, have an advantageous administration by different mucosal routes such as, but not limited to, ocular mucosa, mucosa of the entire gastro-intestinal tract including the buccal or oral, mucosa of the respiratory tract. , including the nasal and bronchial mucosa, the urinary mucosa and the vaginal mucosa. Therefore, a preferred embodiment of this aspect of the invention relates to a pharmaceutical composition comprising the nanoparticles of the invention for administration via ocular mucosa, mucosa of the entire gastro-intestinal tract, mucosa of the respiratory tract, urinary mucosa and the vaginal mucosa.

The nanoparticles can undergo a lyophilization process to allow their preservation during storage while maintaining their original characteristics and allowing the handling of smaller volumes. Therefore, in another preferred embodiment the pharmaceutical composition is in lyophilized form.

The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as age, weight, sex or tolerance, of the mammal. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of the pharmaceutical composition of the invention that produces the desired effect and, in general, will be determined, among other causes, by the characteristics of said pharmaceutical composition and the therapeutic effect to be achieved.

The "pharmaceutically acceptable vehicles" that can be used in the compositions are the vehicles known in the state of the art.

Another aspect of the invention relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament or alternatively to the pharmaceutical composition of the invention for use as a medicament.

Another aspect of the invention relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, or alternatively the pharmaceutical composition. of the invention for use as a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency. A preferred embodiment of this aspect of the invention relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that occurs with a mucin deficiency, wherein the Mucosa is the ocular mucosa, or alternatively to the pharmaceutical composition for use as a medicine for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, where the mucosa is the ocular mucosa.

Another preferred embodiment of this aspect of the invention relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that occurs with a mucin deficiency, wherein the Mucin is MUC5AC, or alternatively to the pharmaceutical composition for use as a medicine for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, where the mucin is MUC5AC. In a more preferred embodiment of the invention, it refers to the use of the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a mucosal disease that occurs with a mucin deficiency, where the mucosa it is the ocular mucosa and the mucin is MUC5AC, or alternatively to the pharmaceutical composition of the invention for use as a medicine for the prevention and / or treatment of a disease of a mucosa that is present with a mucin deficiency, where the mucosa is The ocular mucosa and the mucin is MUC5AC.

Another preferred embodiment relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency where the disease of a mucosa that has a a mucin deficiency is selected from the list comprising: limbic insufficiency syndrome, neurotrophic keratitis, herpetic keratitis, alkali burn, thermal burn, radiation burn, Steven-Johnson syndrome, toxic epidermal necrosis, pemphigus, membrane pemphigoid mucous membranes, eye allergies, vernal keratoconjunctivitis, atopic keratoconjunctivitis, keratoconjunctivitis sicca or dry eye syndrome, infectious, immune and irritant-toxicotoxicomedication, Sjögren's syndrome, primary or secondary to other autoimmune diseases (such as scleroderma, lcleroderma, lcleroderma rheumatoid arthritis, myopathies or primary viral cirrhosis), atopy, graft-versus-host disease, salivary glandular hypofunction, xerostomia, or ichthyosis, or alternatively to the pharmaceutical composition of the invention for use as a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency, where the disease of a mucosa that has a mucin deficiency is selected from the list comprising: limbic insufficiency syndrome, neurotrophic keratitis, herpetic keratitis, alkali burn, thermal burn, burn by radiation, Steven-Johnson syndrome, toxic epidermal necrosis, pemphigus, mucous membrane pemphigoid, eye allergies, vernal keratoconjunctivitis, atopic keratoconjunctivitis, sicca keratoconjunctivitis or dry eye syndrome, infectious conjunctivitis, immune-irritating-toxic-syndrome Sjögren primary or secondary to other autoimmune diseases (c such as scleroderma, mesenchymopathy, lupus, rheumatoid arthritis, myopathies or primary viral cirrhosis), atopy, graft-versus host disease, salivary glandular hypofunction, xerostomia, or ichthyosis. A more preferred embodiment of this aspect of the invention relates to the use of the pharmaceutical composition of the invention for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that occurs with a mucin deficiency where the A mucosal disease that occurs with a mucin deficiency is dry eye syndrome, or alternatively to the pharmaceutical composition of the invention for use as a medicament for the prevention and / or treatment of a mucosal disease that courses with a deficiency of mucin, where the disease of a mucosa that has a mucin deficiency is dry eye syndrome.

Another aspect of the present invention relates to a process for the preparation of nanoparticles comprising:

1) prepare an aqueous solution of at least one ionic polymer,

2) prepare a solution of an ionic crosslinker,

3) Mix by stirring the solutions of (1) and (2) which results in the formation of the nanoparticles.

This production method is a fast, economical, reproducible and scalable method, which allows its industrial implementation. In addition, the absence of chemical elements other than the constituents of the nanoparticles, as well as the use of mild conditions for production, allows greater safety of the resulting particles. In order to carry out the association of the DNA molecule, it is dissolved in the dissolution of the polymer or the crosslinking agent, depending on the electrical charge of each of these components. The DNA is incorporated into the solution that has a negative charge.

Optionally, a polymer with a charge opposite to that of step (1) could be incorporated, either in step (3), or subsequently after forming the nanoparticles.

The incorporation of the polymers is carried out by an aqueous solution thereof at a concentration of between 0.1 and 6 mg / mL, preferably between 0.1 and 5 mg / mL. On the other hand, the crosslinking agent is incorporated by an aqueous solution thereof at a concentration of between 0.0625 and 1.5 mg / mL, preferably between 0.25 and 1.5mg / mL.

Through this procedure the formation of the nanoparticles is carried out, allowing the control of both the cross-linking process and the size and surface charge of the resulting particles. In addition the reproducibility of the resulting particles is allowed.

After the formation of the nanoparticles, they can undergo a lyophilization process to allow their preservation during storage while maintaining their original characteristics and allowing the handling of smaller volumes. This lyophilization can increase the crosslinking of the particles. For lyophilization, the prior addition of molecules that act as cryoprotectants or lioprotectors is necessary, for example, although sugars such as glucose, sucrose or trehalose are not limited to a concentration of between 1 and 5%.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. Scheme of plasmids A: pIRES2-AcGFP1-MUC5AC-280; and B: pMUCIN5AC-29. MAO151 and MAO153: primers; PMCMV: cytomegalovirus promoter; pUC ori: origin of pUC replication; HSV TK poly A: thymidine kinase polyadenylation signal from herpex simplex virus; KanR / NeoR: resistance to kanamycin and neomycin; SV40 ori / p: SV40 origin of replication; f1 ori: origin of replication f1; AcGFP1: Aequorea coerulescens green fluorescent protein; IRES: internal ribosome entry site; MUC5AC ORF: reading frame of MUC5AC; SacI: cut-off point of the SacI restriction enzyme; EcoRV: cut-off point of the EcoRV restriction enzyme.

Figure 2. Image of agarose gel. Results of the digestion of 0.5 microliters of the preparation of pMUCIN5AC-29 with the SacI and EcoRV enzymes and their respective negative controls (-) without digesting with the enzyme.

Figure 3. Morphological characterization of nanoparticles by transmission electron microscopy. (A) GCspm137 nanoparticles: TPP; (B) GCspm137: CS: TPP nanoparticles; (C) GCspm137 nanoparticles: DS: TPP.

Figure 4. Image of the 1% agarose electrophoresis gel loaded with: (A) Free plasmid pMUC5AC-280; (B) GCspm137: TPP nanoparticles, (C) GCspm137: CS: TPP nanoparticles; (D) GCspm nanoparticles: DS: TPP.

Figure 5. Cell viability values obtained using the XTT test in A: human cornea cells (HCE) or B: conjunctiva (IOBA-NHC). (C) Control not treated; (C +) positive control of cell death (treated with 1% benzalkonium chloride solution); (F1) GCspm137 nanoparticles: TPP; (F2) GCspm137 nanoparticles: CS: TPP; (F3) GCspm137 nanoparticles: DS: TPP.

Figure 6. Expression of MUC5AC relative to the control, at the mRNA level in 72h post-transfection cells in A: human cornea cells (HCE) or B: human conjunctiva cells (IOBA-NHC). (Control) Control not treated; (JetPEI + pEGFP) Negative transfection control; (JetPEI + pMUC5AC) Positive transfection control; (F1) GCspm137 nanoparticles: TPP; (F2) GCspm137 nanoparticles: CS: TPP; (F3) GCspm137 nanoparticles: DS: TPP ”

Figure 7. Expression of the MUC5AC protein relative to the control in 72h post-transfection cells in A: human cornea cells (HCE) or B: human conjunctiva cells (IOBA-NHC). Untreated Control (Control); Negative transfection control (JetPEI + pEGFP); Positive transfection control (JetPEI + pMUC5AC); GCspm137 nanoparticles: TPP (F1); GCspm137 nanoparticles: CS: TPP (F2); GCspm137 nanoparticles: DS: TPP (F3).

Figure 8. Production of tear in animals not subjected to induction of dry and untreated eye (Control), in animals with induced dry eye and untreated (Dry Eye Control), in animals with induced dry eye and treated with free pMUC5AC (Eye Dry + free pMUC5AC), in animals with induced dry eye and treated with white nanoparticles (without associated plasmid) (Dry Eye + white NPs), in animals with induced dry eye and treated with nanoparticles associating pMUC5AC (Dry Eye + pMUC5AC-NPs) and in animals with induced dry eye and treated with commercially available formulation for the treatment of dry eye (Dry Eye + Restasis®). Baseline tear production values are shown for each treatment (zero time; first column); those obtained after induction of dry eye (day 5; second column) and those obtained at the end of treatment (day 10; third column). * significant difference with respect to baseline values (p <0.05). # significant difference with respect to values in animals with induced dry eye (p <0.05) (n = 8, divided into two independent groups of 4 animals each).

EXAMPLES

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

Materials and methods.

As a common procedure to the examples detailed below, the nanoparticles have been characterized in terms of size, zeta potential (or surface charge) and encapsulation efficiency.

During the presentation of some of the following examples, reference is made to results obtained by the following techniques:

Gelatin with spermine has been synthesized according to the method described by Seki et al, (2006. Journal of Pharmaceutical Sciences. 95,1393-1401). For this, a solution of 1% w / v gelatin (100 mg of 10 ml of 0.1 M phosphate buffer, pH 5.3) was prepared, to which 1620 mg of spermine and 267.5 mg of N were added. - (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC). The pH (pH = 4.5) was then adjusted to a pH value = 5 with NaOH, and the whole was allowed to react for 18 h in a thermostated bath, at 37 ± 1 ° C. Subsequently, the whole was dialyzed and lyophilized, thus obtaining the cationized gelatin with spermine that was used in the experiments set out in the corresponding examples.

The determination of the isoionic or isoelectric point is to measure the concentration of hydrogen ions of a solution of the polymer that has been demineralized by contact with ion exchange resins (Commission on Methods for Testing Photographic Gelatin. PAGI METHOD 10th Ed. 2006). The process consists in placing a 1% solution of the cationized protein in contact with a mixture of cationic acid and anionic basic resins in 1: 2 proportions. Previously, these exchange resins were treated by washing with milliQ water at 35 ° C and then contacted with the modified protein solution under stirring at 35 ° C for 30 minutes. The solution is then decanted and the pH is measured at 35 ° C. The value obtained indicates the isoionic or isoelectric point of the protein. As control commercial jellies of isoelectric points 9 and 5 have been used.

The particle size has been determined by the technique of photonic correlation spectroscopy (PCS) and using, for this, a Zeta Sizer (Zeta Sizer, Nano series, Nano-ZS, Malvern Instruments, UK) obtaining the average size of the population and the polydispersion index thereof. For this, the samples were conveniently diluted in milli-Q water.

The Zeta particle potential has been determined by the laser dispersion anemometry (LDA) technique and using a Zeta Sizer (Zeta Sizer, Nano series, Nano-ZS, Malvern Instruments, UK). For this, the samples were conveniently diluted in a millimolar solution of KCl.

For the evaluation of the morphology of the nanoparticles, transmission electron microscopy (TEM) has been used in a JEOL JEM-1011 (Jeol, Japan) using 1% phosphotungstic acid as a contrast agent.

The efficiency of association of genetic material with nanoparticles has been determined by the agarose gel electrophoresis technique. For this, a 1% agarose gel was prepared in TAE buffer (Tris-Acetate-EDTA, 40mM Tris, 1% acetic acid, 1mM EDTA) pH 8 with ethidium bromide (10 mg / mL, 5 μL) and used a loading buffer and migration marker composed of glycerin (30%). A potential difference of 100 V was applied for 120 minutes and free genetic material was used as a control.

For the quantitative analysis of the association of the plasmid to the nanostructures, the Picogreen® method (Invitrogen, ES) was used, in accordance with the manufacturer's instructions, in supernatants of formulations previously centrifuged in Microfuge 22R centrifuge (Beckman Coultern, US ) at 12,000 rpm at 4 ° C for 30 minutes.

Two cell lines were used for cell culture experiments: human corneal HIT (Human Corneal Epithelium) and human IOBA-NHC (Normal Human Conjunctive) conjunctiva cells. HCE cells were cultured in DMEM / F-12 medium supplemented with 15% fetal bovine serum (FBS), 0.5% DMSO, cholera toxin (1 mg / mL), endothelial growth factor (EGF; 100 μg / mL), insulin (4 mg / mL), streptomycin (0.1 mg / mL) and penicillin (100 U / mL) (Invitrogen, ES). IOBA-NHC cells were cultured in DMEM / F-12 medium supplemented with 10% fetal bovine serum (FBS), cholera toxin (1 mg / mL), endothelial growth factor (EGF; 2 ng / mL), insulin ( 10 mg / mL), hydrocortisone (50 μg / mL), fungizone (250 μg / mL), streptomycin / penicillin (5000 U / mL) (Invitrogen, ES). The cells were maintained at 37 ° C under a humidified atmosphere of 5% CO2.

For the cell viability assays, the XTT test was used, which is based on the conversion of tetrazole salt into a soluble formazan by viable cells (n = 3, independent experiments performed in octuplicates). The absorbance measurement was carried out at 450 nm with the reference wavelength of 620 nm, in a SpectraMax M5 plate multilector (Molecular Devices, US), expressing the results in% of viability relative to the control.

For the relative quantification of the mRNA expression corresponding to MUC5AC-, RT-PCR was used, using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as a reference. The mRNA was extracted and purified with RNeasy Mini Kit (Qiagen, Germany) and quantified with Quant-it RNA Assay Kit (Invitrogen, US) according to the manufacturer's instructions. The cDNA was constructed with SuperScript® Villo cDNA Synthesis Kit (Invitrogen, US) following the manufacturer's instructions. The parameters of the RT-PCR were: an initial denaturation step at 95 ° C for 10 minutes; a second denaturation and extension step, with 40 cycles, from 15 seconds at 95 ° C and 60 seconds at 60 ° C, using 7500 RT-PCR System (Applied Biosystems, US).

The MUC5AC was quantified by ELISA. Analog B of the MUC5AC (AnaSpec, US) was used as standard. For this purpose, 50μL of cell lysates were incubated in 96-well plates with 50μL of 0.05M bicarbonate buffer pH 9.6 at 37 ° C over night. The plates were washed three times with PBS and blocked with 2% bovine serum albumin (BSA fraction V) (Sigma-Aldrich, Spain) for 1 h at room temperature. The plates were again washed three times with PBS and incubated with 100μL of anti-MUC5AC mouse antibody (Chemicon, US) (1: 500) which was diluted in PBS containing 0.05% Tween 20. After one hour, the wells were washed three times with PBS and then 100 µL of anti-mouse donkey IgG conjugated with dilute peroxidase (1: 10,000) in PBS containing 0.05% Tween 20 and 0.1% of BSA After one hour, the plates were washed three times with PBS. The color reaction was developed with the 3,3', 5,5'-tetramethylbenzidine peroxide reagent (TMB), as described by the corresponding manufacturer (Invitrogen, ES). The absorbance was read at 450 nm in SpectraMax M5 plate reader (Molecular Devices, US). The values obtained were normalized with respect to the total amount of protein and the results were expressed as mucin production relative to the control.

The sequence of the plasmid construct encoding MUC5AC was verified by sequencing.

The identification of the mucin encoding plasmid exogenously administered, alone or associated with the nanoparticles, inside human cells of the ocular surface was done by fluorescence microscopy, evaluating the expression of the green GFP fluorescent protein. The assessment of a possible toxic effect on human cells as a result of this administration was carried out by means of a standard toxicity measurement test (XTT).

The measurement of the amount of mucin produced in the cells as a result of the administration of the invention was done by two complementary techniques: real-time RT-PCR and an ELISA test.

Reagents:

The following polymers, as used in the examples, were purchased from different commercial houses: gelatin (Nitta Gelatin Canada Inc., Canada), chondroitin sulfate (Calbiochem, US), dextran sulfate (Sigma-Aldrich, Spain) . The pEGFP DNA plasmid was purchased from Elim Biopharmaceuticals (US). The pIRES2-AcGFP1-Mucin-5AC-280 DNA plasmid was synthesized in Biomedal (Spain). Sodium tripolyphosphate and sodium citrate were purchased from Sigma Aldrich (Spain).

Example 1. Development of a plasmid DNA encoding a mucin and an easily identifiable protein.

A DNA plasmid encoding a mucin, specifically the MUC5AC mucin, and an easily identifiable protein, specifically the green fluorescent GFP protein, was developed. This took place from synthetic oligonucleotides, of the DNA sequence containing the coding region of a sequence polypeptide derived from the human MUC5AC protein. The DNA sequence, optimized for expression in mammalian cells, was cloned into a bacterial cloning vector, sequenced and transferred to the expression vector pIRES2-AcGFP1, which already contains the sequence encoding GFP (Clontech).

Specifically, from the designed ORF (Open Reading Frame) designed, a sequence of 7660 base pairs was synthesized, which also incorporated a Kozak sequence (GCCACCATG), two additional stop codons (TAATAG) and targets for the SacI restriction enzyme (GAGCTC) at both ends. This sequence was inserted into the E. coli pUC57 cloning vector, giving rise to the 10437 base pair plasmid pUC57-MUC5AC (SEQ ID NO: 3). Finally, once the region of pUC57-MUC5AC corresponding to MUC5AC was sequenced, the 7754 base pair SacI fragment containing it was cleaved and it was cloned, in the appropriate orientation, into the vector linearized with SacI pIRES2-AcGFP1, obtaining the plasmid pIRES2-AcGFP1-Mucin-5AC-280 (SEQ ID NO: 4). The sequence around the insertion points on the SacI targets was verified by sequencing with the primers:

MAO151 5´-GTAGGCGTGTACGGTGGGAGG-3´ (SEQ ID NO: 5).

MAO153 5´-CCTTATTCCAAGCGGCTTCGGCC-3´ (SEQ ID NO: 6).

Plasmid pIRES2-AcGFP1-Mucin-5AC-280, was obtained from a culture of a transformant of strain DH5 alpha. The DNA was extracted and purified using Sigma's "GenElute ™ HP Plasmid Megaprep" kit, and finally lyophilized. Figure 1A shows a scheme of said plasmid.

The plasmid obtained, pIRES2-AcGFP1-Mucin-5AC-280, was tested by transfecting human corneal epithelium (HCE) cells (Araki-Sasaki et al, 1995. Invest Ophthalmol Vis Sci. 36: 614– 621) and conjunctiva (IOBA-NHC) (Diebold Y et al, 2003. Invest Ophthalmol Vis Sci. 44: 4263-4274) using the naked plasmid and associated with standard transfection agents: Lipectamine ™ 2000 (Invitrogen) and Jet -PEI ™ -RGD (Polyplustransfection). For this, 80,000 cells / well were seeded in 24-well plates and 24 hours after incubation with the plasmid complexes (1μg / well) - transfection agent for 4 hours, after which complete fresh medium was added. At 72 hours after transfection, the cell viability of the transfected cultures was studied using the XTT cell toxicity test. Transfection efficiency was analyzed by fluorescence microscopy, observing the expression of the green fluorescent protein. The production of MUC5AC was studied by ELISA and RT-PCR in real time.

Both HCE and IOBA-NHC cells are successfully transfected with the plasmid pIRES2-AcGFP1-Mucin-5AC-280, with green fluorescent cells being observed, corresponding to GFP expression.

MUC5AC expression is detected in transfected HCE and IOBA-NHC cells, by immunofluorescence and Western blotting. This expression is greater in corneal epithelial cells than conjunctival epithelium.

The amount of MUC5AC detected by ELISA in transfected HCE cells is ~ 2500% higher than in untransfected cells. In the case of IOBA-NHC cells, this percentage is ~ 1500%.

The real-time RT-PCR data support those obtained at the protein level, with an expression of MUC5AC of the order of 10,000 times higher in cells transfected with JetPei-RGD than in control cells, in both cell lines. In the case of lipofectamine, the expression of MUC5AC was still higher.

Therefore, the HCE and IOBA-NHC eye cells, after transfection with the plasmid pIRES2-AcGFP1-MUC5AC280, are able to express the plasmid, synthesize the transduced protein (MUC5AC) and secrete it.

Example 2. Development of a plasmid DNA encoding a mucin.

From plasmid pIRES2-AcGFP1-MUC5AC-280 a plasmid DNA encoding the mucin MUC5AC was developed, eliminating the region corresponding to the expression of the green GFP fluorescent protein. Figure 1B shows a scheme of said plasmid. The procedure consisted of the removal of the IRES-GFP region from the pIRES2-AcGFP1-MUC5AC-280 construction by digestion and religation. The deleted region was that contained between the SacI site located from the base at position 8370, and the NotI site located from position 9728 of the sequence of pIRES2-AcGFP1-MUC5AC-280.

The strategy used was the one described below:

one.

Partial digestion with SacI of plasmid pIRES2-AcGFP1-MUC5AC-280 and purification of the linear form of the plasmid (13,061 base pairs)

2.

Digestion with NotI and treatment with T4 polymerase to create blunt ends. Purification of the resulting 11706 base pair NotI-SacI fragment.

3.

Ligation, transformation into E. coli DH5 alpha and PCR identification of candidate colonies with the desired pMUC5AC construct.

Four.

Selection of two positive clones, plasmid DNA extraction and sequencing to verify the elimination of the IRES-GFP sequence.

Plasmid DNA from clone pMUCIN5AC-29 (SEQ ID NO: 7) was prepared, which was eluted in distilled and sterilized water, and digested separately with SacI and EcoRV enzymes, obtaining digestion patterns compatible with those expected (cut with SacI: 1 fragment of 11,702 base pairs; cut with EcoRV: 3 fragments of 6,692 base pairs, 3,135 base pairs and 1,875 base pairs, respectively). Photograph 2 shows the results of digesting 0.5 microliters the preparation of pMUCIN5AC-29 with these enzymes (negative controls of the undigested plasmid are included).

Example 3: Preparation of nanoparticles associating plasmid pIRES2-AcGFP1-MUC5AC-280 made from cationized gelatin and combinations with anionic polymers.

Nanoparticles of cationized gelatin were prepared according to an ionic crosslinking process in the presence of a polyanion. For this purpose, gelatin of type A (MW = 137 kDa) was previously cationized with spermine (SPM), in the presence of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC). The final isoelectric point of the gelatin was 11.08. The association to the nanoparticles of the plasmid pIRES2-AcGFP1-MUC5AC-280 was carried out. Is about

a negatively charged macromolecule, so it was incorporated together with the anionic crosslinker, to avoid the appearance of interactions prior to the formation of the particles. The crosslinking agent used was the anionic tripolyphosphate molecule (TPP). For this, aqueous solutions were prepared in milli-Q water of gelatin previously cationized with spermine (1.0 mg / mL). The crosslinking agent was added by using an aqueous solution of TPP (0.125-0.25 mg / mL) in milli-Q water. Optionally, the anionic polymers of dextran sulfate (DS) (0.1 mg / mL) or chondroitin sulfate (CS) (0.125 mg / mL) were incorporated into the composition. The corresponding genetic material was incorporated in a concentration of 0.05 μg / μL resulting in a mass ratio between 7.3% and 9% with respect to the constituents of the particles). For this, the bioactive molecule was incorporated into the crosslinker solution and the resulting solution was mixed with the cationized gelatin solution under magnetic stirring, which was maintained for half an hour allowing the complete evolution of the system towards a stable nanoparticular form. The formulations developed and their physical-chemical characteristics (average diameter of the nanoparticles obtained as well as surface electric charge) are shown in Table 1.

Formulation composition

Component Ratio Size (nm) POI Zeta Potential (mV) Association (%)

GCspm137: TPP

1: 0.125 261.5 ± 5.8 0.11 +21.6 ± 0.9 99.2 ± 0.5

GCspm137: CS: TPP

1: 0.0625: 0.625 268.5 ± 28.1 0.12 +33.4 ± 3.1 97.6 ± 1.3

GCspm137: DS: TPP

1: 0.05: 0.625 211.3 ± 8.2 0.12 +30.6 ± 2.0 95.7 ± 3.2

Table 1. Physicochemical characteristics of nanoparticles associating pMUC5AC-280 made from cationized jelly with spermine (GCspm137) using sodium tripolyphosphate (TPP) as a crosslinking agent and combinations with chondroitin sulfate (CS) or dextran sulfate (DS ) (n = 3).

The association of the plasmid with the developed nanoparticles was determined by agarose gel electrophoresis. In figure 3 the spherical morphology and homogeneous nanometric size of the systems formed can be verified. As can be seen in Figure 4, the bands corresponding to the plasmid incorporated in the elaboration of the nanoparticles do not migrate in the gel as is the case with the free plasmid control, which indicates that it is effectively associated with the nanoparticles.

Example 4. The nanoparticular systems associating plasmid DNA do not exhibit significant cytotoxicity in human cornea and conjunctiva cells.

The viability evaluation of cells in contact with nanoparticles made from cationized gelatin and combinations with anionic polymers was carried out in human cornea and conjunctiva cells. For this, the cells were seeded in 96-well plates Costar ® (Corning, US) at a confluence of 10,000 cells per well and allowed to grow for 24 hours before testing. The cells were incubated for 4h with 20 μL of formulation (nanoparticles), either with 20 μL of 1% benzalkonium chloride solution (positive control of cell death) or with 20 μL of culture medium (negative control), completing said volumes with DMEM / F-12 medium up to a total volume of 200 μL. After that time, the cells were washed and 200 μL of the respective culture medium was added. The cells were then incubated with 200 μL of RPMI medium without phenol red with XTT (0.2 mg / mL) for 15 h. The results were expressed as a percentage of cell viability relative to the untreated control. As can be seen in Figure 5, the systems do not show significant toxicity in any of the cell lines studied.

Example 5. The nanoparticular systems associating plasmid DNA are sufficient and necessary to give rise to the production of MUC5AC mRNA in human cornea (HCE) and conjunctiva (IOBA-NHC) cells.

For transfection studies the cells were seeded in 24-well plates Costar ® (Corning, US) at a density between 50,000 and 80,000 cells per well and allowed to grow for 24 hours before transfection. They were then incubated with nanoparticles made from cationized gelatin and combinations with anionic polymers. As a positive control, JetPEI-RDG complexes (Polyplustransfections, US) were used with pIRES2-AcGFP1-MUC5AC-280 (1/10 ratio) or with pEGFP (1/5 ratio), according to the manufacturer's instructions. The cells were incubated with the systems and controls (1-5 μg of plasmid per well) for 4h in pure DMEM / F-12 medium. The relative quantification of the MUC5AC mRNA, according to the previously described procedure, was expressed, expressing the results in MUC5AC mRNA log, normalized with GAPDH, relative to the untransfected control. As can be seen in Figure 6, in both the cell lines and with all the formulations tested, significant levels of MUC5AC mRNA were detected, indicating a successful transfection.

Example 6. The nanoparticular systems associating plasmid DNA are sufficient and necessary to give rise to the production by human cornea and conjunctiva cells of the mucin MUC5AC.

For transfection studies the cells were seeded in 24-well plates Costar ® (Corning, US) at a density between 50,000 and 80,000 cells per well and allowed to grow for 24 hours before transfection. They were then incubated with nanoparticles made from cationized gelatin and combinations with anionic polymers. As a positive control, JetPEI-RDG complexes (Polyplustransfections, US) were used with pIRES2-AcGFP1-MUC5AC-280 (1/10 ratio) or with pEGFP (1/5 ratio), according to the manufacturer's instructions. The cells were incubated with the systems and controls (1-5 μg of plasmid per well) for 4h in pure DMEM / F-12 medium. The expression of MUC5AC at the protein level was evaluated 72h post-transfection. As can be seen in Figure 7 for both cell lines, a significant expression of MUC5AC was obtained, confirming that the systems are effective for transfection in cornea and conjunctiva cells.

Example 7. The nanoparticular systems associating plasmid DNA give rise to a biological response capable of restoring functionality in tear production in animals with induced dry eye, similar to that achieved with commercially available formulation for this purpose.

Dry eye was induced in C57BL / 6 mice using a technique called drying stress. Specifically, the animals were treated with scopolamine hydrobromide subcutaneously (0.5mg / 0.2mL; Sigma-Aldrich, US) three times a day alternating administrations between the right and left leg and subjected to a low air flow humidity. For this, the animals were kept in cages with perforated side walls to allow a flow of air provided by fans (one on each side of the cage). These cages were kept inside a flow hood (AirClean Systems, US) in which the air humidity was maintained below 40%. The tear production in animals was determined by using a sterile cotton thread with a red phenol pH indicator (Zone-Quick, Lacrimedics, US). For this, the contact time of the thread with the eye was thirty seconds. The migration distance of the tear was measured in millimeters, based on the color change that said thread experiences when the tear rises by capillarity. Dry eye induction began five days before administration of the formulations and processing of the controls, and was maintained during the five days of treatment.

For treatment, the F2 formulation (GC137spm: CS: TPP) was concentrated 4 times by centrifugation (10,000g, 30 min, 4 ° C, CR22 Beckman Coulter, US) to a final concentration of 0.2μg / µL of pMUCIN5AC-29 . The nanoparticles were administered in a volume of 5μL, 4 times a day for 5 days, in each of the eyes, with an interval of 1h between administrations (4μg per day of pMUCIN5AC-29; 20μg pMUCIN5AC-29 in total). Once the formulations were administered, the animals were immobilized manually for 1 min.

Five groups of control animals were used in the study:

-

Animals not subjected to dry eye induction and untreated.

-

Animals with induced dry eye and untreated.

-

Animals with induced dry eye and treated with free pMUC5AC.

-

Animals with induced dry eye and treated with white nanoparticles (without associated plasmid).

-

Animals with induced dry eye and treated with commercially available formulation for the treatment of dry eye (Restasis®, Allergan Inc., USA).

For the evaluation of the efficacy of the treatment, the tear production was determined according to the previously described procedure, quantifying the basal tear production values (zero time), those obtained after induction of dry eye (day 5) and those obtained at the end of the treatment (day 10). The results were expressed as mean ± standard error of the mean tear migration in the thread (mm) of experimentally independent groups of 4 animals (total of 8 animals). For the statistical treatment of the results, a one-way variance analysis (ANOVA) was used with repeated measures. The differences were considered significant when (p <0.05).

As can be seen in Figure 8, after induction of dry eye all animals show a decrease in the level of tear production in relation to their basal levels (p <0.05). The functionality in tear production is only restored by treatment with free pMUCIN5AC-29 nanoparticles, nanoparticles associating pMUCIN5AC-29 and with the commercial formulation Restasis ® (0.05% cyclosporine emulsion), although the restoration is better in the case of nanoparticles or commercial formulation.

Claims (30)

one.

Polymeric nanoparticles comprising a polynucleotide encoding a modified MUC5AC protein that has the N-terminal end of MUC5AC attached to the dimerization domain located at the C-terminal end.

2.

Nanoparticles according to claim 1 wherein the polynucleotide has a 90% identity with respect to SEQ ID NO: 2.

3.

Nanoparticles according to claim 2 wherein the polynucleotide has a 95% identity with respect to SEQ ID NO: 2.

Four.

Nanoparticles according to claim 3 wherein the polynucleotide has a 98% identity with respect to SEQ ID NO: 2.

5.

Nanoparticles according to claim 4 wherein the polynucleotide is SEQ ID NO: 2.

6.

Nanoparticles according to any one of claims 1 to 5 wherein the polynucleotide is included in a genetic construct or a vector.

7.

Nanoparticles according to any one of claims 1 to 6 formed by at least:

i) at least one polymer with an electric charge, and ii) an ionic crosslinking agent.

8.

Nanoparticles according to claim 7 wherein the polymer of (i) is cationized gelatin.

9.

Nanoparticles according to any one of claims 7 or 8 wherein the ionic crosslinking agent is tripolyphosphate.

10.

Nanoparticles according to any of claims 7 to 9, further comprising an element (iii) which is at least one polymer provided with an electric charge opposite to that of the polymer of (i).

eleven.

Nanoparticles according to claim 10 wherein the polymer of the element (iii) is dextran sulfate or chondroitin sulfate.

12.

Nanoparticles according to any one of claims 1 to 11 wherein the nanoparticles are lyophilized.

13.

Use of nanoparticles as described in any one of claims 1 to 12 for the preparation of a medicament.

14.

Use of nanoparticles as described in any one of claims 1 to 12 for the preparation of a medicament for the prevention and / or treatment of a disease of a mucosa that has a mucin deficiency.

fifteen.

Use of nanoparticles according to claim 14 wherein the mucosa is the ocular mucosa.

16.

Use according to any of claims 14 or 15 wherein the mucin deficiency is a deficiency in MUC5AC.

17.

Use according to any of claims 14 to 16 wherein the disease is selected from the list comprising limbic insufficiency syndrome, neurotrophic keratitis, herpetic keratitis, alkali burn, thermal burn, radiation burn, Steven-Johnson syndrome, toxic epidermal necrosis , pemphiguses, mucous membrane pemphigoid, eye allergies, vernal keratoconjunctivitis, atopic keratoconjunctivitis, keratoconjunctivitis sicca or dry eye syndrome, infectious conjunctivitis, immune and irritative-toxic-medicated drugs, Sjögren syndrome, primary or secondary autoimmune diseases, secondary immune disease - against host, salivary glandular hypofunction, xerostomia or ichthyosis.

18.

 Use according to claim 17 wherein the disease is dry eye syndrome.

19.

Pharmaceutical composition comprising nanoparticles as described in any one of claims 1 to 12.

twenty.

Pharmaceutical composition according to claim 19 further comprising a pharmaceutically acceptable carrier.

twenty-one.

Pharmaceutical composition according to any of claims 19 or 20 for administration via ocular mucosa, mucosa of the entire gastrointestinal tract, mucosa of the respiratory tract, urinary mucosa and vaginal mucosa.

22. Pharmaceutical composition according to any of claims 19 to 21, which is in lyophilized form.

2. 3.

Use of a pharmaceutical composition as described in any of claims 19 to 22 for the preparation of a medicament.

24.

Use of a composition as described in any of claims 19 to 22 for processing

of a medicament for the prevention and / or treatment of a mucosal disease that has a mucin deficiency.

25.

Use according to claim 24 wherein the mucosa is the ocular mucosa.

26.

Use according to any of claims 24 or 25 wherein the mucin deficiency is a deficiency in MUC5AC.

27.

Use according to any of claims 24 to 26 wherein the disease is selected from the list that

15 includes limbic insufficiency syndrome, neurotrophic keratitis, herpetic keratitis, alkali burn, thermal burn, radiation burn, Steven-Johnson syndrome, toxic epidermal necrosis, pemphigus, mucous membrane pemphigoid, eye allergies, vernal keratoconjunctivitis, keratoconjunctivitis , keratoconjunctivitis sicca or dry eye syndrome, infectious, immune and irritative-toxic-conjunctivitis conjunctivitis, Sjögren's syndrome primary or secondary to other autoimmune diseases, atopy, 20 graft-versus host disease, salivary glandular hypofunction, xerostomia or ichthyosis. 28. Use according to claim 27 wherein the disease is dry eye syndrome. FIG. 1A FIG. 1B FIG. 2 FIG. 3 FIG. 4 FIG. 5A Do  175.00 150.00 125.00 100.00 75.00 50.00 25.00 0.00   C C + F1 F2 F3 FIG. 5B  IOBA-NHC 175.0 %> viability> 150.0 125.0 100.0 75.0 50.0 25.0 0.0 FIG. 6A 100000 10000 1000 100 10 1 Do log Relative expression of the MUC5AC mRNA JetPEI + JetPEI + F1 F2 F3 pGFP pMUC5AC control FIG. 6B log Relative Expression of the MUC5AC mRNA 100000 10000 1000 100 10 1  IOBA-NHC   JetPEI + JetPEI + F1 F2 F3 pGFP pMUC5AC control FIG. 7A Do Relative expression of MUC5AC (%) JetPEI + JetPEI + F1 F2 F3 pEGFP pMUC5AC control FIG. 7B  IOBA-NHC Relative expression of MUC5AC (%)

Control

JetPEI + JetPEI + F1 F2 F3

pEGFP

pMUC5AC

24

FIG. 8 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201031678 SPAIN Date of submission of the application: 15.11.2010 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

Y

WO 2010/049562 A1 (UNIVERSIDADE DE SANTIAGO) 06.05.2010, the whole document. 1-4, 6-8, 10-17, 19

27

Y

WO 2004/069136 A2 (APPLIED RESEARCH SYSTEMS.) 19.08.2004, the whole document. 1-4, 6-8, 10-17, 19

27

TO

KUMARI A. et al. "Biodegradable polymeric nanoparticles based drug delivery systems" Colloids and Surfaces B: Biointerfaces (08 September 2009); Vol. 75, pages 1-18; DOI: 10.1016 / j.colsurf.2009.09.001; ISSN 0927-7765; whole document. 1-28

TO

ANNICK LUDWIG "The use of mucoadhesivepolymers in ocular drug delivery" AdvancedDrug Delivery Reviews (November 3, 2005) Vol. 57, No. 11, pages 1595-1639; DOI: 10.1016 / j.addr.2005.07.005; ISSN 0169-409X; whole document. 1-28

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 08.05.2012

Examiner M. d. Garcia Coca Page 1/5

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201031678 CLASSIFICATION OBJECT OF THE APPLICATION B82Y5 / 00 (2011.01) A61K9 / 51 (2006.01) A61K48 / 00 (2006.01) A61P27 / 02 (2006.01) A61K47 / 36 (2006.01) A61K47 / 22 (2006.01) Minimum documentation sought (classification system followed by classification symbols) A61K, A61P Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, WPI, XPESP, BIOSIS, EMBL, EMBASE, MEDLINE State of the Art Report Page 2/5 WRITTEN OPINION Application number: 201031678 Date of Written Opinion: 08.05.2012 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-28 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 5, 9, 18 and 28 1-4, 6-8, 10-17 and 19-27 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986). Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/5 WRITTEN OPINION Application number: 201031678 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

WO 2010/049562 A1 (UNIVERSIDADE DE SANTIAGO) 06.05.2010

D02

WO 2004/069136 A2 (APPLIED RESEARCH SYSTEMS.) 19.08.2004

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The object of the invention, as set forth in claims 1-28, are polymeric nanoparticles comprising a polynucleotide encoding a modified MUC5AC protein. The nanoparticles are formed by cationized gelatin, tripolyphosphate and dextran sulfate or chondroitin sulfate, and are presented in lyophilized form (reiv. 1-12). The use of nanoparticles for the preparation of a medicament for the prevention and / or treatment of a disease of the ocular mucosa (dry eye syndrome) (reiv. 13-18) is also object of the invention. a pharmaceutical composition comprising said nanoparticles (reiv. 19-22) and the use of said composition (reiv. 23-28). 1. Novelty (art. 6.1 of Patent Law 11/1986) Document D02 discloses a sequence (SEQ ID NO: 6) that presents a 98.5% identity with SEQ ID NO: 2 of the international application, however, SEQ ID NO: 2 as such has not been found in the state of the art. Document D01 discloses nanoparticles formed by an active ingredient (polynucleotide, among others), an anionic polymer, an anionic crosslinking agent and a cationic polymer. Although the anionic polymer (chondroitin sulfate, dextran sulfate) and the cationic polymer (cationized gelatin) are the same as the polymers claimed in the international application, the crosslinking agent is different. In the case of the one disclosed in document D01, it is about spermine, which is a cationic crosslinking agent, while the one claimed in the international application is tripolyphosphate. Document D02 discloses mucin modified peptides, useful for the diagnosis or treatment of various diseases that are related to a mucin deficiency, such as allergic conjunctivitis. However, it does not disclose the specific case of dry eye syndrome. Therefore, although in the aforementioned state of the art documents, part of the object of the invention set forth in the international application is disclosed, in these documents there is no evidence that directs the person skilled in the art towards the invention defined in claims 5 , 9, 18 and 28. Accordingly, the object of the invention as set forth in claims 5, 9, 18 and 28, meets the requirement of novelty within the meaning of article 6.1 of Patent Law 11/1986, and It implies inventive activity within the meaning of Article 8.1 of Patent Law 11/1986. 2. Inventive Activity (art. 8.1 of Law 11/1986 on Patents). Document D01 is considered the closest in the state of the art. This document discloses nanoparticles in lyophilized form, for the administration of active ingredients (such as polynucleotides, vectors) comprising an anionic polymer (dextran sulfate, chondroitin sulfate), a cationic crosslinking agent (spermine) and a cationic polymer (cationized gelatin) , where the components of said nanoparticles are crosslinked by electrostatic interactions. This document also discloses the use of said nanoparticles for the preparation of a drug for gene therapy and for diagnostic purposes, and a pharmaceutical composition containing said nanoparticles for administration by oral, ocular, nasal and vaginal route among others. The difference between document D01 and the object of the invention, as set forth in claims 1-4, 6-8, 10-17, 19-27, is that the active ingredient present in the nanoparticles is a polynucleotide encoding for a modified MUC5AC protein, which is useful in the treatment of ocular mucosal diseases that present with mucin deficiencies, specifically with MUC5AC deficiency. However, document D02 discloses a sequence (SEQ ID NO: 6) that presents a 98.5% identity with SEQ ID NO: 2 of the international application. This document discloses mucin modified peptides, useful for the diagnosis or treatment of various diseases, such as allergic conjunctivitis. Both the peptides, such as polynucleotides or the compounds disclosed, are useful in the therapy or prevention of such diseases, which are related to a mucin deficiency. State of the Art Report Page 4/5 WRITTEN OPINION Application number: 201031678 Therefore, as disclosed in documents D01 and D02, it is obvious to one skilled in the art to prepare polymeric nanoparticles where their components are crosslinked by electrostatic type interactions, as disclosed in document D01, which comprise the polynucleotide encoding a modified MUC5AC protein, to develop a medicament for treating ocular mucosal diseases that are deficient in mucins (specifically MUC5AC deficiency), as disclosed in document D02. Accordingly, the invention as set forth in claims 1-4, 6-8, 10-17, 19-27, of the application, although it is new within the meaning of article 6.1 of Patent Law 11/1986 , lacks inventive activity in the sense of art. 8.1 of Law 11/1986 on Patents. State of the Art Report Page 5/5