EP3154573A1 - Nanoparticulate systems for use in gene transfer or gene delivery - Google Patents

Abstract

The present invention provides a nanoparticulate system or composition which comprises nanoparticles comprising a core of a sorbitan ester, a surface layer of a cationic substance and a gene construct consisting of plasmid DNA, wherein the gene construct is physically bound to the cationic substance, for use in a method of treatment by gene therapy, in particular for medical applications by using gene therapy in the ophthalmic sector.

Description

NANOPARTICULATE SYSTEMS FOR USE IN GENE TRANSFER OR GENE

DELIVERY-

FIELD OF THE INVENTION

The present invention relates to systems comprising nanoparticles capable of associating genes. More specifically, it relates to nanoparticulate systems comprising sorbitan esters for in vivo gene therapy, in particular for applications in the ophthalmic sector.

BACKGROUND OF THE INVENTION

Micelles, mixed micelles, emulsions, nanoparticles and liposomes stand out among colloidal systems proposed for gene transport, as an alternative to viral vectors for use in treatment methods by gene therapy.

However, some of systems mentioned above have a serious problem concerning stability. Vesicular systems and emulsions are known to experience aggregation phenomena, and the difficulty in obtaining more stable formulations by means of processes such as lyophilization without significantly changing their initial characteristics is also known. In this sense, a considerable energy input and/or the use of specific combinations of surface-active agents is necessary for the formation of such systems, so that the obtained product is not in an energetically unfavorable situation or unstable. Furthermore, these systems are particularly sensitive to variations in the surrounding environment, such as temperature.

Therefore, there is still a need to provide alternative and efficient systems to the systems referred to above for use in treatment methods by gene therapy, preferably in human subjects.

SUMMARY OF THE INVENTION

The present invention solves the above mentioned problem by providing, in a first aspect of the invention, a nanoparticulate system or composition which comprises nanoparticles comprising a core of a sorbitan ester, a surface layer of a cationic substance and a gene construct consisting of plasmid DNA, wherein the gene construct is physically bound to the cationic substance.

This system or composition is particularly suitable for use in a method of treatment by gene transfer or gene delivery in a mammalian subject, preferably in a human subject.

In a preferred embodiment of the first aspect of the invention, the sorbitan ester is selected from the group consisting of sorbitan mono-, di-, tri- or sesquioleate; sorbitan mono-, di-, tri- or sesquilaurate; sorbitan mono-, di-, tri- or sesquipalmitate; sorbitan mono-, di-, tri- or sesquistearate; and sorbitan mono-, di-, tri- or sesquiisostearate or any of their combinations thereof; and the cationic substance is a polyamino acid or oleylamine (OA). Preferably, the cationic substance is polyarginine (PA).

In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the gene construct, physically bound to the cationic substance, comprises a polynucleotide sequence comprising at least a nucleotide sequence encoding a protein with biological activity encoded by any of the genes selected from the list consisting of: PLA2G5, NMNAT1, RPE65, ABCA4, PRPF3, PRPF31, PRPF8, RD3, EFEMP1, KCNJ13, RGR, RBP4, BEST1, RDH5, RLBP1, CLN3, or any combinations thereof. Preferably, the selected gene is PRPF31. Preferably, the cationic substance is polyarginine and the gene construct, physically bound to the cationic substance, comprises a polynucleotide sequence comprising at least a nucleotide sequence encoding a protein with biological activity encoded by any of the genes selected from the list consisting of: PLA2G5, NMNAT1, RPE65, ABCA4, PRPF3, PRPF31, PRPF8, RD3, EFEMP1, KCNJ13, RGR, RBP4, BEST1, RDH5, RLBP1, CLN3, or any combinations thereof. Preferably, the selected gene is PRPF31.

In a second aspect of the invention, the nanoparticulate system or composition as defined in the first aspect of the invention, is a pharmaceutical composition which optionally further comprises pharmaceutically acceptable excipients, carriers and/or adjuvants.

In a third aspect of the invention, the nanoparticulate system or composition as defined in the second or third aspect of the invention is use in a method of treatment of abnormalities or dysfunctions of the retinal pigment epithelium. Preferably, the abnormality or dysfunction of the retinal pigment epithelium is selected from the list consisting of: age related macular degeneration, retinitis pigmentosa, Leber's congenital amaurosis, Stargardt's disease, diabetic retinopathy, macular dystrophy, cataracts, disruption of retinal pigment epithelium cells, deterioration of retinal pigment epithelium cells, death of retinal pigment epithelium cells, drusden, pigment clumping, RPE detachment, geographic atropy, sub-retinal neovascularization and disciform scar, vitelliform macular dystrophy, butterfly macular dystrophy, Sjogren's syndrome, fundus flavimaculatus, dominant foveal dystrophy, central areolar pigment epithelial dystrophy, North Carolina dystrophy, familiar drusen, pattern dystrophy of Marmor and Byers, foveomacular dystrophy (adult type), dominant slowly progressive macular dystrophy of Singerman-Berkow-Patz,macroreticular dystrophy of the RPE, pigment epithelial dystrophy of Noble-Carr-Siegel, benign concentric annular dystrophy, or combinations thereof. More preferably, the abnormality or dysfunction of the retinal pigment epithelium is selected from the list consisting of Retinitis Pigmentosa, Age-Related Macular Degeneration and/or Leber's congenital amaurosis. Still more preferably, the abnormality or dysfunction of the retinal pigment epithelium is Retinitis Pigmentosa.

In a preferred embodiment of the third aspect of the invention, the administration of the system or composition is via subretinal injection, in particular, in the subretinal space between the photoreceptors and the retinal pigment epithelium (RPE).

BRIEF DESCRIPTION OF THE FIGURES Figure 1. A) Retinal sections were obtained 72 hours after the subretinal injection of blank SP-OA nanoparticles (AC) or SP-OA-GFP nanoparticles (DL). Confocal images of a retinal section are shown in images D-F, and images G-L correspond to enlarged images of the pigmented epithelium of the retina (G-l) and ganglion cells (J-L). The cell nuclei were visualized using DAPI stain (left panel; blue) and the GFP signal was enhanced with anti-GFP antibodies (central panel; green). The right panel shows merged images. (RPE = retinal pigment epithelium; ONL = outer nuclear layer; INL = inner nuclear layer; GLC = ganglion cell layer). The scale bars represent 75 (A-F) and 20 m (G-L). B) Western blot using an anti-GFP antibody in three independent samples from each group. Figure 2. GFP evaluation in mice one month after the subretinal injection of: AAV2- GFP, SP-PA-GFP nanoparticles, SP-OA-GFP nanoparticles, PBS and 5% glucose (image on the left) or blank SP-PA nanoparticles and blank SP-OA nanoparticles (image on the right). Image on the left: in vivo scanning laser ophthalmoscopy for ocular fundus (A-E) and GFP signal (F-J); Confocal images of retinal sections showing the characteristic GFP signal (K-O) or GFP signal improved with anti-GFP antibodies (P-T); merged images with the cell nuclei stained with DAPI stain (UY; Blue). Image on the right: Confocal images of retinal sections showing the characteristic GFP signal (AD) or GFP signal improved with anti-GFP antibodies (BE) and merged images with the cell nuclei stained with DAPI stain GFP signal (CF; blue). (RPE = retinal pigment epithelium; ONL = outer nuclear layer; INL = inner nuclear layer; GLC = ganglion cell layer). The scale bars represent 50 micron.

Figure 3. A) Western Blot using the anti-GFP antibody in three independent samples from each group of animals one month after the injection of AAV2-GFP, SP-PA-GFP and SP-OA-GFP nanoparticles. B) Quantification of the expression levels obtained by image analysis. The bars represent the mean ratio between good farming practices and alpha-tubulin ± SEM of three samples. The results were not statistically significant (p>0.05). Alpha-tubulin was used for verifying load control.

Figure 4. Electroretinograms of mice injected in dark- and light-adapted conditions one month after the administration of PBS, 5% glucose, AAV2-GFP, SP-PA, SP-OA, SP-PA- GFP and SP-OA-GFP nanoparticles. Figure 5. Quantification of rods (A, D), joint cone-rod response (B, E) and cone responses (C, F) obtained from electroretinogram. The results are expressed in mean ± SEM and represent the a-wave or b-wave amplitude (mV) of both eyes in 3 mice for each group. The statistical differences between the treated groups and their respective controls were estimated using the t-test. P = 0.052 (A), P = 0.057 (B) ^*P<0.05 (C, E), ^**P<0.01 (D, F).

Figure 6. Positive optomotor responses observed at a frequency of 0.067 one month after the subretinal injections. The results are expressed in mean ± SEM and represent the percentage of positive responses in 2 mice for each group evaluated at two different times. The statistical differences between the treated groups and their respective controls were estimated using the t-test. ^*P<0.05.

Figure 7. In the in vivo laser ophthalmoscopy examination for ocular fundus and GFP signal one month after the subretinal injection of a 5% glucose solution in wild-type mice (WT 5% Glu control- -negative) or after the injection of SP-PA-GFP nanoparticles and SP-OA PRPF31 nanoparticles in Prpf31 ^,A2 '^6P/+ mice (Kl).

Figure 8. Optomotor test establishing 6 frequencies from 0.031 to 0.272 cycles/degree (c/d) at 100% (A), 75% (B) and 50% (C) contrast sensitivity in healthy mice (WT) injected with a 5% glucose negative control solution (WT 5% Glu) (n = 3), and the mouse model of the disease, Prpf31^A216P/+ mice (Kl), injected with a control formulation of nanoparticles associating the GFP plasmid (Kl SP-PA-GFP) (n = 3) or the developed SP-PA nanoparticles associating the human gene PRPF31 (Kl SP-PA-PRPF31 ) (n = 4). The bars represent ± SEM. ^*P<0.05, ^**P<0.01 represents the statistically significant difference between SP-PA-GFP and SP-PA-PRPF31 .

Figure 9. A) Optical coherence tomography scanner and retinal map of healthy WT mice treated with a 5% glucose negative control solution (WT 5% Glu), and Kl mice, control of the disease, treated with a control formulation of nanoparticles associating the GFP plasmid (Kl SP-PA-GFP) or a formulation of SP-PA-based nanoparticles associating the PRPF31 plasmid (Kl SP-PA-PRPF31 ). The color bar represents a retinal thickness scale. B) Graph depicting the mean thickness of the retina in each group. The bars represent ± SEM. ^*P<0.01 , statistical comparisons were made by means of one-way ANOVA with post hoc Dunnett comparing the two experimental groups with the control separately.

Figure 10. GFP evaluation in Prpf31^A216P/+ mice retinas four months after the subretinal injection of 1 μΙ_ of a viral suspension containing 10⁹ pv/ml of AAV2-GFP, the AAV2- PRPF31 viral construct or PBS: Laser ophthalmoscopic scanning for ocular fundus and GFP signal (A, B); Confocal images of retinal sections of the GFP signal with the cell nuclei stained with DAPI stain (C, D; Blue). Image on the right (E): Western Blot in three independent samples from each group. The expression of the PRPF31 protein in RPE cells was visualized through the fluorescence signal of GFP. GAPDH antibodies were used as load control. RPE: retinal pigment epithelium, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. The scale bar represents 50 micron. Figure 11. Evaluation of retinal function after the injection of AAV2-PRPF31 : Electroretinogram (ERG) of Prpf31^A216P/+ mice (Kl) was done under dark- and light- adapted conditions 4 months after the subretinal injection of PBS (negative control), AAV2-GFP or AAV2-PRPF31 (4 animals per group). The a-wave and b-wave amplitude and frequency were quantified in different conditions. The amplitude of different ERG waves corresponding to rod responses (B), cone-rod responses (C) or cone responses (D) decreased in injected AAV2-PRPF31 mice when compared with the group with PBS injection, respectively. Bars in Figures 1 1 B, 1 1 C and 1 1 D represent mean ERG wave amplitudes ± SEM. (^*P<0.05, one-way ANOVA). The scale bars in Figure 1 1A represent 100 ms and 600 mV.

Figure 12. Positive optomotor responses observed at a frequency 0.067 one month after the subretinal injections of a 5% glucose solution (negative control group), SP-OA- based nanoparticles (blank formulation) or SP-OA-based nanoparticles associating the PRPF31 gene. Positive responses decreased in mice injected with SP-OA-PRPF31 when compared with the negative control group. The results are expressed in mean ± SEM and represent the percentage of positive responses in 3 mice for each group evaluated at two different times. The statistical differences between the treated groups and their respective controls were estimated using the t-test. ^*P<0.05.

Figure 13. Test for evaluating the visual function in mutant mice (Prpf31^A216P/+) 8 months of age compared with healthy mice. Prpf31^A216P/+ mice show impairment of spatial vision (visual acuity and contrast perception). Optomotor adjustment test establishing 6 frequencies from 0.031 to 0.272 cycles/degree (c/d) at 100% (A), 75% (B) and 50% (C) contrast sensitivity in wild-type mice (WT) (n = 5) and Prpf31^A216P/+ mice (n = 5). The bars represent ± SEM. ^*P<0.05, ^**P<0.01 represents the statistically significant difference between WT and Prpf31^A216P/+ DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term "nanoparticle" refers to solid colloidal materials, the mean size of which ranges between 1 and 999 nm, having a solid matrix structure and further characterized by being stable structures having perfectly homogenous, reproducible and modulable characteristics.

As used herein, "Sorbitan esters" are interpreted to mean esterified sorbitan derivatives where the ester groups have a substituent selected from alkyl, alkenyl and alkynyl. Preferably, alkyl, alkenyl and alkynyl have a chain of between 6 and 24 carbon atoms, more preferably between 10 and 16 carbon atoms. Sorbitan esters include derivatives in which one, two, three or four hydroxyl groups are esterified, and they even include esterified derivatives in which one ester molecule is present for every two sorbitan molecules (in which case they are referred to with the "sesqui-" prefix). In that sense, for example, sorbitan monooleate is the sorbitan ester resulting from esterifying a hydroxyl group with oleic acid; sorbitan trioleate is the sorbitan ester resulting from esterifying three sorbitan hydroxyl groups with oleic acid. As used herein, "Alkyl" is interpreted to mean a linear or branched hydrocarbon chain that contains no instauration, of 1 to 24 carbon atoms, optionally substituted with one to three substituents selected from -OR^b, -SR^b, -NR^aR^b, -C(O)R^b, -CO₂R^b, -C(O)NR^aR^b, - NR^aC(O)R^b, -NR^aC(O)OR^b, -NR^aC(O)NR^aR^b, -CF₃, -OCF₃; where R^a and R^b are independently selected from hydrogen, alkyl, alkenyl and alkynyl.

As used herein, "Alkenyl" and "alkynyl" in the compounds of the present invention refer to a linear or branched hydrocarbon chain containing at least one instauration, of 2 to 24 carbon atoms, optionally substituted with one to three substituents selected from -OR^b, - SR^b, -NR^aR^b, -C(O)R^b, -CO₂R^b, -C(O)NR^aR^b, -NR^aC(O)R^b, -NR^aC(O)OR^b, - NR^aC(O)NR^aR^b, -CF₃, -OCF₃; where R^a and R^b are as previously defined.

As used herein, the term "cationic substance" is interpreted to mean that molecule provided with a positive electric charge, for example ammonium salts, cationic polymers and lipophilic or fatty amines.

As used herein the term "cation ized" refers to the presence of a positively charged group, which may be present naturally or may be introduced by means of a chemical reaction.

As used herein, "Gene transfer" or "gene delivery" refers to methods or systems for reliably introducing a particular nucleotide sequence (e. g., DNA) into targeted cells. The introduced nucleotide sequences may persist in vivo in episomal forms, or integrate into the genome of the target cells. Gene transfer provides a unique approach for the treatment of acquired and inherited diseases, and a number of systems have been developed in the art for gene transfer into mammalian cells. See, e. g., U. S. Pat. No. 5, 399,346.

As used herein the term "expression control element" or "regulatory element" or particularly "plasmid" refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

A used herein, "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

As used herein, the term "promoter" refers to a set of control nucleic acid sequences which are controlling transcription of a target nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription. Also, a promoter may optionally include an enhancer or repressor element. A "constitutive promoter" is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an "inducible promoter" is regulated by an external signal or molecule (such as a transcription factor).

A used herein, the term "active ingredient" refers to an ingredient or cell used in the treatment, cure, prevention or diagnosis of a disease, or used for improving the physical and mental wellbeing of humans and animals, as well as that ingredient or cell intended for destroying, blocking the action of, counteracting or neutralizing any harmful entity or organism, or any ingredient or cell used as a cosmetic or for hygiene, as well as that ingredient or cell intended for regenerating tissues in tissue engineering or in cell and gene therapy.

As used herein, the term "PRPF31 " is as defined in table 1 or as defined as follows by its gene bank sequences. In particular, the Genebank (ncbi) nucleotide sequence of PRPF31 is NM_015629 (SEQ ID NO 1 ), and the aminoacidic sequence is NP_056444.3 (SEQ ID NO 2). Variant sequences substantially identical to SEQ ID NO 2 are also included within the boundaries of the present invention. The terms "identical" or "identity," in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same. Sequences are "substantially identical" if they have a percentage of amino acid residues or nucleotides that are the same {i.e., about 60% identity, optionally about 65%, about 70%>, about 75%, about 80%), about 85%), about 90%>, or about 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

SEQ ID NO. 1 : tagtttcctgtttccggcttcgcttcggcccacccccacgtccaccccgaatccctgcttaaaggccttgctttcttgtctaacgc cgcaaccagtcctctgagttgccaacgtctttcttcttgtctcgacgccccgtcgtccggccacagcgattctctgcttagcag gatcggtccacagcgggacgtgagtccctttcctcctcgcggcttaccgcctctctccgcctagtgccaggtgctaataaagt tgttgtttcaaatgcggccaggaacatcgcgagcggggaccaatcagagagtagctttgcctctataacggcgcgagagt gagacgtcatcggtgagcgactaacgctagaaacagtggtgcgcggagaggagaggcctcgggatgtctctggcagat gagctcttagctgatctcgaagaggcagcagaagaggaggaaggaggaagctatggggaggaagaagaggagcca gcgatcgaggatgtgcaggaggagacacagctggatctttccggggattcagtcaagaccatcgccaagctatgggata gtaagatgtttgctgagattatgatgaagattgaggagtatatcagcaagcaagccaaagcttcagaagtgatgggacca gtggaggccgcgcctgaataccgcgtcatcgtggatgccaacaacctgaccgtggagatcgaaaacgagctgaacatc atccataagttcatccgggataagtactcaaagagattccctgaactggagtccttggtccccaatgcactggattacatcc gcacggtcaaggagctgggcaacagcctggacaagtgcaagaacaatgagaacctgcagcagatcctcaccaatgc caccatcatggtcgtcagcgtcaccgcctccaccacccaggggcagcagctgtcggaggaggagctggagcggctgg aggaggcctgcgacatggcgctggagctgaacgcctccaagcaccgcatctacgagtatgtggagtcccggatgtccttc atcgcacccaacctgtccatcattatcggggcatccacggccgccaagatcatgggtgtggccggcggcctgaccaacct ctccaagatgcccgcctgcaacatcatgctgctcggggcccagcgcaagacgctgtcgggcttctcgtctacctcagtgct gccccacaccggctacatctaccacagtgacatcgtgcagtccctgccaccggatctgcggcggaaagcggcccggct ggtggccgccaagtgcacactggcagcccgtgtggacagtttccacgagagcacagaagggaaggtgggctacgaac tgaaggatgagatcgagcgcaaattcgacaagtggcaggagccgccgcctgtgaagcaggtgaagccgctgcctgcg cccctggatggacagcggaagaagcgaggcggccgcaggtaccgcaagatgaaggagcggctggggctgacgga gatccggaagcaggccaaccgtatgagcttcggagagatcgaggaggacgcctaccaggaggacctgggattcagcc tgggccacctgggcaagtcgggcagtgggcgtgtgcggcagacacaggtaaacgaggccaccaaggccaggatctc caagacgctgcagcggaccctgcagaagcagagcgtcgtatatggcgggaagtccaccatccgcgaccgctcctcgg gcacggcctccagcgtggccttcaccccactccagggcctggagattgtgaacccacaggcggcagagaagaaggtg gctgaggccaaccagaagtatttctccagcatggctgagttcctcaaggtcaagggcgagaagagtggccttatgtccac ctgaatgactgcgtgtgtccaaggtggcttcccactgaagggacacagaggtccagtccttctgaagggctaggatcgggt tctggcagggagaacctgccctgccactggccccattgctgggactgcccagggaggaggccttggaagagtccggcct ggcctcccccaggaccgagatcaccgcccagtatgggctagagcaggtcttcatcatgccttgtcttttttaactgagaaag gagattttttgaaaagagtacaattaaaaggacattgtcaagatctgtcaaaaaaaaaaaaaaaaaa

SEQ ID NO. 2:

MSLADELLADLEEAAEEEEGGSYGEEEEEPAIEDVQEETQLDLSGDSVKTIAKLWDSK

MFAEIMMKIEEYISKQAKASEVMGPVEAAPEYRVIVDANNLTVEIENELNIIHKFIRDKYS

KRFPELESLVPNALDYIRTVKELGNSLDKCKNNENLQQILTNATIMVVSVTASTTQGQQ

LSEEELERLEEACDMALELNASKHRIYEYVESRMSFIAPNLSIIIGASTAAKIMGVAGGLT

NLSKMPACNIMLLGAQRKTLSGFSSTSVLPHTGYIYHSDIVQSLPPDLRRKAARLVAAK

CTLAARVDSFHESTEGKVGYELKDEIERKFDKWQEPPPVKQVKPLPAPLDGQRKKRG

GRRYRKMKERLGLTEIRKQANRMSFGEIEEDAYQEDLGFSLGHLGKSGSGRVRQTQV

NEATKARISKTLQRTLQKQSWYGGKSTIRDRSSGTASSVAFTPLQGLEIVNPQAAEKK

VAEANQKYFSSMAEFLKVKGEKSGLMST

Detailed description

The present invention provides a nanoparticulate system or composition for use in a method of treatment by gene transfer or gene delivery, which comprises nanoparticles comprising a core of a sorbitan ester (SP), a surface layer of a cationic substance and a gene construct consisting of plasmid DNA, wherein the gene construct is physically bound to the cationic substance. As already stated and as used herein, the term "nanoparticle" refers to solid colloidal materials, the mean size of which ranges between 1 and 999 nm, having a solid core or matrix structure and further characterized by being stable structures having perfectly homogenous, reproducible and modulable characteristics. The nanoparticles of the systems of the invention are perfectly distinguishable from other colloidal systems due to their structural characteristics; for example, the nanoparticles of the invention do not have lipid bilayers characteristics of liposomes nor do they have an oily core, which is characteristic of nanoemulsions or nanocapsules. The nanoparticles of the invention do not comprise oils or oily components.

The system or composition of the present invention comprises a core or matrix of sorbitan esters. Sorbitan consists of a mixture of cyclic anhydrides of sorbitol, such as for example and without being limited to, 1 ,4-anhydrosorbitol, 1 ,5-anhydrosorbitol and 1 ,4,3,6-dianhydrosorbitol. In a particular embodiment, the sorbitan ester is selected from the group consisting of sorbitan mono-, di-, tri- or sesquioleate; sorbitan mono-, di-, trior sesquilaurate; sorbitan mono-, di-, tri- or sesquipalmitate; sorbitan mono-, di-, tri- or sesquistearate; and sorbitan mono-, di-, tri- or sesquiisostearate; and their combinations. Sorbitan esters are non-ionic surfactants given that they contain two localized regions, a hydrophilic region and another hydrophobic region. These non-ionic surfactants have the advantage of being less irritating than anionic or cationic surfactants. Furthermore, they are generally compatible with both anionic and cationic substances, since they are not ionized in solution.

In a particular embodiment, the invention relates to nanoparticles comprising a core of a sorbitan ester in a proportion by weight (w/w) with respect to the total weight of the nanoparticle components greater than 60%, preferably greater than 80%, more preferably between about 60% and about 100%, still more preferably between about 80% and about 100% characterized by being a solid homogenous matrix and by having a mean size between 1 and 999 nm. More preferably, the nanoparticles of the invention comprise a core of a sorbitan ester in a proportion by weight greater than 90% or between about 90% and about 100%. In a particular embodiment, the nanoparticles of the invention are further characterized by not comprising oils or oily components.

The nanoparticles of the system of the invention have an average particle size of between 1 and 999 nm, preferably between 50 and 600 nm, even more preferably between 100 and 400 nm. The average particle size is primarily influenced by the composition and the conditions for forming the particles established in the selected production method. The term "average size" is thus interpreted to mean the average diameter of the population of nanoparticles moving together in an aqueous medium. The average size of these systems can be measured using standard methods known to the person skilled in the art. In particular, the average size or mean particle size and the size distribution of the nanoparticles is determined by photon correlation spectroscopy (PCS). For this purpose the sample can be diluted with filtered Milli-Q water at the appropriate concentration, performing the analysis at 25°C with an angle of detection of 173°.

Preferably, the system of the invention comprises a homogenous size distribution and the population of nanoparticles of the system moves together in an aqueous medium having a polydispersity index of less than 0.2, more preferably between 0 and 0.1 .

The nanoparticles of the systems of the invention have a core or matrix structure that allows incorporating additional components increasing and improving their stability. In particular, the nanoparticles of the invention comprise a cationic substance, in particular a surface layer of a cationic substance, in a proportion by weight greater than 0% and about 40% with respect to the total weight of the nanoparticle components. In a particular embodiment, the proportion by weight of the cationic substance is greater than 0% and about 20% with respect to the total weight of the nanoparticle components; more particularly greater than 0% and about 10%. The cationic substance allows modulating the characteristics of nanoparticulate systems, such as particle size, electrical surface charge and composition.

In a particular embodiment, the cationic substance is selected from protamine, polyglutamic acid, cationized dextran, polyamino acids and cationized proteins, and their salts. Preferably, the polyamino acids are selected from polylysine and polyarginine. Preferably, the cationized proteins are selected from gelatin, albumin, collagen and atelocollagen, and their cationized derivatives. Preferably, the ammonium salts are selected from cetyltrimethylammonium bromide and benzalkonium chloride. Preferably, the fatty amine is oleylamine (c/s-1 -amino-9-octadecene). The nanoparticles of the invention can optionally comprise an ethylene oxide derivative. For the purposes of the present invention, "ethylene oxide derivative" is interpreted to mean a compound in which a -CH₂CH₂O- unit is repeated. In a particular embodiment, the ethylene oxide derivative is a compound of formula I

Formula I where Ri is a hydrogen or carbonyl group; F½ is an alkyl, alkenyl or alkynyl group having between 2 to 24 carbon atoms; R3 is hydrogen or an alkyl group having between

1 to 6 carbon atoms; n is a value between 1 and 100. In a particular embodiment, n has a value of between 1 and 50, more preferably between 1 and 24.

Examples of ethylene oxide derivatives, without being limited to said examples, are polyethylene glycol dodecyl ether (Brij 30), polyethylene glycol hexadecyl ether (Brij 56), polyethylene glycol 2-octadecyl ether (Brij 72), polyethylene glycol 8-octadecyl ether

(Brij 78), polyethylene glycol 8-stearate (Myrj 45), 2-hydroxyethyl octadecanoate (Myrj

52), ethylene glycol monostearate, triethylene glycol monostearate.

In addition, the nanoparticles have an electric charge (measured by means of Z potential), the magnitude of which can be modulated by means of a suitable system composition selection. Specifically, said electric charge can have positive or negative values depending on the system components and the proportion existing between them. The zeta potential of the nanoparticles of the systems of the invention can be measured using standard methods known to the person skilled in the art and described, for example, in the experimental part of the present specification.

In a particular embodiment of the invention, the nanoparticles have a charge ranging between -50 mV and +60 mV, even more preferably between -40 mV and +50 mV, depending on the proportion of the components.

In addition, the possibility offered by the present invention of modulating the electric charge of nanoparticles has enormous advantages. In that sense, a negative charge is particularly suitable for assuring nanoparticle stability after parenteral administration. The nanoparticles of the systems of the invention comprises a core or matrix of sorbitan esters, a cationic substance, in particular a surface layer of a cationic substance, and a gene construct consisting of plasmid DNA, wherein the gene construct is physically bound to the cationic substance. Preferably, the gene construct, physically bound to the cationic substance, comprises a plasmid polynucleotide sequence which in turn comprises at least a nucleotide sequence encoding a protein with biological activity encoded by any of the genes selected from table I below. Table 1 . Genes and Mapped Loci Causing Retinal Diseases

213300, 614423, 614424

KCNJ13, LCA16, SVD;

2q37.1 3769 204000, 193230, 603208, 614186

SAG, RP47;

2q37.1 6295 181031 , 258100, 268000

USH2B;

not 3p24.2-p23 7400

605472

GNAT1 , CSNBAD3;

3p21 .31 2779 139330, 310500, 610444

LZTFL1 , BBS17;

3p21 .31 54585 606568

TREX1 , AGS1 , CHBL, CRV, RVCL;

3p21 .31 1 1277 192315, 225750, 606609, 610448

ATXN7, ADCA2, OPCA3, SCA7;

3p14.1 6314 164500

ARL6, BBS3, RP55;

3q1 1 .2 84100 209900, 268000, 608845, 613575

IMPG2, RP56, SPARCAN;

3q12.3 50939 268000, 607056, 613581

IQCB1 , NPHP5, SLSN5;

3q13.33 9657 609237, 609254

NPHP3, SLSN3;

3q22.1 27031 604387, 606995, 608002

RHO, CSNBAD1 , OPN2, RP4;

3q22.1 6010 180380, 268000, 310500, 610445

RP5 3q22.1 6010

CLRN1 , RP61 , USH3, USH3A;

3q25.1 7401 268000, 276902, 606397, 614180

SLC7A14, RP68; 3q26.2 57709 200100, 157147

LRIT3, FIGLER4;

4q25 345193 615004

BBS7, BBS2L1 ;

4q27 55212 209900, 607590

BBS12, FLJ35630;

4q27 166379 209900, 610683

RP29;

4q32-q34 541 10

268000, 612165

LRAT, LCA14;

4q32.1 9227 204000, 604863, 613341

TLR3;

4q35.1 7098

603029

CYP4V2, BCD;

4q35.2 285440 210370

MCDR3;

5p15.33-p13.1 317668

608850

GPR98, FEB4, MASS1 , USH2C, j

VLGR1 ; 5q14.3 84059

602851 , 604352, 605472

VCAN, CSPG2, ERVR, WGN1 ;

5q14.3 1462 1 18661 , 143200

NR2F1 , EAR3;

5q15 7025 132890, 615722

BSMD;

5q21 .2-q33.2 5961

608970

HARS, HRS, USH3B;

5q31 .3 3035 614504, 142810

PDE6A, RP43; 5q33.1 5145 276900,601386,601067,605516

! CDHR1, CORD15, PCDH21, RP65;

10q23.1 92211 120970,268000,609502,613660

RGR, RP44;

10q23.1 5995 268000,600342,613769

I KIF11, EG5, HK5P, KNSL1, MCLMR, j

TRIP5; 10q23.33 3832

148760, 152950

PDE6C, COD4, PDEA2;

10q23.33 5146 600827,613093

RBP4;

10q23.33 5950

180250

PAX2, ONCR;

10q24.31 5076 120330, 167409

PDZD7, PDZK7;

10q24.31 79955 612971

BBIP1, BBIP10, BBS18;

10q25.2 92482 613605

CORD17 10q26 101409267

ARMS2, ARMD8, LOC387715;

10q26.13 387715 603075,611313

HTRA1, ARMD7, PRSS11;

10q26.13 5654 602194,603075,610149

OAT;

10q26.13 4942

258870

TUB;

11p15.4 7275

601197

TEAD1, AA, TCF13, TEF1;

11p15.3 7003 108985, 189967 USH1C, DFNB18;

11 p15.1 10083 276900, 276904, 602092, 605242

EVR3;

11p13-p12 81864

133780,605750

CORS2, JBTS2;

11p12-q13.3 373067 608091

BEST1 , RP50, TU15B, VMD2;

11q12.3 7439 153700, 268000, 607854, 613194

ROM1;

11q12.3 6094

180721

BBS1;

11q13 582

209900, 209901

CABP4, CSNB2B;

11 q13.1 57010 310500,608965,610427

LRP5, EVR4, HBM, OPPG;

133780, 259770, 601813, 601884, 11q13.2 4041

603506

CAPN5, ADNIV, HTRA3, VRNI;

11q13.5 726 602537, 193235

MY07A, DFNB2, USH1B;

11q13.5 4647 276900, 276903, 600060

TMEM126A, OPA7;

11q14.1 84233 165500,612988,612989

FZD4, EVR1 , FEVR;

11q14.2 8322 133780,604579

C1QTNF5, CTRP5;

11q23.3 114902 605670, 608752

CEP164, NPHP15;

11q23.3 22897 256100,614845,614848

312600

PGK1;

Xq21.1 5230

300653, 311800

CHM;

Xq21.2 1121

303100

TIMM8A, DDP, DDP2, DFN1;

Xq22.1 1678 300356, 304700, 311150

RP24;

Xq26-q27 6116

300155

COD2, CORDX2;

Xq27 1275 300085

RP34;

Xq28-qter 777642

300605

OPN1LW, BCM, CBP, COD5, RCP;

Xq28 5956 303700, 303900

OPN1MW, CBD, GCP;

Xq28 2652 303700, 303800

KSS;

mitochondrion 3894

530000

LHON;

mitochondrion 3974

535000

MT-TL1, DMDF, TRNL1;

mitochondrion 4567 520000, 590050

MT-ATP6, ATP6, NARP;

mitochondrion 4508 516060, 551500

MT-TH, TRNH;

mitochondrion 4564 590040

MT-TS2, TRNS2;

mitochondrion 4575 500004, 590085 MT-TP, TRNP;

mitochondrion 4571

590075

More preferably, the gene construct, physically bound to the cationic substance, comprises a plasmid polynucleotide sequence which in turn comprises at least a nucleotide sequence encoding a protein with biological activity encoded by any of the genes selected from the list consisting of: PLA2G5, NMNAT1, MERTK, RPE65, ABCA4, PRPF3, PRPF31, PRPF8, RD3, EFEMP1, KCNJ13, RGR, RBP4, BEST1, RDH5, RLBP1, CLN3, or any combinations thereof. Preferably, the gene construct encodes a biological active PRPF31 protein.

In one embodiment of the invention, the nanoparticulate system or composition as described throughout the present invention is a pharmaceutical composition which optionally further comprises pharmaceutically acceptable excipients, carriers and/or adjuvants.

In a preferred embodiment of the present invention, the nanoparticulate system or composition as described throughout the present invention is use in a method of treatment of abnormalities or dysfunctions of the retinal pigment epithelium. Preferably, the abnormality or dysfunction of the retinal pigment epithelium is selected from the list consisting of: age related macular degeneration, retinitis pigmentosa, Leber's congenital amaurosis, Stargardt's disease, diabetic retinopathy, macular dystrophy, cataracts, disruption of retinal pigment epithelium cells, deterioration of retinal pigment epithelium cells, death of retinal pigment epithelium cells, drusden, pigment clumping, RPE detachment, geographic atropy, sub-retinal neovascularization and disciform scar, vitelliform macular dystrophy, butterfly macular dystrophy, Sjogren's syndrome, fundus flavimaculatus, dominant foveal dystrophy, central areolar pigment epithelial dystrophy, North Carolina dystrophy, familiar drusen, pattern dystrophy of Marmor and Byers, foveomacular dystrophy (adult type), dominant slowly progressive macular dystrophy of Singerman-Berkow-Patz,macroreticular dystrophy of the RPE, pigment epithelial dystrophy of Noble-Carr-Siegel, benign concentric annular dystrophy, or combinations thereof. More preferably, the abnormality or dysfunction of the retinal pigment epithelium is selected from the list consisting of Retinitis Pigmentosa, Age-Related Macular Degeneration and/or Leber's congenital amaurosis. Still more preferably, the abnormality or dysfunction of the retinal pigment epithelium is Retinitis Pigmentosa.

In a preferred embodiment, the administration of the system or composition of the invention is via subretinal injection. In particular, the administration of said system or composition is via subretinal injection in the subretinal space between the photoreceptors and the retinal pigment epithelium (RPE).

A further aspect of the invention, refers to a nanoparticulate system or composition, or pharmaceutical compositions comprising said system or composition, which comprises nanoparticles comprising a core of a sorbitan ester, a surface layer of a cationic substance and a gene construct consisting of plasmid DNA, wherein the gene construct is physically bound to the cationic substance, and wherein the gene construct comprises a polynucleotide sequence comprising at least a coding sequence for a protein with biological activity encoded by any of the genes selected from the list consisting of the genes of table 1 or preferably from the list consisting of: PLA2G5, NMNAT1 , RPE65, ABCA4, PRPF3, PRPF31 , PRPF8, RD3, EFEMP1 , KCNJ13, RGR, RBP4, BEST1 , RDH5, RLBP1 , CLN3, or any combinations thereof. Preferably, the selected gene is PRPF31 . More preferably, the cationic substance is polyarginine and the gene construct, physically bound to the cationic substance, comprises a polynucleotide sequence comprising at least a coding sequence for PRPF31 .

Another further aspect of the invention refers to an expression control element, such as a DNA plasmid, comprising a coding sequence for the protein PRPF31 and further comprising the appropriate control elements for the coding sequence to be replicated, transcribed and translated in the appropriate host cells for use in a method of treatment of abnormalities or dysfunctions of the retinal pigment epithelium as defined in any of claims 10 to 12.

Finally, the nanoparticulate system or composition as described throughout the present invention may be in a lyophilized form.

To better understand the features and advantages of the present invention, reference will be made below to a series of examples, which, in an explanatory manner, complete the preceding description without meaning in any way that said invention is limited to such examples. EXAMPLES EXAMPLE 1 . Materials and methods Materials

Sorbitan monooleate (Span® 80) (SP), oleylamine (OA) (purity > 70%) and poly-L- arginine (PA) were acquired from Sigma (Spain). The pEGFP-C3 and pEGFP-C3- PRPF31 plasmids were produced by the research center, CABIMER. The pEGFP-N1 plasmid (Clontech Laboratories, Inc.) with GenBank accession number #U55762 was used to construct the plasmids. pEGFP-N1 encodes a red-shifted variant of wild-type GFP which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm). pEGFP-N1 encodes the GFPmutl variant containing the double amino acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes, which correspond to human codon usage preferences. Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site to further increase the translation efficiency in eukaryotic cells.

The MCS in pEGFP-N1 is between the immediate early promoter of CMV (P CMV IE) and the EGFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of EGFP if they are in the same reading frame as EGFP and there are no intermediate stop codons. SV40 polyadenylation signals downstream of the EGFP gene suitably and directly process the 3' end of the EGFP mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance (Neor) cassette, consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and herpes simplex virus thymidine kinase (HSV TK) gene polyadenylation signals, allows stably transfecting eukaryotic cells for being selected using G418. A bacterial promoter upstream of this kanamycin-resistance cassette is expressed in E. coli. The pEGFP-N1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production. Preparation of DNA-associated Span® 80-based nanoparticles

To produce SP-OA nanoparticles, 6.6 mg/ml of SP and 0.33 mg/ml of OA were dissolved in 30 ml of an organic phase (ethanol) added subsequently under magnetic stirring to 60 ml of an aqueous phase, which leads to the spontaneous formation of nanoparticles. To produce SP-PA nanoparticles, 6.6 mg/ml of SP were dissolved in the organic phase and 0.16 mg/ml of PA were added to the aqueous phase. Ethanol was subsequently eliminated under reduced pressure in a rotary evaporator, and the nanoparticles were concentrated to a final volume of 10 ml. The SP-PA nanoparticles were isolated by filtration-centrifugation (Amicon® Ultra-0.5 Centrifugal Filter Devices, Merck Millipore, Ireland).

The genetic material (pEGFP-C3-GFP or pPRPF31 -PRPF31 ) was associated with the surface of the nanoparticles at a concentration of 0.2 mg/ml or 0.3 mg/ml, respectively, by means of incubation with nanoparticles at a ratio of 1 :1 (v/v) under magnetic stirring at room temperature. For the in vivo study of the formulations, they were administered in a 5% glucose solution.

Characterization ofpDNA nanoparticles

The mean particle size and the nanoparticle size distribution were determined using photon correlation spectroscopy (PCS). To that end, the samples were diluted with filtered Milli-Q water at the suitable concentration. Each analysis was conducted at 25°C with a detection angle of 173°. The values of the zeta potential of the nanoparticles were obtained using laser Doppler anemometry (LDA), measuring the mean electrophoretic mobility. PCS and LDA analyses were performed with a Zetasizer® 3000HS (Malvern Instruments, UK).

In vivo experiments

Animal handling

All the experiments described herein have been conducted in compliance with the Spanish and European Union laws on care of experimental animals. Animal handling and laboratory experimental methods have been analyzed and approved by the Animal Experimentation Committee of CABIMER, Seville, Spain. All efforts were made to reduce the number of animals used and their suffering to a minimum. The animals were housed in the Biological Resource Unit of CABIMER and kept in an environment with controlled temperature (21 ± 1 °C), relative humidity of 55 ± 5%, 08h00-20h00 light/dark cycle, and they were given standard food for mice and water ad libitum.

Subretinal injection

The surgical procedures were performed under general anesthesia by subcutaneous injection of ketamine hydrochloride and xylazine solution (80/12 mg/Kg of body weight). The eyes were topically anesthetized with 0.1 % tetracaine and 0.4% oxybuprocaine. The pupils were dilated with one drop each of 10% phenylephrine and 1 % tropicamide. A 32-gauge needle was used for gently opening the choroids 1 mm posterior to the sclerocorneal limbus. A 10 μΙ syringe (Hamilton, Switzerland) with a 33-gauge needle attached to an UltraMicroPump (World Precision Instruments, Sarasota, FL) was used for slowly injecting 1 μΙ of nanoparticles (200 ng of the GFP plasmid or 300 ng of the PRPF31 plasmid) in the subretinal space. Finally, a drop of antibiotic (0.3% ciprofloxacin) was put in each eye and the animals were kept on a cushion at 37°C until they recovered from the anesthesia. Adeno-associated serotype 2 viral vectors (AAV2) carrying the GFP plasmid, PBS or 5% glucose were also injected in the subretinal space and used as controls. The GFP signals were evaluated using a mouse imaging system (Micron III).

Tested groups

Both eyes of the four mice were injected in each group. The mice were sacrificed by cervical dislocation 72 hours or one month after the subretinal injections. The right eyes were collected for immunofluorescence (IF) studies and the left eyes were collected and analyzed by Western blot (WB). Histology

After cervical dislocation, the right eyes of the injected mice were extirpated and quickly fixed in ice with 4% paraformaldehyde (4% PFA) in PBS overnight at 4°C. The fixed eyes were cryoprotected for 8 hours at room temperature in 20% sucrose-PBS overnight at 4°C and in 30% sucrose-PBS for cryotome sections. 18 μιτι thick serial sections were mounted in five parallel series and processed for IF, incubating with rabbit anti-GFP antibody (1 :100) to improve the GFP signal and prevent the autofluorescence of normal tissue. The sections were mounted with Vectashield mounting medium with DAPI (Vector H-1201 ). Confocal images were captured in a Leica TCS SP5 confocal microscope with an HCX PL APO Lambda blue 63 x 1 .4 OIL lens at 22°C.

Western Blot For neural retina, the crystalline lens, the vitreous humor and aqueous humor are extracted, and the retina was peeled from the RPE. Tissues were rapidly frozen in liquid nitrogen and stored at -80°C until use. Proteins were extracted in cold RIPA buffer plus protease and phosphatase inhibitor cocktails. The protein content was measured using the Bradford assay and the samples were stored at -80°C. 30 g of each extract were separated in a denaturing 10% SDS-PAGE gel and proteins were transferred to a PVDF membrane and it was blocked using 5% NFDM or Superblock (Thermo) for 1 hour. The primary antibody was incubated at 1 mg/ml overnight at 4°C. The membrane was probed with HRP-conjugated secondary antibody for 1 hour at room temperature. The immunoreactive bands were detected by means of chemiluminescence using ECL plus (Amersham) and registered by film exposure and automatic development.

Statistical analysis

All the experimental measurements were taken in triplicate. The values are expressed as the mean ± standard deviation (SD) or the standard error of the mean (SEM). The statistically significant differences between the treatments were evaluated by means of variance analysis (ANOVA) and Fisher's least significant difference (LSD) was evaluated using Statgraphics X64 software. EXAMPLE 2. RESULTS

Preparation and characterization of Span (SP)-based nanoparticles

Span (SP)-based nanoparticles with positive surface charge were developed using oleylamine (OA) or polyarginine (PA) as cationic residues, as described in WO2013068625 A1 . As shown in Table 2, nanometric sized SP-OA or SP-PA nanopartides (180 nm and 230 nm, respectively) having a positive surface charge (43 mV and 32 mV, respectively) were obtained. As expected, the surface charge of these nanopartides changes from positive to negatives values when these nanopartides were incubated with the GFP plasmid (200 g/ml) or PRPF31 plasmid (300 mg/ml), due to the inherent negative charge of these pDNA molecules. These results indicate an effective association of pDNA which was corroborated by the absence of signal bands characteristic of free DNA when agarose gel electrophoresis assays are performed.

Table 2. Physicochemical characteristics of Span-oleylamine (SP-OA)- or Span- polyarginine (SP-PA)-based nanopartides as blank nanopartides (without associated bioactive molecule) or with the association of a GFP plasmid (200 g/ml) or a PRPF31 plasmid (300 mg/ml). (The data are mean ± SD, n = 3).

GFP transfection studies

Blank SP-OA nanopartides (without associated plasmid) or SP-OA nanopartides associated with GFP (SP-OA-GFP) were injected subretinally in mice and the expression of GFP was evaluated 72 hours after administration. As shown in Figure 1 A, the retinal pigment epithelium and ganglion cells showed positive signals for GFP in both cell layers of the retina. Protein expression in these levels was confirmed using Western blot (Figure 1 B). Therefore, the SP-OA-GFP nanopartides were capable of transfecting the pigmented epithelium of the retina in an efficient manner and also the ganglion cells of the mouse retina 72 hours after injection, whereas the viral vectors require more time to provide in vivo transfection (Bennett et al., 1997 Invest. Ophthalmol. Vis. Sci. 38, 2857-2863; Bennett et ai, 1999. Proc. Natl. Acad. Sci. U.S.A., 96, 9920-9925).

For the purpose of further corroborating the efficiency of the developed nanosystems, their transfection capacity was compared with that provided by the adeno-associated serotype 2 viral vectors (AAV2). For this purpose, the three viral and non-viral vectors associating the GFP plasmid (AAV-GFP, SP-OA-GPF and SP-PA-GFP) and the control formulations (PBS, 5% glucose, blank SP-OA nanoparticles and blank SP-PA nanoparticles) were administered subretinally and GFP expression was evaluated one month after injection. As can be seen in Figure 2 - image on the left- evaluation of the ocular fundus of mice treated with AAV2-GFP, SP-PA-GFP and SP-OA-GFP showed a scar in the area of subretinal injections. The GFP signal was also detected in the same injected area, being more intense in mice injected with SP-OA-GFP nanoparticles. Retinal sections of mice treated with these three vectors show GFP signals both in photoreceptors and in RPE cells. Although GFP expression was higher in the SP-OA- GFP injected group, the number of nuclei in the ONL decreased or nuclei were even completely absent in the same injected zone. Therefore, these results suggest that a toxic event occurs in the case of SP-OA-GFP nanoparticles. Taking into account that mice injected with blank SP-OA nanoparticles (without GFP plasmid) do not show any change in retinal layers, as can also be seen in Figure 2 (image on the right), such toxic events can be due to an intense and unexpected high transfection efficiency of these non-viral vectors and the subsequent overexpression of the plasmid mediated by these formulations.

Western blot analysis and the corresponding quantitative study (Figure 3) also suggest a higher transfection efficacy of the non-viral vectors compared with viral vectors. Specifically, these results suggest the following order in the GFP expression levels provided by the different systems that have been studied: SP-OA-GFP> AAV2-GFP> SP-PA-GFP, although statistically significant differences were not observed. In any case, it must be pointed out that this is the first in vivo study comparing transfection levels obtained with viral and non-viral vectors.

To evaluate the developed systems in vivo, the mice were subjected to electroretinogram. Figure 4 shows the rod responses and cone-rod responses determined in dark-adapted mice by means of b-wave and a-wave amplitude, respectively, and cone responses were evaluated in light-adapted mice by means of b- wave amplitude. It can be seen that cone-rod responses were significantly affected by administering these SP-OA-GFP nanoparticles. Specifically, the a-wave and b-wave amplitude decreased considerably in mice treated with SP-OA-GFP nanoparticles. Quantification of the amplitude thereof showed statistically significant differences in rod responses (A, D), cone-rod responses (B, E) and cone responses (C, F) (Figure 5) when compared with the respective control group (treated with a 5% glucose solution), whereas mice injected with blank SP-PA and SP-OA nanoparticles, AAV2-GFP or SP- PA-GFP nanoparticles (associating the GFP plasmid) did not show statistically significant differences. These results can once again be attributed to the intense and unexpected overexpression of GFP after treatment with the SP-OA-GFP non-viral vectors or, in other words, to the high unexpected transfection efficacy resulting in unwanted events.

The conclusions mentioned above concerning the performance of the developed nanoparticles were also supported by the determination of optomotor reflex which involves turning the head in response to moving lines. As shown in Figure 6, although both SP-OA and SP-PA nanoparticles are capable of transfecting mouse retina in vivo, positive optomotor test responses decreased significantly in mice injected with SP-OA- GFP nanoparticles, whereas mice injected with AAV2-GFP and SP-PA-GFP did not show statistically significant differences when compared with control groups. This data also support the explanation provided above by correlating the effect of a high transfection efficiency provided by SP-OA-based nanoparticles instead of the nanoparticles themselves as the cause of potential toxic events. In fact, immunofluorescence images and Western blot results demonstrated that SP-OA-GFP nanoparticles had a higher rate of transfection than AAV2-GFP, which could lead to the aforementioned overexpression of GFP, being strong enough to cause toxic effects, also proven by the reduction described in the a-wave and b-wave amplitude of the electroretinogram and positive optomotor responses. In vivo efficacy in a model of the disease

Once the transfection efficacy provided by the developed nanoparticles being evaluated has been proven, their in vivo potential in a model of the disease is evaluated in a second step. For this purpose, Prpf31^A216P/+ mice (Kl) were selected (Bujakowska et al., 2009. Invest. Ophthalmol. Vis. Sci., 50, 5927-5933) as a mouse model for hereditary retinal disease, for the purpose conducting a preclinical study on the therapeutic activity of Span-based nanoparticles associating the plasmid with PRPF31 as a new nanomedicine for the treatment of haploinsufficiency mentioned above. The animal model used in this study carries the A216P mutation in the human PRPF31 gene, which is known to cause retinitis pigmentosa (Vithana, et al., 2001 . Mol. Cell, 8, 375-381 ). Furthermore, mutations in this gene for producing juvenile macular degeneration are also known (Lu, et al., 2013. PLoS ONE, 8, e78274). Earlier studies conducted by the research group showed that even when the AAV2 vectors are capable of efficiently transfecting RPE cells with the PRPF31 plasmid (Figure 10), no improvement in visual function can be observed by using this viral vector in mutant mice. In fact, the amplitude of different waves of the electroretinogram corresponding to rod responses, cone-rod responses or cone responses decreased in mice injected with AAV2-PRPF31 when compared with the respective negative control group (injected with PBS) (Figure 1 1 ). In order to clarify the efficacy of the developed non-viral vectors (SP-OA and SP-PA nanoparticles), the animals were subjected to different evaluation tests one month after subretinal injection. Once again, the aforementioned high transfection efficacy of SP- OA-based nanoparticles and the subsequent overexpression of the gene can explain the significant reduction in positive optomotor responses that are observed (Figure 12). In addition, SP-PA-based nanoparticles provide a suitable rate of transfection (Figure 7) both for PRPF31 or GFP plasmids (used as control).

The optomotor test was used for evaluating visual function in healthy wild-type mice injected with 5% glucose (WT 5% Glu) and Prpf31^A216P/+ mutant mice treated with SP- PA-PRPF31 nanoparticles (Kl SP-PA-PRPF31) or SP-PA-GFP control nanoparticles (Kl SP-PA-GFP). The graphs of Figure 8 depict the mean positive responses to the optomotor test contrasted at different intensities. The gray line represents the positive response trend of the optomotor tests in mouse models of the disease (untreated mutant Kl) used as control (Figure 13). Different spatial frequencies (cycles/degree = c/d) from 0.031 -0.272 were used to evaluate visual acuity. A statistically significant improvement in visual acuity (100% contrast in each test frequency, p = 0.02 at 0.272 c/d) can be seen in mouse models of the disease after treatment with SP-PA-PRPF31. In fact, with a 100% contrast, the mean positive responses in mouse models of the disease treated with SP-PA-PRPF31 is similar to the control curve of healthy mice injected with a negative control solution (WT 5% Glu). Specifically, a positive optomotor response of 75 ± 2.8% was observed in healthy mice (WT 5% Glu), whereas untreated mutant mice injected with a control formulation associating the GFP plasmid (Kl SP-PA- GFP) showed a positive optomotor response value of only 33.3 ± 4.4%. An unexpected high value of 68.5 ± 8.6% was observed in Prpf31^A216P/+ mutant mice when they were treated with SP-PA-based nanoparticles associating the PRPF31 plasmid. Although the number of positive responses in mice treated with SP-PA-PRPF31 decreases as the contrast intensity decreases, said response is still more favorable than that observed in mice injected with the SP-PA-GFP control formulation and are even above the trend line of mutant mice models of the disease that were not treated. Therefore, it can be affirm that the spatial vision of Kl mouse models of the disease treated with SP-PA-PRPF31 improved only one month after treatment.

For the in-depth study of the performance of the developed nanomedicines, retinal structure and thickness were also determined by means of optical coherence tomography. As can be seen in Figure 9A, the color scales of healthy WT mice treated with a 5% glucose negative control solution and Kl mice, control of the disease, injected with SP-PA-PRPF31 are similar, whereas the levels cannot be reached in mouse models of the disease treated with the SP-PA-GFP control formulation. Furthermore, after determining retinal thickness (Figure 9B), a mean value of 216.6 ± 6.9 μιτι was observed for healthy control mice (WT 5% Glu) and 198.5 ± 1 .9 μιτι for untreated mutant mice (Kl SP-PA-GFP). However, it must be emphasized that when comparing retinal thickness of these untreated Kl mice with that of Kl mice treated with SP-PA- PRPF31 nanoparticles (Kl SP-PA-PRPF31), it was found that retinal thickness increased significantly (p = 0.04) in the treated animals up to a value of 21 1 .9 ± 13.5 microns. In other words, only one month after treatment, the developed nanomedicines not only prevent retinal degeneration, but were also capable of giving rise to a significant and unexpected increase in retinal thickness.

Therefore, the polyarginine-based nanoparticles of the invention were capable of efficiently transfecting PRPF31 at the retinal level in a mouse model of retinal degeneration without causing adverse effects (i.e., toxicity or immunogenicity). Furthermore, visual acuity and retinal thickness of treated mice increased after treatment.

Claims

1 . A nanoparticulate system or composition which comprises nanoparticles comprising a core of a sorbitan ester, a surface layer of a cationic substance and a gene construct consisting of plasmid DNA, wherein the gene construct is physically bound to the cationic substance, and wherein the sorbitan ester is selected from the group consisting of sorbitan mono-, di-, tri- or sesquioleate; sorbitan mono-, di-, tri- or sesquilaurate; sorbitan mono-, di-, tri- or sesquipalmitate; sorbitan mono-, di-, tri- or sesquistearate; and sorbitan mono-, di-, tri- or sesquiisostearate or any of their combinations thereof,

for use in a method of treatment by gene transfer or gene delivery in a mammalian subject.

2. The nanoparticulate system or composition of claim 1 for use according to claim 1 , wherein the cationic substance is a polyamino acid or oleylamine.

3. The nanoparticle system or composition of claim 1 for use according to claim 1 , wherein the cationic substance is polyarginine.

4. The nanoparticulate system or composition according to any of claims 1 to 3 for use according to claim 1 , wherein the gene construct, physically bound to the cationic substance, comprises a polynucleotide sequence comprising at least a coding sequence for a protein with biological activity encoded by a gene selected from the list consisting of: PLA2G5, NMNAT1, RPE65, ABCA4, PRPF3, PRPF31, PRPF8, RD3, EFEMP1, KCNJ13, RGR, RBP4, BEST1, RDH5, RLBP1, CLN3, or any combinations thereof.

5. The nanoparticulate system or composition according to any of the precedent claims for use according to claim 1 , wherein the gene construct comprises a coding sequence for the PRPF31 protein.

6. The nanoparticulate system or composition according to claim 5 for use according to claim 1 , wherein the cationic substance is polyarginine.

7. The nanoparticulate system or composition of any of claims 1 to 3 for use according to claim 1 , wherein said composition is a pharmaceutical composition further comprising pharmaceutically acceptable excipients, carriers and/or adjuvants.

8. The nanoparticulate system or composition of any of claims 4 to 6 for use according to claim 1 , wherein said composition is a pharmaceutical composition further comprising pharmaceutically acceptable excipients, carriers and/or adjuvants.

9. The nanoparticulate system or composition as defined in any of claims 4 to 6 or

8, for use in a method of treatment of abnormalities or dysfunctions of the retinal pigment epithelium.

10. The nanoparticulate system or composition of claim 9 for use according to claim 9, wherein the abnormality or dysfunction of the retinal pigment epithelium is selected from the list consisting of: age related macular degeneration, retinitis pigmentosa, Leber's congenital amaurosis, Stargardt's disease, diabetic retinopathy, macular dystrophy, cataracts, disruption of retinal pigment epithelium cells, deterioration of retinal pigment epithelium cells, death of retinal pigment epithelium cells, drusden, pigment clumping, RPE detachment, geographic atropy, sub-retinal neovascularization and disciform scar, vitelliform macular dystrophy, butterfly macular dystrophy, Sjogren's syndrome, fundus flavimaculatus, dominant foveal dystrophy, central areolar pigment epithelial dystrophy, North Carolina dystrophy, familiar drusen, pattern dystrophy of Marmor and Byers, foveomacular dystrophy (adult type), dominant slowly progressive macular dystrophy of Singerman-Berkow-Patz,macroreticular dystrophy of the RPE, pigment epithelial dystrophy of Noble-Carr-Siegel, benign concentric annular dystrophy, or combinations thereof.

1 1 .The nanoparticulate system or composition of claim 9 for use according to claim

9, wherein the abnormality or dysfunction of the retinal pigment epithelium is selected from the list consisting of Retinitis Pigmentosa, Age-Related Macular Degeneration and/or Leber's congenital amaurosis.

12. The nanoparticulate system or composition of claim 9 for use according to claim 9, wherein the abnormality or dysfunction of the retinal pigment epithelium is Retinitis Pigmentosa.

13. The nanoparticulate system or composition of any of claims 9 to 12 for use according to any of claims 9 to 12, wherein the administration of said system or composition is via subretinal injection.

14. The nanoparticulate system or composition of claim 13 for use according to claim 13, wherein the administration of said system or composition is via subretinal injection in the subretinal space between the photoreceptors and the retinal pigment epithelium (RPE).

15. The system or composition as defined in any of claims 1 to 9 for use according to claim 1 or claims 9 to 12, which is in lyophilized form.

16. The nanoparticulate system or composition as defined in any of claims 4 to 8.

17. The nanoparticulate system or composition as defined in claim 6.

18. The nanoparticulate system or composition according to claim 17, wherein said composition is a pharmaceutical composition further comprising pharmaceutically acceptable excipients, carriers and/or adjuvants.

19. An expression control element, such as a DNA plasmid, comprising a coding sequence for the protein PRPF31 and further comprising the appropriate control elements for the coding sequence to be replicated, transcribed and translated in the appropriate host cells for use in a method of treatment of abnormalities or dysfunctions of the retinal pigment epithelium as defined in any of claims 10 to 12.