TEAR FILM COMPOSITION TO TREAT CORNEAL DEFECTS

FIELD OF THE INVENTION:

The present invention relates to pharmaceutical fied. More particularly, the invention relates to a method for modulating tear film composition in a subject in need thereof.

BACKGROUND OF THE INVENTION:

To ensure clear vision, the anterior parts of the eye, namely the cornea and lens, need to be fully transparent. While the lens is protected by being located inside the eye, the cornea is the most external tissue of the eye, and thus prone to environmental aggressions. This ectodermal organ is subjected to life-long cell renewal, which relies on stem and progenitor cells ¹ . To coordinate epithelium homeostasis, corneal microenvironment is composed of epithelial cell-cell communication, dense innervation, and tear film. The latter is the source of corneal hydration and nutrients for the epithelium ² . Moreover, after wounding, the tear composition changes to support corneal wound healing, through a modification of the factors secreted by the lacrimal gland (LG) ³ . The tear composition change requires an efficient sensory network of the cornea. The dense corneal innervation is essential to maintain corneal physiology. First, blinking and tear composition adaptation depends on the activity of corneal sensory nerves. Furthermore, the neurotrophic factors released by the nerves for the epithelium are crucial for homeostasis and wound healing ⁴ .

Among the most prominent causes for corneal defects, three are making most of the influx of patients in hospital. First cause, physical wounds, such as abrasions ⁵ , are mostly caused by small foreign objects that scratch the epithelium. This injury is painful, and the subsequent oedema provokes photophobia and impairs visual acuity. If the particle gets embedded within the epithelium, corneal irregularities might form, resulting in continuous pain and significant visual disruption ⁶ . Dry eye diseases account for the second cause of corneal defects. Dry eye diseases can arise from genetic disease, such as Gougerot-Sjbrgren syndrome ⁷ , or from ageing, as up to 1/3 of the elder population can be affected ^8,9 . The altered tear film has an imbalanced composition, offering less nutrients and growth factors to the corneal epithelium, which in turn affects corneal homeostasis. Consequently, persistent epithelial defects appear, such as ulceration, melting and perforation, impairing sight ¹⁰ . Third cause, neurotrophic keratitis is due to a partial or complete loss of corneal innervation, causing a defected corneal homeostasis ¹¹ . Neurotrophic keratitis results from defected corneal wound healing after abrasion or transplant, from neurodegenerative diseases, or from chronic metabolic diseases, such as diabetes. Currently 415 million adults globally are diagnosed with diabetes, and the World Health Organization projected that there will be 640 million adults by 2040 ¹² . While being underdiagnosed, diabetic keratopathy affects 47 to 64% of diabetic adults ¹³ . The main symptom of neurotrophic keratitis is corneal ulceration and perforation.

The current treatments for these corneal defects are topical and consist in eye drops, which can be supplemented with autologous serum ¹⁴ , or nerve growth factor (NGF) in the case of neurotrophic keratitis ¹⁵ . Not only these treatments are heavy for patients, especially autologous serum, but the frequent lack of patient compliance to a prescribed eyedrop regimen can lead to increased sight defects ¹⁶ . Their costs are also a burden for the society. There is thus a need to identify novel treatment for these corneal defects.

These treatments solely rely on the addition of an external eye drop solution mimicking the tear film composition without considering the LG which produces, secretes, and modulates the tear film composition. Consequently, using LG directly as a bioreactor would constitute an appealing alternative strategy to modulate the tear film composition. This could be achieved by adenovirus-associated virus (AAV) vector-mediated gene transfer into LG. Indeed, AAV vectors present many advantages for gene delivery. They efficiently transduce a broad range of cells in which they allow long-lasting transgene expression. Importantly, they trigger limited/mild immunogenic responses in vivo which overall asserts their biosafety ¹⁷ . Numerous AAV-based gene therapies have emerged as evidenced by many ongoing clinical trials, namely for neurodegenerative, neuromuscular, cardiovascular, ocular genetic diseases and cancer ^18,19 . Only three AAV-based gene therapies have been approved by the FDA, among which two are still used. Luxturna® (voretigene neparvovec) is used for the treatment of biallelic RPE65 mutation-associated retinal dystrophy ²⁰ while Zolgensma® (onasemnogene abeparvovec-xioi) is delivered to paediatric patients under two years of age suffering from spinal muscular atrophy ²¹ .

In this study, we present a novel strategy to modulate corneal physiology through targeted tear film modification by transferring a gene of interest into LG.

The inventor herein present a novel strategy to rescue corneal physiology through targeted tear film modification by transferring a chosen gene in the lacrimal gland.

SUMMARY OF THE INVENTION: In a first aspect, the present invention relates to methods and pharmaceutical compositions for modulating tear film composition in a subject in need thereof, comprising administering into the lacrimal gland of said subject a vector comprising a nucleic acid encoding a protein of interest. The methods of the invention are particularly suitable to treat corneal defects in subject in need thereof.

In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors confirm that AAV-mediated gene transfer was feasible in murine LG using AAV2/5 or AAV2/9. After demonstrating that all epithelial cell types are prone to AAV- mediated gene transfer, they used AAV2/5-mNGF and AAV2/9-mNGF, as proof of concept, to establish the parameters for efficient gene transfer, allowing a targeted modification of tear composition. They investigated the impact of AAV2 serotype impact on secreted protein level and chose AAV2/9-mNGF to investigate the duration of tear film modulation, as well as the safety of such an approach on the cornea. They show that a single AAV2/9 CAG-mNGF injection in the lacrimal gland does increase the volume of tears. Altogether, their results demonstrate that a single AAV2/9 injection into murine LG could be used to specifically modify the tear film and consequently to support corneal physiology in pathological contexts, such as neurotrophic keratitis, or recurring abscesses. The same kind of targeted tear film modification is obtained after injecting mRNA into murine lacrimal gland. They show that a single injection of mRNA encoding GFP or mNGF is enough to lead to the protein secretion within the tear film for a short period of time.

Their findings provide a new clinically applicable approach to heal corneal pathologies.

Therapeutic methods and uses

Accordingly, in a first aspect the present invention relates to a method for modulating tear film composition in a subject in need thereof, comprising administering into the lacrimal gland of said subject a nucleic acid encoding a protein of interest.

As used herein, the term “tear film”, has its general meaning in the art and refers to a thin fluid layer covering the cornea and acting as barrier between the eye and environment. This film is transparent and has an aqueous/mucin phase, decreasing in mucin concentration towards a distinct superficial lipid layer. The outermost layer of the tear film is composed predominantly of lipids and is referred to as the Tear Film Lipid Layer (TFLL). The most significant role of the lipid layer is in retarding evaporation of tears from the ocular surface. Most of the aqueous fluid is secreted from the lacrimal glands, which also secretes a specific variety of proteins, electrolytes, and water (Dartt D.A et al, 2013).

Alterations of tear lipids composition and properties are related to dry eye syndrome. After wounding, the tear composition changes to support corneal wound healing, through a modification of the factors secreted by the lacrimal gland (Kuony, 2019). The tear composition change requires an efficient sensory network of the cornea. The dense corneal innervation is essential to maintain corneal physiology.

As used herein, the term “subject” refers to any mammals, such as a human, a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with corneal defects. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with disease associated with corneal defects. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with physical wounds corneal, dry eye disease, diabetic keratopathy, corneal thinning disorders such as keratoconus, ICE syndrome or neurotrophic keratitis (NK). Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with drye eye diseases. Particularly, in the present invention, the subject is a human on which an eye surgery (such as corneal transplantation) has been perfomed or will be performed.

As used herein, the term “nucleic acid” has its general meaning in the art and refers to a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).

DNA is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of all known organisms and many viruses. Each strand has a backbone made of alternating sugar (deoxyribose) and phosphate groups. Attached to each sugar is one of four bases: adenine (A), cytosine (C), guanine (G) or thymine (T). RNA is a single-stranded biopolymer which consists of ribose nucleotides (nitrogenous bases appended to a ribose sugar) attached by phosphodiester bonds, forming strands of varying lengths. The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil, which replaces thymine in DNA. RNA includes messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA. mRNA refers to RNA made from a DNA template during the process of transcription. mRNA is read by a ribosome in the process of synthesizing a protein. In particular embodiment, the nucleic acid is a messenger RNA (mRNA).

Nucleid acid (DNA or RNA) as used herein encompasses both modified and unmodified nucleic acid. DNA or RNA (such mRNA) may contain one or more coding and non-coding regions. DNA or RNA (such as mRNA) can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, DNA mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An nucleic acide sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, the mRNA comprises natural nucleosides {e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs {e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5- fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases {e.g., methylated bases); intercalated bases; modified sugars {e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups {e.g., phosphorothioates and 5'-N-phosphoramidite linkages). In some embodiments, the DNA comprises natural nucleosides {e.g., adenosine, guanosine, cytidine, thymine), 4- acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl -2 -thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine,

1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine,

5- methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,

2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine. As used herein, the term “lacrimal gland” (LG) has its general meaning in the art and refers to exocrine gland that secret the aqueous layer of the tear film onto the surface of the conjunctiva and cornea of the eye. In some embodiments, the nucleic acid encoding a protein of interest, in particular the messenger ribonucleic acid (mRNA) is administered into the lacrimal gland which has been previously anesthetized. In some embodiments, the vector is administered into the lacrimal gland which has been previously anesthetized.

In some embodiment, according to all the methods of the invention, the protein of interest is a growth factor. According to the invention, the term “protein of interest” includes natural and recombinant analog of the protein of interest.

As used herein, the term “growth factor” has its general meaning in the art, and refers to a naturally occurring substance capable of stimulating cell proliferation, wound healing, and occasionally cellular differentiation. In some embodiment, the protein of interest is a growth factor promoting healing of corneal. According to the invention, the term “growth factor” includes natural and recombinant analog of the growth factor.

Growth factors promoting healing of corneal are very well known in the art and include adrenomedullin, angiopoietin, vascular Endothelial Growth Factor (VEGF) such as VEGF-A, epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), autocrine motility factor, ephrins, hepatocyte growth factor (HGF), neuropilin 2 (NRP2), semaphorin (SEMA) such as semaphorin 3C (SEMA3C), pigment epithelium-derived factor (PEDF) also known as serpin Fl (SERPINF1), thyroglobuline , insulin, insulin-like growth factors (IGF) such as insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF -2), interleukins, neuregulins, neurotrophins such as nerve growth factor (NGF), brain-derived neutrophic factor (BDNF), neurothropin-3 (NT-3) and neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), transforming growth factor such as transforming growth factor alpha (TGF-a) and transforming growth factor beta (TGF-P), tumor necrosis factor-alpha (TNF- a), glial-cell derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), such as fibroblast growth factor 7 (FGF-7) also know as keratinocyte growth factor (KGF), and bone morphogenesis protein (BMP), such as bone morphogenesis protein 6 (BMP-6).

In some embodiment, according to all the methods of the invention, the protein of interest is selected in the group consisting of nerve growth factor (NGF), vascular Endothelial Growth Factor A (VEGF-A), epidermal growth factor (EGF), brain-derived neutrophic factor (BDNF), ciliary neurotrophic factor (CNTF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), neuropilin 2 (NRP2), semaphorin 3C SEMA3C, pigment epithelium- derived factor (PEDF) also known as serpin Fl (SERPINF1), thyroglobuline, insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), neurothropin-3 (NT-3) , neurothropin-4 (NT -4), tumor necrosis factor-alpha (TNF- a), insulin, bone morphogenesis protein 6 (BMP-6), glial-cell derived neurotrophic factor (GDNF) and transforming growth factor (TGF).

In some embodiment, according to all the methods of the invention, the protein of interest is selected in the group consisting of nerve growth factor (NGF), neuropilin 2 (NRP2), semaphorin 3C (SEMA3C), pigment epithelium-derived factor (PEDF), vascular Endothelial Growth Factor A (VEGF-A), thyroglobuline, epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), glial-cell derived neurotrophic factor (GDNF) and transforming growth factor (TGF).

As used the term “nerve growth factor” (NGF) has its general meaning in the art and refers to a neurotrophic factor and neuropeptide primarily involved in the regulation of growth, proliferation of nerve cells. The NGF gene is located in chromosome 1. Its Entrez reference is 4803. Its Unit Prot is P01138. Exemplary amino acid sequences of NGF include sequences a set forth in SEQ ID NO: 1. Exemplary acid nucleic coding for the nerve growth factor is NM 002506. In particular embodiment, the nucleic acid encoding NGF comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO:2. In particular embodiment, the nucleic acid encoding NGF comprises or consists of SEQ ID NO:2. In particular embodiment, the nerve growth factor is a recombinant human nerve growth factor. In particular embodiment the nerve growth factor is cemergermin.

According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

SEQ ID NO: 1 : amino acid sequence of the human NGF

MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSA PAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAP FNRTHRSKRS S SHPIFHRGEF S VCD S VS VW VGDKTT ATDIKGKEVMVLGEVNINNS VF KQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDT ACVCVLSRKAVRRA SEQ ID NO :2 : nucleic acid sequence coding for human NGF atgtccatgttgttctacactctgatcacagcttttctgatcggcatacaggcggaacca cactcagagagcaatgtccctgca ggacacaccatcccccaagcccactggactaaacttcagcattcccttgacactgccctt cgcagagcccgcagcgccccggcagcg gcgatagctgcacgcgtggcggggcagacccgcaacattactgtggaccccaggctgttt aaaaagcggcgactccgttcaccccgt gtgctgtttagcacccagcctccccgtgaagctgcagacactcaggatctggacttcgag gtcggtggtgctgcccccttcaacaggac tcacaggagcaagcggtcatcatcccatcccatcttccacaggggcgaattctcggtgtg tgacagtgtcagcgtgtgggttggggata agaccaccgccacagacatcaagggcaaggaggtgatggtgttgggagaggtgaacatta acaacagtgtattcaaacagtactttttt gagaccaagtgccgggacccaaatcccgttgacagcgggtgccggggcattgactcaaag cactggaactcatattgtaccacgact cacacctttgtcaaggcgctgaccatggatggcaagcaggctgcctggcggtttatccgg atagatacggcctgtgtgtgtgtgctcag caggaaggctgtgagaagagcctga

As used the term “fibroblast growth factor” (FGF), has its general meaning in the art and refers to a growth factor produced by macrophages; they are involved in a wide variety of processes, most notably as crucial elements for normal development in animal cells. Any irregularities in their function lead to a range of developmental defects. FGF family include FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF- 12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, and FGF -23. In particular, the FGF is FGF-7 (or KGF). In particular embodiment, the fibroblast growth factor is a recombinant human fibroblast growth factor.

As used the term “keratinocyte growth factor” (KGF), also known as FGF7, has its general meaning in the art and refers to a growth factor present in the epithelialization-phase of wound healing that binds to fibroblast growth factor receptor 2b (FGFR2b). The FGF7 gene is located in chromosome 15. Its Entrez reference is 2252. Its Unit Prot is P21781. Exemplary acid nucleic coding for the keratinocyte growth factor is NM 002009. Exemplary amino acid sequences of NGF include sequences a set forth in SEQ ID NO:3. In particular embodiment, the nucleic acid encoding KGF comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO:4. In particular embodiment, the nucleic acid encoding KGF comprises or consists of SEQ ID NO:4. In particular embodiment, the keratinocyte growth factor is a recombinant human fibroblast growth factor.

SEQ ID NO:3: amino acid sequence of the human KGF

MHKWILTWILPTLLYRSCFHIICL VGTISLACNDMTPEQMATNVNC S SPERHTR SYDYMEGGDIRVRRLFCRTQWYLRIDKRGKVKGTQEMKNNYNIMEIRTVAVGIVAI KGVESEFYLAMNKEGKLYAKKECNEDCNFKELILENHYNTYASAKWTHNGGEMFV

ALNQKGIPVRGKKTKKEQKTAHFLPMAIT

SEQ ID NO :4 nucleic acid sequence coding for human KGF atgcacaaatggatactgacatggatcctgccaactttgctctacagatcatgctttcac attatctgtctagtgggtactatatct ttagcttgcaatgacatgactccagagcaaatggctacaaatgtgaactgttccagccct gagcgacacacaagaagttatgattacatg gaaggaggggatataagagtgagaagactcttctgtcgaacacagtggtacctgaggatc gataaaagaggcaaagtaaaagggac ccaagagatgaagaataattacaatatcatggaaatcaggacagtggcagttggaattgt ggcaatcaaaggggtggaaagtgaattct atcttgcaatgaacaaggaaggaaaactctatgcaaagaaagaatgcaatgaagattgta acttcaaagaactaattctggaaaaccatt acaacacatatgcatcagctaaatggacacacaacggaggggaaatgtttgttgccttaa atcaaaaggggattcctgtaagaggaaaa aaaacgaagaaagaacaaaaaacagcccactttcttcctatggcaataacttaa

As used the term “insulin-like growth factor” (IGF-1), also known as somatomedin C, has its general meaning in the art and refers to a hormone similar to insulin. The IGF1 gene is located in chromosome 12. Its Entrez reference is 3479. Its Unit Prot is P05019. Exemplary of acid nucleic coding for the insulin-like growth factor is NM_000618, NM_001111283, NM_001111284 or NM_001111285. Exemplary amino acid sequences of IGF-1 include sequences a set forth in SEQ ID NO: 5. In particular embodiment, the nucleic acid encoding IGF-1 comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO:6. In particular embodiment, the nucleic acid encoding IGF-1 comprises or consists of SEQ ID NO:6. In particular embodiment, the insulin-like growth factor is a recombinant human insulin-like growth factor.

SEQ ID NO:5: amino acid sequence of the human IGF-1

MGKIS SLPTQLFKCCFCDFLKVKMHTMS S SHLF YLALCLLTFTS S AT AGPETLC GAELVDALQF VCGDRGF YFNKPTGYGS S SRRAPQTGIVDECCFRSCDLRRLEMYC AP LKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSAGNKNYRM

SEQ ID NO:6: nucleic acid sequence coding for human IGF-1 atgggaaaaatcagcagtcttccaacccaattatttaagtgctgcttttgtgatttcttg aaggtgaagatgcacaccatgtcctc ctcgcatctcttctacctggcgctgtgcctgctcaccttcaccagctctgccacggctgg accggagacgctctgcggggctgagctgg tggatgctcttcagttcgtgtgtggagacaggggcttttatttcaacaagcccacagggt atggctccagcagtcggagggcgcctcag acaggcatcgtggatgagtgctgcttccggagctgtgatctaaggaggctggagatgtat tgcgcacccctcaagcctgccaagtcag ctcgctctgtccgtgcccagcgccacaccgacatgcccaagacccagaaggaagtacatt tgaagaacgcaagtagagggagtgca ggaaacaagaactacaggatgtag

As used the term “hepatocyte growth factor” (HGF), also known as scatter factor (SF) has its general meaning in the art and refers to a paracrine cellular growth, motility and morphogenic factor secreted by mesenchymal cells. The HGF gene is located in chromosome 7. Its Entrez reference is 3082. Its Unit Prot is P14210. Exemplary acid nucleic coding for the hepatocyte growth factor is NM_000601, NM_001010931, NM_001010932, NM_001010933 or NM 001010934. Exemplary amino acid sequences of HGF include sequences a set forth in SEQ ID NO:7. In particular embodiment, the nucleic acid encoding HGF comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO: 8. In particular embodiment, the nucleic acid encoding HGF comprises or consists of SEQ ID NO:8. In particular embodiment, the hepatocye growth factor is a recombinant human hepatocyte growth factor.

SEQ ID NO:7: amino acid sequence of the human HGF

MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKID PALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKK EFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRG KDLQENYCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDH TESGKICQRWDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWE YCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEH DMTPENFKCKDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRG NGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAH GPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIG WMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHGRGDEKCK QVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGY TGLINYDGLLRVAHLYIMGNEKCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGP LVCEQHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS

SEQ ID NO: 8: nucleic acid sequence coding for human HGF atgtgggtgaccaaactcctgccagccctgctgctgcagcatgtcctcctgcatctcctc ctgctccccatcgccatcccctat gcagagggacaaaggaaaagaagaaatacaattcatgaattcaaaaaatcagcaaagact accctaatcaaaatagatccagcactga agataaaaaccaaaaaagtgaatactgcagaccaatgtgctaatagatgtactaggaata aaggacttccattcacttgcaaggcttttgt ttttgataaagcaagaaaacaatgcctctggttccccttcaatagcatgtcaagtggagt gaaaaaagaatttggccatgaatttgacctct atgaaaacaaagactacattagaaactgcatcattggtaaaggacgcagctacaagggaa cagtatctatcactaagagtggcatcaaa tgtcagccctggagttccatgataccacacgaacacagctttttgccttcgagctatcgg ggtaaagacctacaggaaaactactgtcga aatcctcgaggggaagaagggggaccctggtgtttcacaagcaatccagaggtacgctac gaagtctgtgacattcctcagtgttcag aagttgaatgcatgacctgcaatggggagagttatcgaggtctcatggatcatacagaat caggcaagatttgtcagcgctgggatcatc agacaccacaccggcacaaattcttgcctgaaagatatcccgacaagggctttgatgata attattgccgcaatcccgatggccagccg aggccatggtgctatactcttgaccctcacacccgctgggagtactgtgcaattaaaaca tgcgctgacaatactatgaatgacactgat gttcctttggaaacaactgaatgcatccaaggtcaaggagaaggctacaggggcactgtc aataccatttggaatggaattccatgtcag cgttgggattctcagtatcctcacgagcatgacatgactcctgaaaatttcaagtgcaag gacctacgagaaaattactgccgaaatcca gatgggtctgaatcaccctggtgttttaccactgatccaaacatccgagttggctactgc tcccaaattccaaactgtgatatgtcacatgg acaagattgttatcgtgggaatggcaaaaattatatgggcaacttatcccaaacaagatc tggactaacatgttcaatgtgggacaagaa catggaagacttacatcgtcatatcttctgggaaccagatgcaagtaagctgaatgagaa ttactgccgaaatccagatgatgatgctcat ggaccctggtgctacacgggaaatccactcattccttgggattattgccctatttctcgt tgtgaaggtgataccacacctacaatagtcaa tttagaccatcccgtaatatcttgtgccaaaacgaaacaattgcgagttgtaaatgggat tccaacacgaacaaacataggatggatggtt agtttgagatacagaaataaacatatctgcggaggatcattgataaaggagagttgggtt cttactgcacgacagtgtttcccttctcgag acttgaaagattatgaagcttggcttggaattcatgatgtccacggaagaggagatgaga aatgcaaacaggttctcaatgtttcccagct ggtatatggccctgaaggatcagatctggttttaatgaagcttgccaggcctgctgtcct ggatgattttgttagtacgattgatttacctaat tatggatgcacaattcctgaaaagaccagttgcagtgtttatggctggggctacactgga ttgatcaactatgatggcctattacgagtgg cacatctctatataatgggaaatgagaaatgcagccagcatcatcgagggaaggtgactc tgaatgagtctgaaatatgtgctggggct gaaaagattggatcaggaccatgtgagggggattatggtggcccacttgtttgtgagcaa cataaaatgagaatggttcttggtgtcattg ttcctggtcgtggatgtgccattccaaatcgtcctggtatttttgtccgagtagcatatt atgcaaaatggatacacaaaattattttaacatat aaggtaccacagtcatag

As used the term “transforming growth factor” (TGF), also known as “tumor growth factor” has its general meaning in the art and refers to a family of polypeptide growth factors. In particular embodiment, the transforming growth factor is a recombinant human transforming growth factor. TGF include TGFa and TGFp. Transforming growth factor alpha (TGF-a) is a protein that in humans is encoded by the TGFA gene, located in chromosome 2. Its Entrez reference is 7039. Exemplary acid nucleic coding for the TGFa is NM 001099691, NM_001308158, NM_001308159 or NM_003236. Its Uniprot reference is P01135. Exemplary amino acid sequences of TGF-a include sequences a set forth in SEQ ID NO:9. In particular embodiment, the nucleic acid encoding TGF-a comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ IDNO: 10. In particular embodiment, the nucleic acid encoding TGF-a comprises or consists of SEQ ID NO: 10. In particular embodiment, the transforming growth factor a is a recombinant human transforming growth factor a. Transforming growth factor beta (TGF-P) is a multifunctional cytokine that include three different mammalian isoforms (TGF-pi, TGF-P2 and TGF-P3). TGF-pi is encoded by the TGFB1 gene. Its Entrez reference is 7040. Exemplary acid nucleic coding for the TGF-pi is NM 000660. Its Uniprot reference is P01137. Exemplary amino acid sequences of TGF-P 1 include sequences a set forth in SEQ ID NO: 11. In particular embodiment, the nucleic acid encoding TGF-pi comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO: 12. In particular embodiment, the nucleic acid encoding TGF- pi comprises or consists of SEQ ID NO: 12. In particular embodiment, the transforming growth factor pi is a recombinant human transforming growth factor pi. TGF-P2 is encoded by the TGFB2 gene. Its Entrez reference is 7042. Exemplary acid nucleic coding for the TGF- P2 is NM_003238 or NM_001135599. In particular embodiment, the transforming growth factor P2 is a recombinant human transforming growth factor P2. Exemplary amino acid sequences of TGF- P2 include sequences a set forth in SEQ ID NO: 13. In particular embodiment, the nucleic acid encoding TGF- P2 comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO: 14. In particular embodiment, the nucleic acid encoding TGF- P2 comprises or consists of SEQ ID NO: 14. TGF-P3 is encoded by the TGFB3 gene. Its Entrez reference is 7043. Exemplary acid nucleic coding for the TGF- P2 is NM_003239, NM_001329938 or NM_001329939. Its Uniprot reference is P61812. Exemplary amino acid sequences of TGF- P2 include sequences a set forth in SEQ ID NO: 15. In particular embodiment, the DNA encoding TGF- P2 comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO: 16. In particular embodiment, the nucleic acid encoding TGF- P2 comprises or consists of SEQ ID NO: 16. In particular embodiment, the transforming growth factor P3 is a recombinant human transforming growth factor P3.

SEQ ID NO:9: amino acid sequence of the human TGF-a

MVPSAGQLALFALGIVLAACQALENSTSPLSADPPVAAAVVSHFNDCPDSHTQ FCFHGTCRFLVQEDKPACVCHSGYVGARCEHADLLAVVAASQKKQAITALVVVSIV ALAVLIITCVLIHCCQVRKHCEWCRALICRHEKPSALLKGRTACCHSETVV

SEQ ID NO: 10: nucleic acid sequence coding for human TGF-a

Atggtcccctcggctggacagctcgccctgttcgctctgggtattgtgttggctgcg tgccaggccttggagaacagcacgt ccccgctgagtgcagacccgcccgtggctgcagcagtggtgtcccattttaatgactgcc cagattcccacactcagttctgcttccatg gaacctgcaggtttttggtgcaggaggacaagccagcatgtgtctgccattctgggtacg ttggtgcacgctgtgagcatgcggacctc ctggccgtggtggctgccagccagaagaagcaggccatcaccgccttggtggtggtctcc atcgtggccctggctgtccttatcatcac atgtgtgctgatacactgctgccaggtccgaaaacactgtgagtggtgccgggccctcat ctgccggcacgagaagcccagcgccct cctgaagggaagaaccgcttgctgccactcagaaacagtggtctga

SEQ ID NO: 11 : amino acid sequence of the human TGF-pi

MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILS KLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVL MVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVEL YQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDS RDNTLQVDINGFTTGRRGDL ATIHGMNRPFLLLMATPLERAQHLQ S SRHRR ALDTN Y CF S STEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCP YIWSLDTQ YSKVLA LYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

SEQ ID NO: 12: nucleic acid sequence coding for human TGF-pi atgccgccctccgggctgcggctgctgccgctgctgctaccgctgctgtggctactggtg ctgacgcctggccggccggc cgcgggactatccacctgcaagactatcgacatggagctggtgaagcggaagcgcatcga ggccatccgcggccagatcctgtcca agctgcggctcgccagccccccgagccagggggaggtgccgcccggcccgctgcccgagg ccgtgctcgccctgtacaacagca cccgcgaccgggtggccggggagagtgcagaaccggagcccgagcctgaggccgactact acgccaaggaggtcacccgcgtg ctaatggtggaaacccacaacgaaatctatgacaagttcaagcagagtacacacagcata tatatgttcttcaacacatcagagctccga gaagcggtacctgaacccgtgttgctctcccgggcagagctgcgtctgctgaggctcaag ttaaaagtggagcagcacgtggagctgt accagaaatacagcaacaattcctggcgatacctcagcaaccggctgctggcacccagcg actcgccagagtggttatcttttgatgtc accggagttgtgcggcagtggttgagccgtggaggggaaattgagggctttcgccttagc gcccactgctcctgtgacagcagggata acacactgcaagtggacatcaacgggttcactaccggccgccgaggtgacctggccacca ttcatggcatgaaccggcctttcctgctt ctcatggccaccccgctggagagggcccagcatctgcaaagctcccggcaccgccgagcc ctggacaccaactattgcttcagctcc acggagaagaactgctgcgtgcggcagctgtacattgacttccgcaaggacctcggctgg aagtggatccacgagcccaagggcta ccatgccaacttctgcctcgggccctgcccctacatttggagcctggacacgcagtacag caaggtcctggccctgtacaaccagcat aacccgggcgcctcggcggcgccgtgctgcgtgccgcaggcgctggagccgctgcccatc gtgtactacgtgggccgcaagccca aggtggagcagctgtccaacatgatcgtgcgctcctgcaagtgcagctga

SEQ ID NO: 13: amino acid sequence of the human TGF-P2

MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPP EDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFP SETVCPVVTTPSGSVGSLCSRQSQVLCGYLDAIPPTFYRPYFRIVRFDVSAMEKNASN LVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSF DVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTY TSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDN CCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPE ASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS

SEQ ID NO: 14: nucleic acid sequence coding for human TGF-P2 atgcactactgtgtgctgagcgcttttctgatcctgcatctggtcacggtcgcgctcagc ctgtctacctgcagcacactcgat atggaccagttcatgcgcaagaggatcgaggcgatccgcgggcagatcctgagcaagctg aagctcaccagtcccccagaagacta tcctgagcccgaggaagtccccccggaggtgatttccatctacaacagcaccagggactt gctccaggagaaggcgagccggaggg cggccgcctgcgagcgcgagaggagcgacgaagagtactacgccaaggaggtttacaaaa tagacatgccgcccttcttcccctcc gaaactgtctgcccagttgttacaacaccctctggctcagtgggcagcttgtgctccaga cagtcccaggtgctctgtgggtaccttgat gccatcccgcccactttctacagaccctacttcagaattgttcgatttgacgtctcagca atggagaagaatgcttccaatttggtgaaagc agagttcagagtctttcgtttgcagaacccaaaagccagagtgcctgaacaacggattga gctatatcagattctcaagtccaaagattta acatctccaacccagcgctacatcgacagcaaagttgtgaaaacaagagcagaaggcgaa tggctctccttcgatgtaactgatgctgt tcatgaatggcttcaccataaagacaggaacctgggatttaaaataagcttacactgtcc ctgctgcacttttgtaccatctaataattacat catcccaaataaaagtgaagaactagaagcaagatttgcaggtattgatggcacctccac atataccagtggtgatcagaaaactataaa gtccactaggaaaaaaaacagtgggaagaccccacatctcctgctaatgttattgccctc ctacagacttgagtcacaacagaccaacc ggcggaagaagcgtgctttggatgcggcctattgctttagaaatgtgcaggataattgct gcctacgtccactttacattgatttcaagag ggatctagggtggaaatggatacacgaacccaaagggtacaatgccaacttctgtgctgg agcatgcccgtatttatggagttcagaca ctcagcacagcagggtcctgagcttatataataccataaatccagaagcatctgcttctc cttgctgcgtgtcccaagatttagaacctcta accattctctactacattggcaaaacacccaagattgaacagctttctaatatgattgta aagtcttgcaaatgcagctaa

SEQ ID NO: 15: amino acid sequence of the human TGF-P3

MKMHLQRALVVL ALLNF ATVSLSLSTCTTLDFGHIKKKRVEAIRGQILSKLRL TSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMI QGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIE LFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCP CHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPP HRLDNPGQGGQRKKRALDTNYCFR

SEQ ID NO: 16: nucleic acid sequence of the human TGF-P3 atgaagatgcacttgcaaagggctctggtggtcctggccctgctgaactttgccacggtc agcctctctctgtccacttgcacc accttggacttcggccacatcaagaagaagagggtggaagccattaggggacagatcttg agcaagctcaggctcaccagcccccct gagccaacggtgatgacccacgtcccctatcaggtcctggccctttacaacagcacccgg gagctgctggaggagatgcatgggga gagggaggaaggctgcacccaggaaaacaccgagtcggaatactatgccaaagaaatcca taaattcgacatgatccaggggctgg cggagcacaacgaactggctgtctgccctaaaggaattacctccaaggttttccgcttca atgtgtcctcagtggagaaaaatagaacca acctattccgagcagaattccgggtcttgcgggtgcccaaccccagctctaagcggaatg agcagaggatcgagctcttccagatcctt cggccagatgagcacattgccaaacagcgctatatcggtggcaagaatctgcccacacgg ggcactgccgagtggctgtcctttgatg tcactgacactgtgcgtgagtggctgttgagaagagagtccaacttaggtctagaaatca gcattcactgtccatgtcacacctttcagcc caatggagatatcctggaaaacattcacgaggtgatggaaatcaaattcaaaggcgtgga caatgaggatgaccatggccgtggagat ctggggcgcctcaagaagcagaaggatcaccacaaccctcatctaatcctcatgatgatt cccccacaccggctcgacaacccgggc caggggggtcagaggaagaagcgggctttggacaccaattactgcttccggtga

As used the term “bone morphogenesis protein” (BMP) has its general meaning in the art and refers to a group of growth factors also known as cytokines and as metabologens. BMPs interact with specific receptors on the cell surface, referred to as bone morphogenetic protein receptors (BMPRs). BMP family include BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP11 and BMP15. In particular, the BMP is BMP-6.

As used the term “Bone morphogenetic protein 6” (BMP-6), also known as VGR, has its general meaning in the art and refers to the key regulator of hepcidin, the small peptide secreted by the liver which is the major regulator of iron metabolism in mammals. Its Entrez reference is 654. Exemplary acid nucleic coding for the keratinocyte growth factor is NM_001718. Its Uniprot reference is P22004. Exemplary amino acid sequences of BMP-6 include sequences a set forth in SEQ ID NO: 17. In particular embodiment, the nucleic acid encoding BMP-6 comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO: 18. In particular embodiment, the nucleic acid encoding BMP-6 comprises or consists of SEQ ID NO: 18. In particular embodiment, the bone morphogenetic protein 6 is a recombinant human bone morphogenetic protein 6.

SEQ ID NO: 17: amino acid sequence of the human BMP-6

MPGLGRRAQWLCWWWGLLCSCCGPPPLRPPLPAAAAAAAGGQLLGDGGSP GRTEQPPPSPQSSSGFLYRRLKTQEKREMQKEILSVLGLPHRPRPLHGLQQPQPPALRQ QEEQQQQQQLPRGEPPPGRLKSAPLFMLDLYNALSADNDEDGASEGERQQSWPHEA ASSSQRRQPPPGAAHPLNRKSLLAPGSGSGGASPLTSAQDSAFLNDADMVMSFVNLV EYDKEFSPRQRHHKEFKFNLSQIPEGEVVTAAEFRIYKDCVMGSFKNQTFLISIYQVLQ EHQHRDSDLFLLDTRVVWASEEGWLEFDITATSNLWVVTPQHNMGLQLSVVTRDGV HVHPRAAGLVGRDGPYDKQPFMVAFFKVSEVHVRTTRSASSRRRQQSRNRSTQSQD VARVSSASDYNSSELKTACRKHELYVSFQDLGWQDWIIAPKGYAANYCDGECSFPL

NAHMNATNHAIVQTLVHLMNPEYVPKPCCAPTKLNAISVLYFDDNSNVILKKYRNM

VVRACGCH

SEQ ID NO: 18: amino acid sequence of the human BMP-6 atgccggggctggggcggagggcgcagtggctgtgctggtggtgggggctgctgtgcagc tgctgcgggcccccgccg ctgcggccgcccttgcccgctgccgcggccgccgccgccggggggcagctgctgggggac ggcgggagccccggccgcacgg agcagccgccgccgtcgccgcagtcctcctcgggcttcctgtaccggcggctcaagacgc aggagaagcgggagatgcagaagga gatcttgtcggtgctggggctcccgcaccggccccggcccctgcacggcctccaacagcc gcagcccccggcgctccggcagcag gaggagcagcagcagcagcagcagctgcctcgcggagagccccctcccgggcgactgaag tccgcgcccctcttcatgctggatct gtacaacgccctgtccgccgacaacgacgaggacggggcgtcggagggggagaggcagca gtcctggccccacgaagcagcca gctcgtcccagcgtcggcagccgcccccgggcgccgcgcacccgctcaaccgcaagagcc ttctggcccccggatctggcagcgg cggcgcgtccccactgaccagcgcgcaggacagcgccttcctcaacgacgcggacatggt catgagctttgtgaacctggtggagta cgacaaggagttctcccctcgtcagcgacaccacaaagagttcaagttcaacttatccca gattcctgagggtgaggtggtgacggctg cagaattccgcatctacaaggactgtgttatggggagttttaaaaaccaaacttttctta tcagcatttatcaagtcttacaggagcatcagc acagagactctgacctgtttttgttggacacccgtgtagtatgggcctcagaagaaggct ggctggaatttgacatcacggccactagca atctgtgggttgtgactccacagcataacatggggcttcagctgagcgtggtgacaaggg atggagtccacgtccacccccgagccg caggcctggtgggcagagacggcccttacgacaagcagcccttcatggtggctttcttca aagtgagtgaggtgcacgtgcgcacca ccaggtcagcctccagccggcgccgacaacagagtcgtaatcgctctacccagtcccagg acgtggcgcgggtctccagtgcttcag attacaacagcagtgaattgaaaacagcctgcaggaagcatgagctgtatgtgagtttcc aagacctgggatggcaggactggatcatt gcacccaagggctatgctgccaattactgtgatggagaatgctccttcccactcaacgca cacatgaatgcaaccaaccacgcgattgt gcagaccttggttcaccttatgaaccccgagtatgtccccaaaccgtgctgtgcgccaac taagctaaatgccatctcggttctttactttg atgacaactccaatgtcattctgaaaaaatacaggaatatggttgtaagagcttgtggat gccactaa

As used the term “epidermal growth factor” (EGF) has its general meaning in the art and refers to a protein that stimulates cell growth and differentiation by binding to its receptor, EGFR. The EGF gene is located in chromosome 4. Its Entrez reference is 1950. Exemplary acid nucleic coding for the epidermal growth factor is NM_001178130, NM_001178131, NM_001963 and NM_001357021. Its Uniprot reference is P01133. Exemplary amino acid sequences of EGF include sequences a set forth in SEQ ID NO: 19. In particular embodiment, the DNA encoding EGF comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO:20. In particular embodiment, the nucleic acid encoding EGF comprises or consists of SEQ ID NO:20. In particular embodiment, the epidermal growth factor is a recombinant human epidermal growth factor. In some embodiments, the methods according to the invention, wherein the vector is a non-viract vector when the acid nucleic encodes for EGF.

SEQ ID NO: 19: amino acid sequence of the human EGF

MLLTLIILLPVVSKFSFVSLSAPQHWSCPEGTLAGNGNSTCVGPAPFLIFSHGNS IFRIDTEGTNYEQLVVDAGVSVIMDFHYNEKRIYWVDLERQLLQRVFLNGSRQERVC NIEKNVSGMAINWINEEVIWSNQQEGIITVTDMKGNNSHILLSALKYPANVAVDPVER FIFWSSEVAGSLYRADLDGVGVKALLETSEKITAVSLDVLDKRLFWIQYNREGSNSLI CSCDYDGGSVHISKHPTQHNLFAMSLFGDRIFYSTWKMKTIWIANKHTGKDMVRINL HS SFVPLGELKVVHPLAQPKAEDDTWEPEQKLCKLRKGNC S STVCGQDLQSHLCMC AEGYALSRDRKYCEDVNECAFWNHGCTLGCKNTPGSYYCTCPVGFVLLPDGKRCH QLVSCPRNVSECSHDCVLTSEGPLCFCPEGSVLERDGKTCSGCSSPDNGGCSQLCVPL SPVSWECDCFPGYDLQLDEKSCAASGPQPFLLFANSQDIRHMHFDGTDYGTLLSQQM GMVYALDHDPVENKIYFAHTALKWIERANMDGSQRERLIEEGVDVPEGLAVDWIGR RFYWTDRGKSLIGRSDLNGKRSKIITKENISQPRGIAVHPMAKRLFWTDTGINPRIESS SLQGLGRLVIASSDLIWPSGITIDFLTDKLYWCDAKQSVIEMANLDGSKRRRLTQNDV GHPFAVAVFEDYVWFSDWAMPSVMRVNKRTGKDRVRLQGSMLKPSSLVVVHPLAK PGADPCLYQNGGCEHICKKRLGTAWCSCREGFMKASDGKTCLALDGHQLLAGGEV DLKNQVTPLDILSKTRVSEDNITESQHMLVAEIMVSDQDDCAPVGCSMYARCISEGE DATCQCLKGFAGDGKLCSDIDECEMGVPVCPPASSKCINTEGGYVCRCSEGYQGDGI HCLDIDECQLGEHSCGENASCTNTEGGYTCMCAGRLSEPGLICPDSTPPPHLREDDHH YSVRNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWE LRHAGHGQQQKVIVVAVCVVVLVMLLLLSLWGAHYYRTQKLLSKNPKNPYEESSR DVRSRRPADTEDGMSSCPQPWFVVIKEHQDLKNGGQPVAGEDGQAADGSMQPTSW RQEPQLCGMGTEQGCWIPVSSDKGSCPQVMERSFHMPSYGTQTLEGGVEKPHSLLSA NPLWQQRALDPPHQMELTQ

SEQ ID NO:20: nucleic acid sequence of the human EGF atgctgctcactcttatcattctgttgccagtagtttcaaaatttagttttgttagtctc tcagcaccgcagcactggagctgtcctg aaggtactctcgcaggaaatgggaattctacttgtgtgggtcctgcacccttcttaattt tctcccatggaaatagtatctttaggattgacac agaaggaaccaattatgagcaattggtggtggatgctggtgtctcagtgatcatggattt tcattataatgagaaaagaatctattgggtgg atttagaaagacaacttttgcaaagagtttttctgaatgggtcaaggcaagagagagtat gtaatatagagaaaaatgtttctggaatggc aataaattggataaatgaagaagttatttggtcaaatcaacaggaaggaatcattacagt aacagatatgaaaggaaataattcccacatt cttttaagtgctttaaaatatcctgcaaatgtagcagttgatccagtagaaaggtttata ttttggtcttcagaggtggctggaagcctttatag agcagatctcgatggtgtgggagtgaaggctctgttggagacatcagagaaaataacagc tgtgtcattggatgtgcttgataagcggc tgttttggattcagtacaacagagaaggaagcaattctcttatttgctcctgtgattatg atggaggttctgtccacattagtaaacatccaac acagcataatttgtttgcaatgtccctttttggtgaccgtatcttctattcaacatggaa aatgaagacaatttggatagccaacaaacacact ggaaaggacatggttagaattaacctccattcatcatttgtaccacttggtgaactgaaa gtagtgcatccacttgcacaacccaaggca gaagatgacacttgggagcctgagcagaaactttgcaaattgaggaaaggaaactgcagc agcactgtgtgtgggcaagacctccag tcacacttgtgcatgtgtgcagagggatacgccctaagtcgagaccggaagtactgtgaa gatgttaatgaatgtgctttttggaatcatg gctgtactcttgggtgtaaaaacacccctggatcctattactgcacgtgccctgtaggat ttgttctgcttcctgatgggaaacgatgtcatc aacttgtttcctgtccacgcaatgtgtctgaatgcagccatgactgtgttctgacatcag aaggtcccttatgtttctgtcctgaaggctcagt gcttgagagagatgggaaaacatgtagcggttgttcctcacccgataatggtggatgtag ccagctctgcgttcctcttagcccagtatc ctgggaatgtgattgctttcctgggtatgacctacaactggatgaaaaaagctgtgcagc ttcaggaccacaaccatttttgctgtttgcca attctcaagatattcgacacatgcattttgatggaacagactatggaactctgctcagcc agcagatgggaatggtttatgccctagatcat gaccctgtggaaaataagatatactttgcccatacagccctgaagtggatagagagagct aatatggatggttcccagcgagaaaggct tattgaggaaggagtagatgtgccagaaggtcttgctgtggactggattggccgtagatt ctattggacagacagagggaaatctctgat tggaaggagtgatttaaatgggaaacgttccaaaataatcactaaggagaacatctctca accacgaggaattgctgttcatccaatggc caagagattattctggactgatacagggattaatccacgaattgaaagttcttccctcca aggccttggccgtctggttatagccagctctg atctaatctggcccagtggaataacgattgacttcttaactgacaagttgtactggtgcg atgccaagcagtctgtgattgaaatggccaat ctggatggttcaaaacgccgaagacttacccagaatgatgtaggtcacccatttgctgta gcagtgtttgaggattatgtgtggttctcaga ttgggctatgccatcagtaatgagagtaaacaagaggactggcaaagatagagtacgtct ccaaggcagcatgctgaagccctcatca ctggttgtggttcatccattggcaaaaccaggagcagatccctgcttatatcaaaacgga ggctgtgaacatatttgcaaaaagaggctt ggaactgcttggtgttcgtgtcgtgaaggttttatgaaagcctcagatgggaaaacgtgt ctggctctggatggtcatcagctgttggcag gtggtgaagttgatctaaagaaccaagtaacaccattggacatcttgtccaagactagag tgtcagaagataacattacagaatctcaac acatgctagtggctgaaatcatggtgtcagatcaagatgactgtgctcctgtgggatgca gcatgtatgctcggtgtatttcagagggag aggatgccacatgtcagtgtttgaaaggatttgctggggatggaaaactatgttctgata tagatgaatgtgagatgggtgtcccagtgtg cccccctgcctcctccaagtgcatcaacaccgaaggtggttatgtctgccggtgctcaga aggctaccaaggagatgggattcactgtc ttgatattgatgagtgccaactgggggagcacagctgtggagagaatgccagctgcacaa atacagagggaggctatacctgcatgtg tgctggacgcctgtctgaaccaggactgatttgccctgactctactccaccccctcacct cagggaagatgaccaccactattccgtaag aaatagtgactctgaatgtcccctgtcccacgatgggtactgcctccatgatggtgtgtg catgtatattgaagcattggacaagtatgcat gcaactgtgttgttggctacatcggggagcgatgtcagtaccgagacctgaagtggtggg aactgcgccacgctggccacgggcagc agcagaaggtcatcgtggtggctgtctgcgtggtggtgcttgtcatgctgctcctcctga gcctgtggggggcccactactacaggact cagaagctgctatcgaaaaacccaaagaatccttatgaggagtcgagcagagatgtgagg agtcgcaggcctgctgacactgaggat gggatgtcctcttgccctcaaccttggtttgtggttataaaagaacaccaagacctcaag aatgggggtcaaccagtggctggtgaggat ggccaggcagcagatgggtcaatgcaaccaacttcatggaggcaggagccccagttatgt ggaatgggcacagagcaaggctgctg gattccagtatccagtgataagggctcctgtccccaggtaatggagcgaagctttcatat gccctcctatgggacacagacccttgaagg gggtgtcgagaagccccattctctcctatcagctaacccattatggcaacaaagggccct ggacccaccacaccaaatggagctgact cagtga

As used the term “glial-cell derived neutrophic factor” (GDNF) has its general meaning in the art and refers to a small protein that potently promotes the survival of many types of neurons.lt signals through GFRa receptors, particularly GFRal.Its Entrez reference is 2668. Exemplary acid nucleic coding for the glial-cell derived neutrophic factor is NM 000514, NM_001190468, NM_001190469, NM_001278098 and NM_199231. Its Uniprot reference is P39905. Exemplary amino acid sequences of GDNF include sequences a set forth in SEQ ID NO:21. In particular embodiment, the nucleic acid encoding GDNF comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO:22. In particular embodiment, the nucleic acid encoding GDNF comprises or consists of SEQ ID NO:22. In particular embodiment, the GDNF is a recombinant human GDNF.

SEQ ID NO:21 : amino acid sequence of the human GDNF

MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSN MPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGR RGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSR NRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI

SEQ ID NO:22: nucleic acid sequence of the human GDNF atgaagttatgggatgtcgtggctgtctgcctggtgctgctccacaccgcgtccgccttc ccgctgcccgccggtaagaggc ctcccgaggcgcccgccgaagaccgctccctcggccgccgccgcgcgcccttcgcgctga gcagtgactcaaatatgccagaggat tatcctgatcagttcgatgatgtcatggattttattcaagccaccattaaaagactgaaa aggtcaccagataaacaaatggcagtgcttcc tagaagagagcggaatcggcaggctgcagctgccaacccagagaattccagaggaaaagg tcggagaggccagaggggcaaaa accggggttgtgtcttaactgcaatacatttaaatgtcactgacttgggtctgggctatg aaaccaaggaggaactgatttttaggtactgc agcggctcttgcgatgcagctgagacaacgtacgacaaaatattgaaaaacttatccaga aatagaaggctggtgagtgacaaagtag ggcaggcatgttgcagacccatcgcctttgatgatgacctgtcgtttttagatgataacc tggtttaccatattctaagaaagcattccgcta aaaggtgtggatgtatctga

As used the term “ciliary neutrophic factor” (CTNF) has its general meaning in the art and refers to a survival factor for various neuronal cell types. Seems to prevent the degeneration of motor axons after axotomy. Its Entrez reference is 1270. Exemplary acid nucleic coding for the ciliary neutrophic factor is NM 000614. Its Uniprot reference is P26441. Exemplary amino acid sequences of CTNF include sequences a set forth in SEQ ID NO:23. In particular embodiment, the DNA encoding CTNF comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO:24. In particular embodiment, the nucleic acid encoding CTNF comprises or consists of SEQ ID NO:24. In particular embodiment, the CTNF is a recombinant human CTNF.

SEQ ID NO:23: amino acid sequence of the human CTNF

MAFTEHSPLTPHRRDLCSRSIWLARKIRSDLTALTESYVKHQGLNKNINLDSA DGMPVASTDQWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAI HTLLLQVAAFAYQIEELMILLEYKIPRNEADGMPINVGDGGLFEKKLWGLKVLQELS Q WT VR S IHDLRF I S SHQTGIPARGSHYIANNKKM

SEQ ID NO:24: nucleic acid sequence of the human CTNF atggctttcacagagcattcaccgctgacccctcaccgtcgggacctctgtagccgctct atctggctagcaaggaagattcg ttcagacctgactgctcttacggaatcctatgtgaagcatcagggcctgaacaagaacat caacctggactctgcggatgggatgccag tggcaagcactgatcagtggagtgagctgaccgaggcagagcgactccaagagaaccttc aagcttatcgtaccttccatgttttgttgg ccaggctcttagaagaccagcaggtgcattttaccccaaccgaaggtgacttccatcaag ctatacatacccttcttctccaagtcgctgc ctttgcataccagatagaggagttaatgatactcctggaatacaagatcccccgcaatga ggctgatgggatgcctattaatgttggagat ggtggtctctttgagaagaagctgtggggcctaaaggtgctgcaggagctttcacagtgg acagtaaggtccatccatgaccttcgtttc atttcttctcatcagactgggatcccagcacgtgggagccattatattgctaacaacaag aaaatgtag.

As used the term “vascular endothelial growth factor” (VEGF) has its general meaning in the art and refers to a sub-family of growth factors, the platelet-derived growth factor family of cystine-knot growth factors which stimulates the formation of blood vessels. Seems to prevent the degeneration of motor axons after axotomy. VEGF includes VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C and VEGF-D. Vascular endothelial growth factor A (VEGF) induces angiogenesis and vascular hyperpermeability in ocular tissues and is therefore a key therapeutic target for eye conditions in which these processes are dysregulated. Its Entrez reference is 7422. Exemplary acid nucleic coding for the ciliary neutrophic factor is NM_003376, NM_001025366, NM_001025367, NM_001025368, NM_001025369. Its Uniprot reference is P15692. Exemplary amino acid sequences of VEGF-A include sequences a set forth in SEQ ID NO:25. In particular embodiment, the nucleic acid encoding VEGF-A comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO:26. In particular embodiment, the nucleic acid encoding VEGF-A comprises or consists of SEQ ID NO:26. In particular embodiment, the VEGF is a recombinant human VEGF.

SEQ ID NO:25: amino acid sequence of the human VEGF-A

MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVA LKLFVQLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQ WRLGARKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPPHSPS RRGSASRAGPGRASETMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHE VVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPT EESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKR KRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNT DSRCKARQLELNERTCRCDKPRR

SEQ ID NO 26: nucleic acid sequence of the human VEGF-A ctgacggacagacagacagacaccgcccccagccccagctaccacctcctccccggccgg cggcggacagtggacgc ggcggcgagccgcgggcaggggccggagcccgcgcccggaggcggggtggagggggtcgg ggctcgcggcgtcgcactgaa acttttcgtccaacttctgggctgttctcgcttcggaggagccgtggtccgcgcggggga agccgagccgagcggagccgcgagaa gtgctagctcgggccgggaggagccgcagccggaggagggggaggaggaagaagagaagg aagaggagagggggccgcagt ggcgactcggcgctcggaagccgggctcatggacgggtgaggcggcggtgtgcgcagaca gtgctccagccgcgcgcgctcccc aggccctggcccgggcctcgggccggggaggaagagtagctcgccgaggcgccgaggaga gcgggccgccccacagcccgag ccggagagggagcgcgagccgcgccggccccggtcgggcctccgaaaccatgaactttct gctgtcttgggtgcattggagccttgc cttgctgctctacctccaccatgccaagtggtcccaggctgcacccatggcagaaggagg agggcagaatcatcacgaagtggtgaa gttcatggatgtctatcagcgcagctactgccatccaatcgagaccctggtggacatctt ccaggagtaccctgatgagatcgagtacat cttcaagccatcctgtgtgcccctgatgcgatgcgggggctgctgcaatgacgagggcct ggagtgtgtgcccactgaggagtccaac atcaccatgcagattatgcggatcaaacctcaccaaggccagcacataggagagatgagc ttcctacagcacaacaaatgtgaatgca gaccaaagaaagatagagcaagacaagaaaaaaaatcagttcgaggaaagggaaaggggc aaaaacgaaagcgcaagaaatccc ggtataagtcctggagcgtgtacgttggtgcccgctgctgtctaatgccctggagcctcc ctggcccccatccctgtgggccttgctcag agcggagaaagcatttgtttgtacaagatccgcagacgtgtaaatgttcctgcaaaaaca cagactcgcgttgcaaggcgaggcagctt gagttaaacgaacgtacttgcagatgtgacaagccgaggcggtga

As used the term “neuropilin 2” (NRP2) has its general meaning in the art and refers to a neuropilin factor that binds to SEMA3C, SEMA3F, VEGF-A, VEGF-C, VEGF-D, TGFp, integrins and ANGPTL4 to promote downstream signaling pathways. Its Entrez reference is 8828. Exemplary acid nucleic coding for the neuropilin-2 is NM_003872, NM_018534, NM_201264, NM_201266 or NM_201267. Its Uniprot reference is 060462. Exemplary amino acid sequences of NRP2 include sequences a set forth in SEQ ID NO:27. In particular embodiment, the nucleic acid encoding NRP2 comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO:28. In particular embodiment, the nucleic acid encoding NRP2 comprises or consists of SEQ ID NO:28. In particular embodiment, the NRP2is a recombinant human NRP2.

SEQ ID NO:27: amino acid sequence of the human NRP2

MDMFPLTWVFLALYFSRHQVRGQPDPPCGGRLNSKDAGYITSPGYPQDYPSH QNCEWIVYAPEPNQKIVLNFNPHFEIEKHDCKYDFIEIRDGDSESADLLGKHCGNIAPP TIISSGSMLYIKFTSDYARQGAGFSLRYEIFKTGSEDCSKNFTSPNGTIESPGFPEKYPH NLDCTFTILAKPKMEIILQFLIFDLEHDPLQVGEGDCKYDWLDIWDGIPHVGPLIGKYC GTKTPSELRSSTGILSLTFHTDMAVAKDGFSARYYLVHQEPLENFQCNVPLGMESGRI ANEQISASSTYSDGRWTPQQSRLHGDDNGWTPNLDSNKEYLQVDLRFLTMLTAIATQ GAISRETQNGYYVKSYKLEVSTNGEDWMVYRHGKNHKVFQANNDATEVVLNKLH APLLTRFVRIRPQTWHSGIALRLELFGCRVTDAPCSNMLGMLSGLIADSQISASSTQEY LWSPSAARLVSSRSGWFPRIPQAQPGEEWLQVDLGTPKTVKGVIIQGARGGDSITAVE ARAFVRKFKVSYSLNGKDWEYIQDPRTQQPKLFEGNMHYDTPDIRRFDPIPAQYVRV YPERWSPAGIGMRLEVLGCDWTDSKPTVETLGPTVKSEETTTPYPTEEEATECGENCS FEDDKDLQLPSGFNCNFDFLEEPCGWMYDHAKWLRTTWAS S S SPNDRTFPDDRNFL RLQSDSQREGQYARLISPPVHLPRSPVCMEFQYQATGGRGVALQVVREASQESKLLW VIREDQGGEWKHGRIILPSYDMEYQIVFEGVIGKGRSGEIAIDDIRISTDVPLENCMEPI SAFAGENFKVDIPEIHEREGYEDEIDDEYEVDWSNSSSATSGSGAPSTDKEKSWLYTL DPILITIIAMS SLGVLLGATC AGLLLYCTC S YSGLS SRSCTTLENYNFELYDGLKHKVK MNHQKCCSEA

SEQ ID NO:28: nucleic acid sequence of the human NRP2 atggatatgtttcctctcacctgggttttcttagccctctacttttcaagacaccaagtg agaggccaaccagacccaccgtgc ggaggtcgtttgaattccaaagatgctggctatatcacctctcccggttacccccaggac tacccctcccaccagaactgcgagtggatt gtttacgcccccgaacccaaccagaagattgtcctcaacttcaaccctcactttgaaatc gagaagcacgactgcaagtatgactttatcg agattcgggatggggacagtgaatccgcagacctcctgggcaaacactgtgggaacatcg ccccgcccaccatcatctcctcgggct ccatgctctacatcaagttcacctccgactacgcccggcagggggcaggcttctctctgc gctacgagatcttcaagacaggctctgaa gattgctcaaaaaacttcacaagccccaacgggaccatcgaatctcctgggtttcctgag aagtatccacacaacttggactgcaccttt accatcctggccaaacccaagatggagatcatcctgcagttcctgatctttgacctggag catgaccctttgcaggtgggagaggggg actgcaagtacgattggctggacatctgggatggcattccacatgttggccccctgattg gcaagtactgtgggaccaaaacaccctct gaacttcgttcatcgacggggatcctctccctgacctttcacacggacatggcggtggcc aaggatggcttctctgcgcgttactacctg gtccaccaagagccactagagaactttcagtgcaatgttcctctgggcatggagtctggc cggattgctaatgaacagatcagtgcctca tctacctactctgatgggaggtggacccctcaacaaagccggctccatggtgatgacaat ggctggacccccaacttggattccaacaa ggagtatctccaggtggacctgcgctttttaaccatgctcacggccatcgcaacacaggg agcgatttccagggaaacacagaatggc tactatgtcaaatcctacaagctggaagtcagcactaatggagaggactggatggtgtac cggcatggcaaaaaccacaaggtatttca agccaacaacgatgcaactgaggtggttctgaacaagctccacgctccactgctgacaag gtttgttagaatccgccctcagacctggc actcaggtatcgccctccggctggagctcttcggctgccgggtcacagatgctccctgct ccaacatgctggggatgctctcaggcctc attgcagactcccagatctccgcctcttccacccaggaatacctctggagccccagtgca gcccgcctggttagcagccgctcgggct ggttccctcgaatccctcaggcccagcccggtgaggagtggcttcaggtagatctgggaa cacccaagacagtgaaaggtgtcatcat ccagggagcccgcggaggagacagtatcactgctgtggaagccagagcatttgtgcgcaa gttcaaagtctcctacagcctaaacgg caaggactgggaatacattcaggaccccaggacccagcagccaaagctgttcgaagggaa catgcactatgacacccctgacatcc gaaggtttgaccccattccggcacagtatgtgcgggtatacccggagaggtggtcgccgg cggggattgggatgcggctggaggtg ctgggctgtgactggacagactccaagcccacggtagagacgctgggacccactgtgaag agcgaagagacaaccaccccctacc ccaccgaagaggaggccacagagtgtggggagaactgcagctttgaggatgacaaagatt tgcagctcccttcgggattcaattgcaa cttcgatttcctcgaggagccctgtggttggatgtatgaccatgccaagtggctccggac cacctgggccagcagctccagcccaaac gaccggacgtttccagatgacaggaatttcttgcggctgcagagtgacagccagagagag ggccagtatgcccggctcatcagcccc cctgtccacctgccccgaagcccggtgtgcatggagttccagtaccaggccacgggcggc cgcggggtggcgctgcaggtggtgc gggaagccagccaggagagcaagttgctgtgggtcatccgtgaggaccagggcggcgagt ggaagcacgggcggatcatcctgc ccagctacgacatggagtaccagattgtgttcgagggagtgatagggaaaggacgttccg gagagattgccattgatgacattcggat aagcactgatgtcccactggagaactgcatggaacccatctcggcttttgcaggtgagaa ttttaaagtggacatcccagaaatacatga gagagaaggatatgaagatgaaattgatgatgaatacgaggtggactggagcaattcttc ttctgcaacctcagggtctggcgccccct cgaccgacaaagaaaagagctggctgtacaccctggatcccatcctcatcaccatcatcg ccatgagctcactgggcgtcctcctggg ggccacctgtgcaggcctcctgctctactgcacctgttcctactcgggcctgagctcccg aagctgcaccacactggagaactacaact tcgagctctacgatggccttaagcacaaggtcaagatgaaccaccaaaagtgctgctccg aggcatga

As used the term “Pigment epithelium-derived factor (PEDF)”, also known as serpin Fl (SERPINF1)” has its general meaning in the art and refers to a multifunctional secreted protein that has anti-angiogenic, anti-tumorigenic, and neurotrophic functions. Its Entrez reference is 5176. Exemplary acid nucleic coding for the PEDF is NM 002615, NM_001329903, NM_001329904 or NM_001329905. Its Uniprot reference is P36955. Exemplary amino acid sequences of PEDF include sequences a set forth in SEQ ID NO:29. In particular embodiment, the nucleic acid encoding PEDF comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO:30. In particular embodiment, the nucleic acid encoding PEFD comprises or consists of SEQ ID NO:30. In particular embodiment, the PEDF is a recombinant human PEDF.

SEQ ID NO:29: amino acid sequence of the human PEDF

MQALVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKL AAAVSNFGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLI S SPDIHGTYKELLDTVTAPQKNLKS ASRIVFEKKLRIKS SF VAPLEKS YGTRPRVLTGN PRLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLE DFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLT LIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKIT GKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGA LLFIGKILDPRGP

SEQ ID NO:30: nucleic acid sequence of the human PEDF atgcaggccctggtgctactcctctgcattggagccctcctcgggcacagcagctgccag aaccctgccagccccccgga ggagggctccccagaccccgacagcacaggggcgctggtggaggaggaggatcctttctt caaagtccccgtgaacaagctggca gcggctgtctccaacttcggctatgacctgtaccgggtgcgatccagcacgagccccacg accaacgtgctcctgtctcctctcagtgt ggccacggccctctcggccctctcgctgggagcggagcagcgaacagaatccatcattca ccgggctctctactatgacttgatcagc agcccagacatccatggtacctataaggagctccttgacacggtcactgccccccagaag aacctcaagagtgcctcccggatcgtctt tgagaagaagctgcgcataaaatccagctttgtggcacctctggaaaagtcatatgggac caggcccagagtcctgacgggcaaccct cgcttggacctgcaagagatcaacaactgggtgcaggcgcagatgaaagggaagctcgcc aggtccacaaaggaaattcccgatga gatcagcattctccttctcggtgtggcgcacttcaaggggcagtgggtaacaaagtttga ctccagaaagacttccctcgaggatttctac ttggatgaagagaggaccgtgagggtccccatgatgtcggaccctaaggctgttttacgc tatggcttggattcagatctcagctgcaag attgcccagctgcccttgaccggaagcatgagtatcatcttcttcctgcccctgaaagtg acccagaatttgaccttgatagaggagagc ctcacctccgagttcattcatgacatagaccgagaactgaagaccgtgcaggcggtcctc actgtccccaagctgaagctgagttatga aggcgaagtcaccaagtccctgcaggagatgaagctgcaatccttgtttgattcaccaga ctttagcaagatcacaggcaaacccatca agctgactcaggtggaacaccgggctggctttgagtggaacgaggatggggcgggaacca cccccagcccagggctgcagcctgc ccacctcaccttcccgctggactatcaccttaaccagcctttcatcttcgtactgaggga cacagacacaggggcccttctcttcattggc aagattctggaccccaggggcccctaa

As used the term “semaphorin’’ (SEMA), has its general meaning in the art and refers to a class of secreted and membrane proteins that were originally identified as axonal growth cone guidance molecules. Semaphorin includes SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SEMA4A, SEMA4B, SEM4C, SEMA4D, SEMA4F, SEMA4G, SEMA5A, SEMA5B, SEMA6A, SEMA6B, SEMA6C, SEMA6D, SEMA7A. In particular embodiment, the SEMA is SEMA3C. Semaphorin-3C is a protein that in humans is encoded by the SEMA3C gene and play a role in corneal epithelial wound closure ⁵⁹ . Its Entrez reference is 10512. Exemplary acid nucleic coding for the SEMA3C is NM_006379, NM_001350120 or NM_001350121. Its Uniprot reference is Q99985. Exemplary amino acid sequences of SEMA3AC include sequences a set forth in SEQ ID NO:31. In particular embodiment, the nucleic acid encoding SEMA3C comprises an amino acid sequence having at least 90% of identity with the sequence as set forth in SEQ ID NO:32. In particular embodiment, the nucleic acid encoding SEMA3C comprises or consists of SEQ ID NO:32. In particular embodiment, the SEMA3C is a recombinant human SEMA3C.

SEQ ID NO: 31 : amino acid sequence of the human SEMA3C

MAFRTICVLVGVFICSICVKGSSQPQARVYLTFDELRETKTSEYFSLSHHPLDY RILLMDEDQDRIYVGSKDHILSLNINNISQEALSVFWPASTIKVEECKMAGKDPTHGC GNFVRVIQTFNRTHLYVCGSGAFSPVCTYLNRGRRSEDQVFMIDSKCESGKGRCSFNP NVNTVSVMINEELFSGMYIDFMGTDAAIFRSLTKRNAVRTDQHNSKWLSEPMFVDA HVIPDGTDPNDAKVYFFFKEKLTDNNRSTKQIHSMIARICPNDTGGLRSLVNKWTTFL KARLVCSVTDEDGPETHFDELEDVFLLETDNPRTTLVYGIFTTSSSVFKGSAVCVYHL SDIQTVFNGPFAHKEGPNHQLISYQGRIPYPRPGTCPGGAFTPNMRTTKEFPDDVVTFI RNHPLMYNSIYPIHKRPLIVRIGTDYKYTKIAVDRVNAADGRYHVLFLGTDRGTVQK VVVLPTNNS VSGELILEELEVFKNHAPITTMKIS SKKQQLYVS SNEGVSQ VSLHRCHIY GTACADCCLARDPYCAWDGHSCSRFYPTGKRRSRRQDVRHGNPLTQCRGFNLKAY RNAAEIVQYGVKNNTTFLECAPKSPQASIKWLLQKDKDRRKEVKLNERIIATSQGLLI RSVQGSDQGLYHCIATENSFKQTIAKINFKVLDSEMVAVVTDKWSPWTWASSVRAL PFHPKDIMGAFSHSEMQMINQYCKDTRQQHQQGDESQKMRGDYGKLKALINSRKSR NRRNQLPES

SEQ ID NO:32: nucleic acid sequence of the human SEMA3C atggcattccggacaatttgcgtgttggttggagtatttatttgttctatctgtgtgaaa ggatcttcccagccccaagcaagagt ttatttaacatttgatgaacttcgagaaaccaagacctctgaatacttcagcctttccca ccatcctttagactacaggattttattaatggatg aagatcaggaccggatatatgtgggaagcaaagatcacattctttccctgaatattaaca atataagtcaagaagctttgagtgttttctgg ccagcatctacaatcaaagttgaagaatgcaaaatggctggcaaagatcccacacacggc tgtgggaactttgtccgtgtaattcagact ttcaatcgcacacatttgtatgtctgtgggagtggcgctttcagtcctgtctgtacttac ttgaacagagggaggagatcagaggaccaag ttttcatgattgactccaagtgtgaatctggaaaaggacgctgctctttcaaccccaacg tgaacacggtgtctgttatgatcaatgaggag cttttctctggaatgtatatagatttcatggggacagatgctgctatttttcgaagttta accaagaggaatgcggtcagaactgatcaacat aattccaaatggctaagtgaacctatgtttgtagatgcacatgtcatcccagatggtact gatccaaatgatgctaaggtgtacttcttcttca aagaaaaactgactgacaataacaggagcacgaaacagattcattccatgattgctcgaa tatgtcctaatgacactggtggactgcgta gccttgtcaacaagtggaccactttcttaaaggcgaggctggtgtgctcggtaacagatg aagacggcccagaaacacactttgatgaa ttagaggatgtgtttctgctggaaactgataacccgaggacaacactagtgtatggcatt tttacaacatcaagctcagttttcaaaggatc agccgtgtgtgtgtatcatttatctgatatacagactgtgtttaatgggccttttgccca caaagaagggcccaatcatcagctgatttcctat cagggcagaattccatatcctcgccctggaacttgtccaggaggagcatttacacccaat atgcgaaccaccaaggagttcccagatg atgttgtcacttttattcggaaccatcctctcatgtacaattccatctacccaatccaca aaaggcctttgattgttcgtattggcactgactac aagtatacaaagatagctgtggatcgagtgaacgctgctgatgggagataccatgtcctg tttctcggaacagatcggggtactgtgca aaaagtggttgttcttcctactaacaactctgtcagtggcgagctcattctggaggagct ggaagtctttaagaatcatgctcctataacaa caatgaaaatttcatctaaaaagcaacagttgtatgtgagttccaatgaaggggtttccc aggtatctctgcaccgctgccacatctatggt acagcctgtgctgactgctgcctggcgcgggacccttattgcgcctgggatggccattcc tgttccagattctacccaactgggaaacg gaggagccgaagacaagatgtgagacatggaaacccactgactcaatgcagaggatttaa tctaaaagcatacagaaatgcagctga aattgtccagtatggagtaaaaaataacaccacttttctggagtgtgcccccaagtctcc gcaggcatctatcaagtggctgttacagaaa gacaaagacaggaggaaagaggttaagctgaatgaacgaataatagccacttcacaggga ctcctgatccgctctgttcagggttctg accaaggactttatcactgcattgctacagaaaatagtttcaagcagaccatagccaaga tcaacttcaaagttttagattcagaaatggtg gctgttgtgacggacaaatggtccccatggacctgggccagctctgtgagggctttaccc ttccacccgaaggacatcatgggggcatt cagccactcagaaatgcagatgattaaccaatattgcaaagacactcggcagcaacatca gcagggagatgaatcacagaaaatgag aggggactatggcaagttaaaggccctcatcaatagtcggaaaagtagaaacaggaggaa tcagttgccagagtcataa

In some embodiments, according to all the method of the invention, wherein the acid nucleic encodes for NGF or EGF.

In particular embodiment, according to all the method of the invention, the nucleic acid encoding a protein of interest is administered into the lacrimal gland simultaneously, separately or sequentially with a transfection reagent.

In particular embodiment, according to all the method of the invention, the nucleic acid encoding a protein of interest is further mixed with a transfection reagent prior to the administration into lacrimal gland. In particular embodiment, according to all the method of the invention, the nucleic acid encoding a protein of interest is administered together with a transfection reagent. In particular embodiment, according to all the method of the invention, the messenger RNA encoding a protein of interest is further mixed with a transfection reagent prior to the administration into lacrimal gland. In particular embodiment, according to all the method of the invention, the messenger RNA encoding a protein of interest is administered together with a transfection reagent. In other words, in particular embodiment, according to all the method of the invention, the messenger RNA encoding a protein of interest is administered into the lacrimal gland simultaneously, separately or sequentially with a transfection reagent. In particular embodiment, according to all the method of the invention, the messenger RNA encoding NGF or EGF is administered into the lacrimal gland simultaneously, separately or sequentially with a transfection reagent.

As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients (i.e the nucleic acid encoding a protein of interest and trasnsfection reagents) by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients (i.e the nucleic acid encoding a protein of interest and trasnsfection reagents) at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients ((i.e the nucleic acid encoding a protein of interest and trasnsfection reagents) at different times, the administration route being identical or different.

Thus, in a particular embodiment, the present invention relates to a method for modulating tear film composition in a subject in need thereof, comprising administering into the lacrimal gland of said subject a messenger ribonucleic acid (mRNA) encoding a protein of interest simultaneously, separately or sequentially with a transfection reagent.

In a particular embodiment, the present invention relates to a method for modulating tear film composition in a subject in need thereof, comprising administering into the lacrimal gland of said subject a messenger ribonucleic acid (mRNA) encoding NGF or EGF simultaneously, separately or sequentially with a transfection reagent.

As used herein, the term “transfection reagent” has its general meaning in the art and refers to any agents suitable to introduction of nucleic acid into eukaryotic cells by non-viral agents. It is well known in the art many various non viral methods to introduce nucleic acid as described in Yamano S et al, Mol Biotechno.2010 ⁵² , Kumar et al, Cold Spring HArb Protoc. 2019 ⁵³ , Feigner et al, Proc Natl Acad Sci.1987 ⁵⁴ , Rahimi et al, Bratisl Lek Listy. 2018 ⁵⁵ , Pardi et al. J Control Release. 2015 ⁵⁶ .

In another embodiment, the transfection reagent is a cationic polymers or cationic lipid reagent. In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin® or Lipofectamine®. In another embodiment, the transfection reagent is in vzvo-jetRNA® or in vzvo-jetPEI®. In another embodiment, the transfection reagent forms a liposome. Liposomes can increase intracellular stability, increase uptake efficiency and improve biological activity. In another embodiment, the transfection reagent forms solid-lipid nanoparticles (SLNs or LNPs), polymers-based nanoparticles such polyethylenimine(PEI)-based nanoparticles; lipopeptides-based nanoparticles such as lipid 1,2- dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA)-based nanoparticles, dilinoleylmethyl-4- dimethylaminobutyrate (DLin-MC3-DMA)-based nanoparticles, ALC-0315-based nanoparticles, ALC-0159-based nanoparticles SM-102-based nanonparticles. In another embodiment, the transfection reagent is any other transfection reagent known in the art. Transfection reagent according to the invention includes also transfection reagents as described in patent WO2017049245, WO2018081480 and WO2021016430.

In particular embodiment, the nucleic acid encoding a protein of interest is comprised in a vector.

Thus, in particular embodiment, the present invention relates also to a method for modulating tear film composition in a subject in need thereof, comprising administering into the lacrimal gland of said subject a vector comprising a nucleic acid encoding a protein of interest.

In particular embodiment, the present invention relates to a method for modulating tear film composition in a subject in need thereof, comprising administering into the lacrimal gland of said subject a vector comprising DNA or RNA encoding a protein of interest, and more particular a messenger ribonucleic acid (mRNA) encoding a protein of interest.

As used herein, the term “vector” has its general meaning in the art and refers to the vehicle or agents by which a nucleic acid molecule can be introduced into cells, so as to transform/transfect the cell and promote expression (e.g. transcription and/or translation) of the introduced sequence.

According to the invention, vectors include viral vectors or non-viral vectors.

In some embodiments, the method according to the invention, wherein the vector is a viral or non-viral vector.

Non-viral vectors mainly comprise chemical systems that are not of viral origin and generally include chemical methods such as cationic liposomes and polymers. Non viral vectors useful in the practice of the present invention has very well known in the art. According to the invention non-viral vector includes transfection agents as defined above.

According to the invention, non viral vectors include but are not limited to liposomes such as cationic liposomes, solid-lipid nanoparticles (SLNs or LNPs) such as [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)-based nanoparticles; niosomes; polymers such as cationic polymers; polymers-based nanoparticles such polyethylenimine(PEI)-based nanoparticles; lipopeptides-based nanoparticles such as lipid 1,2- dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA)-based nanoparticles, dilinoleylmethyl-4- dimethylaminobutyrate (DLin-MC3-DMA)-based nanoparticles, ALC-0315-based nanoparticles, ALC-0159-based nanoparticles SM- 102-based nanonparticles and ; and chitosans as described in Toualbi L, et al. International Journal of Molecular Sciences, Maier.M et al. Molecular Therapy (2013), Shriane D et al.Biol Pharm Bull (2018). Non viral vectors according to the invention include also the non-viral vectors described in patent WO2017049245, W02018081480 and W02021016430.

In some embodiments, the method according to the invention, wherein the non-viral vector is cationic a polymers-based nanoparticle, and more particularly is a polyethylenimine(PEI)-based nanoparticle.

In some embodiments, the method according to the invention, wherein the non-viral vector is in vzvo-jetRNA® or in vzvo-jetPEI®.

In some embodiments, the method according to the invention, wherein the vector is a non-viral vector comprising ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) encoding a protein of interest and more particularly mRNA encoding a protein of interest. In some embodiments, the method according to the invention, wherein the vector is a non-viract vector when the acid nucleic encodes for NGF or EGF.

In some embodiments, the method according to the invention, wherein the vector is a non-viral vector comprising messenger ribonucleic acid (mRNA) encoding EGF or NGF.

Thus, in a particular embodiment, the present invention relates to a method for modulating tear film composition in a subject in need thereof, comprising administering into the lacrimal gland of said subject a non-viral vector comprising a messenger ribonucleic acid (mRNA) encoding a protein of interest, and in particular mRNA encoding EGF or NGF.

In some embodiments, the method according to the invention, the vector is a viral vector. Viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology. Typically, viral vectors carrying nucleic acide encoding the protein of interest, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction. Examples of viral vector include but are not limited to retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, pox virus, human foamy virus (HFV), and lentivirus.

In some embodiments, the method according to the invention, wherein the viral vector is a lentivirus (LV) vector, adenovirus vector or an adeno-associated virus (AAV) vector .

As used herein, the term “lentivirus” refers to enveloped RNA particles measuring approximately 120 nm in size are efficient drug delivery tools and more particularly gene delivery tools. The LV binds to, and enters into target cells through its envelope proteins which confer its pseudotype. Once the LV has entered into the cells, it releases its capsid components and undergoes reverse transcription of the lentiviral RNA before integrating the proviral DNA into the genome of target cells. Non-integrative lentiviral vectors have been generated by modifying the properties of the vector integration machinery and can be used for transient gene expression. Virus-like particles lacking a provirus have also been generated and can be used to deliver proteins or messenger RNA. LV can be used for example, for gene addition, RNA interference, exon skipping or gene editing. All of these approaches can be facilitated by tissue or cell targeting of the LV via its pseudotype.

Lentivirus-like particles are described for example in (Aoki et al., 2011; Kaczmarczyk et al., 2011; McBurney et al., 2006; Muratori et al., 2010). Examples of lentivirus-like particles are VLPs generated by co-expressing in producer cells, a syncytin protein with a gag fusion protein (Gag fused with the gene of interest). The drug and/or syncytin may be, either displayed on the surface of the particles, or enclosed (packaged) into the particles. The syncytin protein is advantageously displayed on the surface of the particles, such as coupled to the particles or incorporated into the envelope of (enveloped) virus particles or virus-like particles to form pseudotyped enveloped virus particles or virus-like particles. The drug is coupled to the particles or packaged into the particles. For example, the drug is coupled to viral capsids or packaged into viral capsids, wherein said viral capsids may further comprise an envelope, preferably pseudotyped with syncytin. In some preferred embodiments, the drug is packaged into the particles pseudotyped with syncytin protein. The drug which is packaged into particles is advantageously a heterologous gene of interest which is packaged into viral vector particles, preferably retroviral vector particles, more preferably lentiviral vector particles.

As used herein, the term “adenovirus” refers to medium-sized (90-100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double stranded DNA genome. In a particular embodiment, the method according to the invention, wherein the viral vector is an adeno-associated virus (AAV) vector.

As used herein, the term “AAV vector” refers to a vector derived from an adeno- associated virus serotype, including without limitation AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAVrhlO or any other serotypes of AAV that can infect humans, monkeys or other species. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e. g., functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e. g by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the nucleic acid molecule of the present invention and a transcriptional termination region. The control elements are selected to be functional in a mammalian cell. The resulting construct which contains the operatively linked components is bounded (5' and 3’) with functional AAV ITR sequences. By "adeno-associated virus inverted terminal repeats " or "AAV ITRs" is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome. The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, 1994; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. As used herein, an "AAV ITR" does not necessarily comprise the wild-type nucleotide sequence, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. In some embodiments, the AAV vector of the present invention is a double-stranded, self-complementary AAV (scAAV) vector. Alternatively to the use of single-stranded AAV vector, self-complementary vectors can be used. The efficiency of AAV vector in terms of the number of genome-containing particles required for transduction, is hindered by the need to convert the single-stranded DNA (ssDNA) genome into double-stranded DNA (dsDNA) prior to expression. This step can be circumvented through the use of self-complementary vectors, which package an inverted repeat genome that can fold into dsDNA without the requirement for DNA synthesis or base-pairing between multiple vector genomes. Resulting self- complementary AAV (scAAV) vectors have increased resulting expression of the transgene. For an overview of AAV biology, ITR function, and scAAV constructs, see McCarty D M. Self-complementary AAV vectors; advances and applications. Mol. Ther. 2008 October; 16 (10): at pages 1648-51, first full paragraph, incorporated herein by reference for disclosure of AAV and scAAV constructs, ITR function, and role of ATRS ITR in scAAV constructs. A rAAV vector comprising a ATRS ITR cannot correctly be nicked during the replication cycle and, accordingly, produces a self-complementary, double-stranded AAV (scAAV) genome, which can efficiently be packaged into infectious AAV particles. Various rAAV, ssAAV, and scAAV vectors, as well as the advantages and drawbacks of each class of vector for specific applications and methods of using such vectors in gene transfer applications are well known to those of skill in the art (see, for example, Choi V W, Samulski R J, McCarty D M. Effects of adeno-associated virus DNA hairpin structure on recombination. J. Virol. 2005 June; 79(11):6801-7; McCarty D M, Young S M Jr, Samulski R J. Integration of adeno-associated virus (AAV) and recombinant AAV vectors. Annu Rev Genet. 2004; 38:819-45; McCarty D M, Monahan P E, Samulski R J. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther. 2001 August; 8(16): 1248-54; and McCarty D M. Self-complementary AAV vectors; advances and applications. Mol. Ther. 2008 October; 16(10): 1648-56; all references cited in this application are incorporated herein by reference for disclosure of AAV, rAAV, and scAAV vectors).

In some embodiment, the AAV vector is an AAV2/9 vector or an AAV2/5 vector. AAV2/9 vector denotes that the AAV2 genome carrying the transgene (i.e nucleic acid encoding for the protein of interest) was packaged in an AAV9 capsid. An AAV2/5 vector refers to a AAV2 genome carrying the transgene (i.e e nucleic acid encoding for the protein of interest) packaged in an AAV5 capsid. Scalable production of such rAAV are well known to those of skill in the art (Blessing D, et al. Scalable production of AAV vectors in orbitally shaken HEK293 cells. Mol Ther Methods Clin Dev. 2019 Jun 14; 13: 14-26.

Thus, in some embodiments, the present invention relates to a method for modulating tear film composition in a subject in need thereof, comprising administering into the lacrimal gland of said subject a viral vector comprising nucleic acid encoding for a protein of interest.

In particular embodiment, the present invention relates to a method for modulating tear film composition in a subject in need thereof, comprising administering into the lacrimal gland of said subject an AAV vector comprising a deoxyribonucleic acid (DNA) encoding for a protein of interest.

In particular embodiment, concerning all the methods of the invention, the AAV vector comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding for a protein of interest is administered alone. In particular embodiment, concerning all the methods of the invention, the AAV vector comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding for a protein of interest is not administered in combination with nicotinic acetylcholine receptor (nAChR) agonist.

In particular embodiment, the present invention relates to a method for modulating tear film composition in a subject in need thereof, comprising administering into the lacrimal gland of said subject an AAV2/9 vector comprising a deoxyribonucleic acid (DNA) encoding for a protein of interest.

In particular embodiment, the present invention relates to a method for modulating tear film composition in a subject in need thereof, comprising administering into the lacrimal gland of said subject an AAV2/9 vector comprising a deoxyribonucleic acid (DNA) encoding for a NGF.

In particular embodiment, concerning all the methods of the invention, the AAV2/9 vector comprising deoxyribonucleic acid (DNA) encoding for NGF is administered alone. In particular embodiment, concerning all the methods of the invention, the AAV2/9 vector comprising deoxyribonucleic acid (DNA) encoding for NGF is not administered in combination with nicotinic acetylcholine receptor (nAChR) agonist.

The inventors shows that a single AAV2/9 CAG-mNGF injection in the lacrimal gland stimulate the reinnervation of the cornea, following an epithelial abrasion.

Thus the methods of the invention are suitable for improving corneal reinnervation.

The methods of the invention are also suitable for improving corneal wound healing.

The methods of the invention are also suitable for increasing tear production. Thus, in another aspect the present invention relates to a method for improving corneal reinnervation in a subject in need thereof, comprising administering into the lacrimal gland of said subject a nucleic acid encoding a protein of interest.

The inventors shows also that a single AAV2/9 CAG-mNGF injection in the lacrimal gland increase the tear volume.

Thus, in another aspect the present invention relates to a method for increasing tear production in a subject in need thereof, comprising administering into the lacrimal gland of said subject a nucleic acid encoding a protein of interest.

Thus, in another aspect the present invention relates to a method for improving corneal wound healing in a subject in need thereof, comprising administering into the lacrimal gland of said subject a nucleic acid encoding a protein of interest.

In some embodiment, regarding the method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and more particularly messenger ribonucleic acid (mRNA).

In some embodiment, regarding the method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production, the protein of interest is selected in the group consisting of nerve growth factor (NGF), neuropilin 2 (NRP2), semaphorin 3C (SEMA3C), pigment epithelium-derived factor (PEDF), vascular Endothelial Growth Factor A (VEGF-A), thyroglobuline, epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), bone morphogenetic protein 6 (BMP6), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), glial-cell derived neurotrophic factor (GDNF) and transforming growth factor (TGF).

In some embodiment, regarding the method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production, the protein of interest is nerve growth factor (NGF).

In some embodiments, regarding the method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production, the messenger ribonucleic acid (mRNA) encoding for a protein of interest such as NGF is administered gland simultaneously, separately or sequentially with a transfection reagent, such as in vzvo-RNAjet.

In some embodiments, regarding the method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production, the messenger ribonucleic acid (mRNA) encoding for a protein of interest such as NGF is further mixed with a transfection reagent prior to the administration into lacrimal gland.

Thus, in particular embodiment, the invention refers to a method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production in a subject in need thereof, comprising administering into the lacrimal gland of said subject a messenger ribonucleic acid (mRNA) encoding for a protein of interest such as NGF, wherein said mRNA is administered simultaneously, separately or sequentially with a transfection reagent.

In particular embodiment, the invention refers to a method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production in a subject in need thereof, comprising administering into the lacrimal gland of said subject a messenger ribonucleic acid (mRNA) encoding a protein of interest, wherein said mRNA is administered simultaneously, separately or sequentially with a transfection reagent, and wherein the protein of interest is selected in the group consisting of nerve growth factor (NGF), neuropilin 2 (NRP2), semaphorin 3C (SEMA3C), pigment epithelium-derived factor (PEDF), vascular Endothelial Growth Factor A (VEGF-A), thyroglobuline, epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), insulin-like growth factor

1 (IGF-1), bone morphogenetic protein 6 (BMP6), insulin-like growth factor 2 (IGF-2), glialcell derived neurotrophic factor (GDNF) and transforming growth factor (TGF).

In particular embodiment, the invention refers to a method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production in a subject in need thereof, comprising administering into the lacrimal gland of said subject a messenger ribonucleic acid (mRNA) encoding a protein of interest, wherein said mRNA is further mixed with a transfection reagent prior to the administration into lacrimal gland, and wherein the protein of interest is selected in the group consisting of nerve growth factor (NGF), neuropilin

2 (NRP2), semaphorin 3C (SEMA3C), pigment epithelium-derived factor (PEDF), vascular Endothelial Growth Factor A (VEGF-A), thyroglobuline, epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), bone morphogenetic protein 6 (BMP6), insulin-like growth factor 2 (IGF-2), glialcell derived neurotrophic factor (GDNF) and transforming growth factor (TGF).

In particular embodiment, the invention refers to a method for corneal reinnervation and/or corneal wound healing and/or increasing tear production in a subject in need thereof, comprising administering into the lacrimal gland of said subject a messenger ribonucleic acid (mRNA) encoding NGF, wherein said mRNA is administered simultaneously, separately or sequentially with a transfection reagent. In some embodiments, regarding the method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production, the nucleic acid is comprised in a vector.

In some embodiments, regarding the method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production, the vector is a viral or non-viral vector.

In some embodiments, regarding the method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production the vector is a non-viral vector comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding NGF and more particularly messenger RNA encoding a protein of interest.

In some embodiments, regarding the method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production, the viral vector is a an adeno- associated virus (AAV) vector and more particularly AAV2/9.

In some embodiments, regarding the method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production, wherein the viral vector is an AAV2/9 vector comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a protein of interest.

Thus, in particular embodiment, the invention refers to a method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production in a subject in need thereof, comprising administering into the lacrimal gland of said subject an AAV2/9 vector comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a protein of interest

Thus, in particular embodiment, the invention refers to a method for corneal reinnervation and/or corneal wound healing and/or increasing tear production in a subject in need thereof, comprising administering into the lacrimal gland of said subject an AAV2/9 vector comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a protein of interest, wherein the protein of interest is selected in the group consisting of nerve growth factor (NGF), neuropilin 2 (NRP2), semaphorin 3C SEMA3C, pigment epithelium-derived factor (PEDF), glial-cell derived neurotrophic factor (GDNF) and thyroglobuline.

Thus, in particular embodiment, the invention refers to a method for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production in a subject in need thereof, comprising administering into the lacrimal gland of said subject an AAV2/9 vector comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding NGF. In particular embodiment, the an AAV2/9 vector comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding aprotein of interest is not administered in combination with nicotinic acetylcholine receptor (nAChR) agonist. In particular embodiment, the an AAV2/9 vector comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a protein of interest is administered alone.

In particular embodiment, the an AAV2/9 vector comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a NGF is not administered in combination with nicotinic acetylcholine receptor (nAChR) agonist. In particular embodiment, the an AAV2/9 vector comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) encoding a NGF is administered alone.

The methods of the invention are also particularly suitable to treat corneal defects in subject in need thereof.

Thus, the present invention relates to a method for treating corneal defects in subject in need thereof comprising administering into the lacrimal gland to said subject an effective amount of a nucleic acid encoding a protein of interest.

In other word, the invention relates to a nucleic acid encoding a protein of interest for use for treating corneal defects in subject in need thereof, wherein said nucleic acid is administered into the the lacrimal gland.

In some embodiment, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and more particularly messenger RNA.

In some embodiments, the nucleic acid is administered into the lacrimal gland simultaneously, separately or sequentially with a transfection reagent.

In some embodiments, the messenger RNA acid is administered into the lacrimal gland simultaneously, separately or sequentially with a transfection reagent.

In some embodiments, the messenger RNA acid is further mixed with a transfection reagent prior to the administration into lacrimal gland.

In some embodiments, the nucleic acid is comprised in a vector.

In some embodiments, the vector is a viral or non-viral vector.

In some embodiments, the viral vector is an adeno-associated virus (AAV) vector comprising deoxyribonucleic acid (DNA) encoding a protein of interest.

In some embodiments, the AAV vector is an AAV2/9 or AAV2/5 vector Thus, in particular embodiments, the inventions relates to an AAV2/9 vector comprising deoxyribonucleic acid (DNA) encoding a protein of interest for use for treating corneal defects in subject in need thereof.

Thus, in particular embodiments, the inventions relates to an AAV2/9 vector comprising deoxyribonucleic acid (DNA) encoding a protein of interest for use for treating corneal defects in subject in need thereof, wherein the AAV2/9 vector is not administered with nicotinic acetylcholine receptor (nAChR) agonist.

In another embodiments, the vector is a non-viral vector comprising messenger ribonucleic acid (RNA) encoding a protein of interest.

Thus, in particular embodiment, the inventions relates to a non-viral vector comprising messenger ribonucleic acid (mRNA) encoding a protein of interest for use for treating corneal defects in subject in need thereof.

In particular embodiment, the inventions relates to a messenger ribonucleic acid (mRNA) encoding a protein of interest for use for treating corneal defects in subject in need thereof, wherein the mRNA encoding a protein of interest is is administered into the lacrimal gland simultaneously, separately or sequentially with a transfection reagent.

In some embodiment, the protein of interest is selected from the group consisting in nerve growth factor (NGF), neuropilin 2 (NRP2), semaphorin 3C (SEMA3C), pigment epithelium-derived factor (PEDF), vascular Endothelial Growth Factor A (VEGF-A), thyroglobuline, epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), bone morphogenetic protein 6 (BMP6), insulin-like growth factor 1 (IGF- 1), insulin-like growth factor 2 (IGF-2), glial-cell derived neurotrophic factor (GDNF) and transforming growth factor (TGF).

In particular embodiment, the protein of interest is selected from the group consisting in nerve growth factor (NGF), vascular Endothelial Growth Factor A (VEGF-A), epidermal growth factor (EGF), neuropilin 2 (NRP2), semaphorin 3C SEMA3C, pigment epithelium- derived factor (PEDF), glial-cell derived neurotrophic factor (GDNF) and thyroglobuline.

In particular embodiment, the protein of interest is NGF and the tear production is increased.

Thus, in particular embodiment, the invention relates to a method for treating corneal defects by increasing tear production in subject in need thereof comprising administering into the lacrimal gland to said subject an effective amount of a nucleic acid encoding a protein of interest. Thus, in particular embodiment, the invention relates to a method for treating corneal defects by increasing tear production in subject in need thereof comprising administering into the lacrimal gland to said subject an effective amount of a nucleic acid encoding NGF

In particular embodiment, the invention relates to a method for treating corneal defects by improving corneal reinnervation and/or corneal wound healing in subject in need thereof comprising administering into the lacrimal gland to said subject an effective amount of a nucleic acid encoding a protein of interest.

Thus, in particular embodiment, the invention relates to a method for treating corneal defects by improving corneal reinnervation and/or corneal wound healing in subject in need thereof comprising administering into the lacrimal gland to said subject an effective amount of a nucleic acid encoding a protein of interest, wherein the protein of interest is selected from the group consisting in nerve growth factor (NGF), vascular Endothelial Growth Factor A (VEGF- A), epidermal growth factor (EGF), neuropilin 2 (NRP2), semaphorin 3C SEMA3C, pigment epithelium-derived factor (PEDF), thyroid hormone, and glial-cell derived neurotrophic factor (GDNF).

As used herein, the term “corneal defects”, has its general meaning in the art and refers to abroad range of diseases, including physical wounds corneal (e.g abrasion), dry eye diseases, diabetes such as diabetic keratopathy, fuchs’ endothelial corneal dystrophy, bullous keratopathy, corneal thinning disorders, ICE syndrome or neurotrophic keratitis (NK).

In some embodiment, the corneal defects is physical wounds corneal, dry eye diseases, diabetic keratopathy, corneal thinning disorders, ICE syndrome or neurotrophic keratitis (NK).

In particular embodiment, the corneal defects is drye eye diseases or physical wouds corneal.

Thus, the methods of the invention are particularly suitable for treating drye eye diseases by increasing tear production.

Thus, in particular embodiment, the invention relates to a method for treating drye eye diseases by increasing tear production in subject in need thereof comprising administering into the lacrimal gland to said subject an effective amount of a nucleic acid encoding a protein of interest.

Thus, in particular embodiment, the invention relates to a method for treating drye eye diseases by increasing tear production in subject in need thereof comprising administering into the lacrimal gland to said subject an effective amount of an AVV2/9 comprising DNA encoding a protein of interest, and more particularly DNA encoding NGF. Thus, in particular embodiment, the invention relates to a method for treating drye eye diseases by increasing tear production in subject in need thereof comprising administering into the lacrimal gland to said subject an effective amount of a mRNA a protein of interest, and more particularly DNA encoding NGF.

As used herein, the term “dry eye disease” or “dry eye symptom”, has its general meaning in the art and refers to a generic ocular disorder. Dry eye disease (DED) has become common on a global scale in recent years. There is a wide prevalence of DED in different countries based on various ethnicities and environment. In addition to advanced age and gender, such factors as living at high altitude, smoking, pterygium, prolonged use of consumer electronics or overingesting of caffeine or multivitamins are considered to be the major risk factors of DED (Kuo et al, 2019). Dry eye symptom can be idiopathic (without apparent cause) Furthermore, a various broad of pathology can caused dry eye symptom. Dry eye symptom can be caused by alacrima, xerophthalmia, lacrimal gland ablation, sensory denervation, collagen vascular diseases and immune disease such as polychondritis, allergic eye diseas, sarcoidosis, thyroid disorders, rheumatoid arthritis, graft vs. host disease, diabetes, neurotrophic keratitis and systemic lupus erythematosus. Sjogren syndrome are mainly associated with drye eye disease. Drugs such as isotretinoin, sedatives, diuretics, tricyclic antidepressants, antihypertensives, oral contraceptives, antihistamines, nasal decongestants, beta-blockers, phenothiazines, atropine, and pain relieving opiates such as morphine can cause or worsen dry eye symptom. Drye eye symptom can also result from nocturnal lagophthalmos or insufficient rate of blinking. Abnormalities of the mucin tear layer caused by vitamin A deficiency, trachoma, diphtheric keratoconjunctivitis, mucocutaneous disorders and certain topical medications are also causes of dry eye symptom. Dry eye symptom can also result after eye surgery, such as refractive surgeries or cornea transplant.

In some embodiment, dry eye symptom is caused by the Sjogren syndrome, diabetic keratopathy, eye surgery or neurotrophic keratitis.

As used herein, the term “Sjogren syndrome”, has its general meaning in the art and refers to an autoimmune disease or disorder in which immune cells attack the glands that produce tears and saliva. The hallmark symptoms of the disorder are dry mouth and dry eyes. In addition, Sjogren’s syndrome may cause skin, nose, and vaginal dryness, and may affect other organs of the body including the kidneys, blood vessels, lungs, liver, pancreas, and brain. Sjogren’s syndrome can exist as a primary disorder ("primary Sjogren’s syndrome") or as a secondary disorder ("secondary Sjogren’s syndrome") that is associated with and/or secondary to other autoimmune disorders including rheumatic disorders such as rheumatoid arthritis, systemic lupus, polymyositis, scleroderma, and autoimmune hepatitis, lymphomas such as nonHodgkin's lymphoma, and endocrine disorders such as thyroiditis. The term " Sjogren’s syndrome" as used herein applies to Sjogren’s syndrome no matter what the stage, including both primary and secondary Sjogren’s syndrome.

As used herein, the term “neurotrophic keratitis” (NK), has its general meaning in the art and refers to a degenerative disease characterized by corneal sensitivity reduction, spontaneous epithelium breakdown, and impairment of corneal healing. Several causes of NK includes herpetic keratitis, diabetes, and ophthalmic and neurosurgical procedures. NK is characterized by decreased or absent corneal sensation, leading to epithelial breakdown, impairment of healing, and ultimately corneal ulceration, melting and perforation.

As used herein, the term “diabetic keratopathy” has its general meaning in the art and refers to the main ocular complications from diabetes mellitus. Diabetic keratopathy exhibits several clinical manifestations, including persistent corneal epithelial erosion, superficial punctate keratopathy, delayed epithelial regeneration, and decreased corneal sensitivity, that may lead to compromised visual acuity or permanent vision loss.

As used herein, the term “keratoconus” has its general meaning in the art and refers to thinning of cornea which gradually bulges outward into a cone shape. A cone-shaped cornea causes blurred vision and may cause sensitivity to light and glare. Keratoconus usually affects both eyes, though it often affects one eye more than the other.

As used herein, the term “ICE syndrome” for “Iridocorneal Endothelial (ICE) Syndrome” has its general meaning in the art and refers to a group of ophthalmic disorder that involves an irregular corneal endothelium that can lead to varying degrees of corneal edema, iris atrophy, and secondary angle-closure glaucoma.

The methods of the invention is also particurlary suitable to prevent or treat corneal defect caused by eye surgery, such as refractive surgery (PRK or LASIK) or cornea transplant.

As used herein, the term “eye surgery” include various corneal surgical procedures. According to the invention, eye surgery includes corneal transplantation, amniotic membrane transplantation, limbal stem cell transplantation, phototherapeutic kerarectomy (PTK), photorefractive keratectomy (PRK) , Laser in Situ Keratomileusis (LASIK) eye surgery and removal of conjunctival lesions. The methods of the invention are particularly suitable for treating physical wounds corneal by improving corenal reinnervation and/or corneal wounds healing.

Thus, in particular embodiment, the invention relates to a method for treating physical wounds corneal by improving corenal reinnervation and/or corneal wounds healing in subject in need thereof comprising administering into the lacrimal gland to said subject an effective amount of a nucleic acid encoding a protein of interest.

Thus, in particular embodiment, the invention relates to a method for treating physical wounds corneal by improving corenal reinnervation and/or corneal wounds healing in subject in need thereof comprising administering into the lacrimal gland to said subject an effective amount of an AVV2/9 comprising DNA encoding a protein of interest, and more particularly DNA encoding NGF.

Thus, in particular embodiment, the invention relates to a method for treating physical wounds corneal by improving corenal reinnervation and/or corneal wounds healing in subject in need thereof comprising administering into the lacrimal gland to said subject an effective amount of a mRNA a protein of interest, and more particularly DNA encoding NGF

As used herein, the term “therapeutically effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease condition, including reducing or eliminating one or more symptoms or manifestations of the disorder or disease. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an “effective amount” in any individual case can be determined by one of skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.

As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. According to the invention, the method of the invention allows to ameliorate/improve corneal reinnervation.

The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms (i.e dry eye) of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

In some embodiment, the vector of the invention is administered by a local injection into lachrymal gland.

Pharmaceutical composition

The nucleic acid encoding a protein of interest according to the invention may be administrated in the form of a pharmaceutical compositions comprising pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers

Accordingly, the invention relates to a pharmaceutical composition comprising a nucleic acid encoding a protein of interest for use for modulating tear film composition in a subject in need thereof.

The invention also relates to a pharmaceutical composition comprising a nucleic acid encoding a protein of interest for use for improving corneal reinnervation and/or increasing tear production in a subject in need thereof.

The invention also relates to a pharmaceutical composition comprising a nucleic acid encoding a protein of interest for use for treating corneal defects in a subject in need thereof. In particular embodiment, the nucleic acid (RNA) is messenger ribonucleic acid, and more particularly a messenger ribonucleic acid encoding for NGF or EGF.

In particular embodiment, the pharmaceutical composition further comprises a transfection reagents

In particular embodiment, the nucleic acid (RNA) is administered with transfection reagent.

Thus, in particular embodiment, the pharmaceutical composition comprises a messenger ribonucleic acid , and more particularly a messenger ribonucleic acid encoding for NGF or EGF, wherein the pharmaceutical composition is administered with transfection reagent.

The vector comprising a nucleic acid encoding a protein of interest according to the invention may be administrated in the form of a pharmaceutical compositions comprising pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers.

Accordingly, the invention relates to a pharmaceutical composition comprising a vector comprising a nucleic acid encoding a protein of interest for use for modulating tear film composition in a subject in need thereof.

The invention also relates to a pharmaceutical composition comprising a vector comprising a nucleic acid encoding a protein of interest for use for improving corneal reinnervation and/or corneal wound healing and/or increasing tear production in a subject in need thereof.

The invention also relates to a pharmaceutical composition comprising a vector comprising a nucleic acid encoding a protein of interest for use for treating corneal defects in a subject in need thereof.

In some embodiments, the vector is a viral or non-viral vector.

In some embodiment, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

In some embodiment, the ribonucleic acid (RNA) is messenger ribonucleic acid.

In some embodiments, the viral vector is an adeno-associated virus (AAV) vector.

In some embodiments, the AAV vector is an AAV2/9 or AAV2/5 vector.

In some embodiments, the AAV vector comprises deoxyribonucleic acid (DNA) encoding a protein of interest. Thus, in particular embodiments, the inventions relates to an AAV2/9 vector comprising deoxyribonucleic acid (DNA) encoding a protein of interest for use for treating corneal defects in subject in need thereof.

In another embodiments, the non-viral vector comprises messenger ribonucleic acid (RNA) encoding a protein of interest.

In some embodiment, the protein of interest is selected in the group consisting of nerve growth factor (NGF), neuropilin 2 (NRP2), semaphorin 3C (SEMA3C), pigment epithelium- derived factor (PEDF), vascular Endothelial Growth Factor A (VEGF-A), thyroglobuline, epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), bone morphogenetic protein 6 (BMP6), insulin-like growth factor 1 (IGF-1), insulinlike growth factor 2 (IGF-2), glial-cell derived neurotrophic factor (GDNF) and transforming growth factor (TGF).

In some embodiment, the protein of interest is NGF.

In some embodiment, the pharmaceutical composition does not compises nicotinic acetylcholine receptor (nAChR) agonist.

In some embodiment, the pharmaceutical composition for use according to the invention is not administered in combination with nicotinic acetylcholine receptor (nAChR) agonist.

In a particular embodiment, according to all the aspects of the invention, the pharmaceutical composition is suitable for a local administration to the subject to be treated, such as is suitable for an administration into the lacrimal gland of the subject to be treated.

As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and/or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically" or "pharmaceutically acceptable" refers to medium and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable medium comprises any of standard pharmaceutically accepted mediums known to those of ordinary skill in the art, in particular in formulating pharmaceutical compositions to be administered to the eye.

In some embodiment, the pharmaceutical composition of the invention is administered by a local injection. In some embodiment, the pharmaceutical composition of the invention is injected into the gland lacrimal In some embodiment, the pharmaceutical composition is injected by needle linked to a syringe, and more particulary by a beveled needle linked to a syringe, and more particularly by a 34-gauge beveled needle linked to a syringe.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. GFP is detected in AAV2/9 and AAV2/5 CAG-GFP- injected LG and dosed by Western blot. Quantification of GFP / total protein ratio in LG lysates (n = 3 mice per group). Results are expressed as the mean ± SD. Statistical analysis using Brown-Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test. ** p = 0.0026 between AAV2/9 empty capsid and AAV2/9 CAG-GFP groups, **p = 0.0077 between AAV2/5 empty capsid and AAV2/5 CAG-GFP groups, ** p = 0.0018 between AAV2/9 CAG- GFP and AAV2/5 CAG-GFP groups; ns, not significant; A. U., arbitrary units.

Figure 2. Injections of AAV2/5 or AAV2/9 CAG-mNGF in the murine LG significantly increase the level of mNGF in tears. mNGF protein levels were analyzed by ELISA in the tears of AAV2/9 or AAV2/5 CAG-mNGF-injected mice in the right LG at 12 weeks old, when compared to AAV2/9 or AAV2/5 empty capsid-injected mice, 1 month post injection respectively (10 ¹⁰ vg / LG in 3 pl, n = 5 mice per group). A. Quantification of the mNGF level in the tears of injected mice. Results are expressed as the mean ± SD. Statistical analysis using Brown-Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test. ** p = 0.0065 between AAV2/9 empty capsid and AAV2/9 CAG- mNGF groups, * p = 0.0464 between AAV2/5 empty capsid and AAV2/5 CAG-mNGF groups, ** p = 0.0085 between AAV2/9 CAG-mNGF and AAV2/5 CAG-mNGF groups.

Figure 3. Dose response and kinetic studies reveal the correlation between mNGF protein level in the tears and the AAV2/9-injected dose, and the detection of mNGF in tears for at least 6 months. For the dose response study, mNGF protein levels were analyzed by ELISA in the tears of AAV2/9 CAG-mNGF-injected mice in the right LG at 12 weeks old by increasing dose of vector (10 ⁹ , 10 ¹⁰ , and 10 ¹¹ vg / LG in 3 pl, n = 5 mice per dose), when compared to the tears of AAV2/9 empty capsid-injected mice 30 days post injection. For the long-term kinetic study, mNGF protein levels were analyzed by ELISA in the tears of AAV2/9 CAG-mNGF-injected mice in the right LG at 12 weeks old (10 ¹¹ vg / LG in 3 pl), when compared to the tears of AAV2/9 empty capsid-injected mice before injection and 7, 15, 30, 60, 90, 120 and 150 days post injection (n = 3 or 5 for AAV2/9 empty capsid-injected mice and AAV2/9 CAG-mNGF -injected mice respectively). A. Quantification by ELISA of the mNGF protein level in the tears 30 days post injection following the indicated increasing dose of injected vector. Results are expressed as the mean ± SD. Statistical analysis using Brown- Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test. * p = 0.0107 and * p = 0.0341 between the doses 10 ⁹ and 10 ¹⁰ vg / LG and between the doses 10 ¹⁰ and 10 ¹¹ vg / LG respectively, ns, not significant. B. Quantification by ELISA of the mNGF protein level in the tears up to 6 months post injection at the indicated days post injection. Statistical analysis using repeated measures two-way ANOVA test followed by Sidak’s multiple comparisons test. ** p = 0.0012, p =0.0574, *p =0.0299, ** p =0.0356, * p = 0.0410, * p = 0.0346, * p = 0.0454, between AAV2/9 empty capsid and AAV2/9 CAG- mNGF groups 7, 15, 30, 60, 90, 120 and 150 days post injection respectively, ns, not significant.

Figure 4. Biodistribution analysis after the injection of AAV2/9 CAG-mNGF in murine LG reveals a limited diffusion to peripheral organs. AAV2/9 CAG-mNGF was injected in the right LG of 12 week-old mice (10 ¹¹ vg / LG in 3 pl, n = 7 mice). 1 month post injection, tears were collected and mNGF protein level was quantified by ELISA. At the same time, mice were sacrificed, LG, liver and heart were collected and analyzed by qPCR. A. Graph showing the transduction rate expressed in vector genome / diploid genome (vg / dg) above the lowest limit of quantification (LLOQ). Results are expressed as the mean ± SD. Statistical analysis using Brown-Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test. **** p < 0.0001 between LG and liver, **** p < 0.0001 between LG and heart, * p = 0.0226 between liver and heart. B. Graph showing the linear correlation for each mouse between the vg / dg in LG and the mNGF protein level (ng/ml) from the statistical linear regression (red line). R ² , R squared.

Figure 5. Injection of AAV2/9 CAG-mNGF in murine LG does not alter tear protein concentration nor corneal integrity. AAV2/9 empty capsid or AAV2/9 C AG- mNGF were injected into murine LG (10 ¹¹ vg / LG in 3 pl, n = 3 and 5 mice respectively). The volume of tears (A), the protein concentration in tears (B) were assessed 1 day before injection and 7, 30, 60, 120 days and 180 post injection. A. Graph showing the volume of tears (pl / min) collected before injection and at the indicated days post injection. Results are expressed as the mean ± SD. Statistical analysis using repeated measures two-way ANOVA test followed by Sidak’s multiple comparisons test. ** p = 0.0036, **** p < 0.0001, ** p = 0.0057, * p = 0.0246 between AAV2/9 empty capsid and AAV2/9 CAG-mNGF groups at 7, 30, 60 and 120 days post injection respectively, ns, not significant. B. Graph showing the protein concentration in tears (mg / ml) collected before injection and at the indicated days post injection. Results are expressed as the mean ± SD. Statistical analysis using repeated measures two-way ANOVA test followed by Sidak’s multiple comparisons test, ns, not significant.

Figure 6. Injection of AAV2/9 CAG-mNGF into murine LG does not affect corneal sensitivity. Quantification of the corneal sensitivity (1 / g) measured by von Frey test performed both on the contralateral side and the injected side of AAV2/9 empty capsid (white plots, n = 7) or AAV2/9 CAG-mNGF (red plots, n = 5)-injected mice. Von Frey analysis was performed two weeks post injection. Results are expressed as the mean ± SD. Statistical analysis using two-way ANOVA test followed by Sidak’s multiple comparisons test, ns, not significant.

Figure 7. Injection mRNA into murine lacrimal gland. 0.85ng of mRNA are diluted in the transfection medium (In vivo-Jet for example) and then are incubated for 15min at room temperature. As complexes are stable Ihr at room temperature, injections are performed swiftly (0.85pg of mRNA in 2pL are injected in each murine LG).

EXAMPLEI:

Material & Methods

Study design

The goal of this study was to assess the transduction pattern of AAV vector serotypes 2/5 and 2/9 after a single injection into murine LG. Then, to evaluate the efficiency and the safety of a AAV2/9-mediated gene transfer of mNGF in the murine LG, for its secretion in the tear film. The main readouts of this study included: the transduction pattern analyzed by H4C and Western blot, the secretion of mNGF measured in tears by ELISA and Western blot, the AAV2/9 biodistribution by qPCR, the injection biosafety by analyzing the corneal integrity and innervation, the volume of tears and the protein concentration in tears. Experimental groups were sized according to the literature to allow statistical analysis. No outliers were excluded from the study, except mice exhibiting spontaneous eye damages after the surgery or during the experiments. Behavioral data obtained from animals displaying eye damages unrelated to the abrasion procedure during the study were excluded. Scientists who performed the experiments and analysis were blinded to the group’s identity. Data were analyzed by those carrying out the experiments and verified by the supervisor.

Cloning and vector production

Cloning of the enhanced GFP (GFP) and the mNGF in pAAV and AAV vector productions were provided by the vector core of the TarGeT (Translational Gene Therapy) Laboratory of Nantes, INSERM UMR 1089 (Nantes University, France). Briefly, single- stranded AAV2/5 and AAV2/9 CAG-GFP, single-stranded AAV2/5 and AAV2/9 CAG-mNGF vectors were obtained from pAAV CAG-GFP and pAAV CAG-mNGF plasmids respectively, containing AAV2 inverted terminal sequences, CAG promoter and BGH polyA signal. AAV2/5 and AAV2/9 CAG-GFP vectors were used to assess the transduction pattern after injection in the murine LG. AAV2/5 and AAV2/9 CAG-mNGF vectors were used to evaluate the efficiency of an AAV-based gene transfer in the LG allowing mNGF secretion in the tear fluid. Singlestranded vectors containing AAV2/5 and AAV2/9 empty capsid served as controls.

Vector production was performed following the protocol of vector core of the TarGeT Laboratory of Nates ⁴⁹ . Briefly, recombinant AAVs were manufactured by co-transfection of HEK293 cells and purified by cesium chloride density gradients followed by extensive dialysis against phosphate-buffered saline (PBS). Vector titers were determined by qPCR and expressed as vector genome (vg) / ml.

Animals included in this study

All mice experiments were approved by the local ethical committee and the “ministere de la recherche et de Fenseignement superieur” (authorization 2016080510211993 version2). All the procedures were performed in accordance with the French regulation for the animal procedure (French decree 2013-118) and with specific European Union guidelines for the protection of animal welfare (Directive 2010/63/EU). Mice were maintained on a 12 h dark, 12 h light cycle with a humidity between 40 and 60 % and an ambient temperature of 21-22 °C.

Surgery and Vector delivery

Twelve-week-old Swiss/CDl female mice (Janvier Labs, France) were injected into the right LG. The LG injection of AAV vectors was performed under anesthesia with a mixture of Ketamine (70 mg / kg, Imalgene® 1000, Centravet, France) and Medetomidine (1 mg / kg, Domitor®, Centravet, France). One drop of Ocry-gel (Centravet, France) was applied to each eye. The skin on the cheek under the right ear was disinfected with vetedine solution (Centravet, France) and ethanol 70% and then cut above the LG location. Next, the viral solution was injected using a 34-gauge beveled needle (Hamilton, reference 207434, Reno, NV, USA) linked to a 10-pl Hamilton syringe (1701 RN serie, Hamilton, reference 7653-01, Reno, NV, USA). Wound were closed by suture wires (Novosyn® 6/0, reference C0068006, B Braun) and then disinfected with vetedine solution (Centravet, France). After surgery, mice were treated with Buprenorphine (100 pg / kg, Brupecare®, Centravet, France) and were woken up with Atipamezole (1 mg / kg, Antisedan®, Centravet, France).

AAV vector solutions were prepared by diluting vectors at the right titer with sterile PBS and 0.01 % of Fast Green (Sigma-Aldrich, reference F7252, France). For all experimental studies, mice were unilaterally injected in the right LG with 3 pl of vectors indicated below. For the transduction pattern study, AAV2/5 and AAV2/9 CAG GFP were injected at

10 ¹⁰ vg / LG. For the dose response study, AAV2/9 CAG-mNGF was injected at 10 ⁹ , IO ¹⁰ or

10 ¹¹ vg / LG. For the kinetic study, AAV2/5 CAG-mNGF was injected at 10 ¹⁰ vg / LG and AAV2/9 CAG-mNGF was injected at IO ¹⁰ and 10 ¹¹ vg / LG. For the biodistribution and the biosafety studies, AAV2/9 CAG-mNGF was injected at 10 ¹¹ vg / LG. Animals injected with AAV2/5 or AAV2/9 empty capsid served as control.

Corneal abrasion

Corneal abrasions were performed as previously described ^1,3 ’ ⁵⁰ . Briefly, an ocular burr (Algerbrush II, reference BR2-5 0,5 mm, Alger company) was used on mice that were anesthetized with a mixture of Ketamine (70 mg / kg, Imalgene® 1000, Centravet, France) and Medetomidine (1 mg / kg, Domitor®, Centravet, France). Abrasions were performed unilaterally. A fluorescein solution (1% in PBS, Sigma-Aldrich) was used to visualize the wound under a cobalt blue light. After abrasion, one drop of Ocry-gel (Centravet, France) was applied to each eye, mice were treated with Buprenorphine (100 pg / kg, Brupecare®, Centravet, France) and were woken up with Atipamezole (1 mg / kg, Antisedan®, Centravet, France).

Tissue collection and processing

Tears were collected using a 1 pl-microcapillary (Sigma-Aldrich, reference P1424, France) for one minute, 1 day before injection and 30 days post injection for the dose response and biodistribution studies; 1 day before injection and 7, 30, 60, 120 and 180 days post injection for the kinetic and biosafety studies; 1 day before abrasion and 1, 3 and 7 days post abrasion for the corneal abrasion study.

For the transduction pattern and the biodistribution studies, mice were euthanized 30 days post injection using pentobarbital (54.7 mg/mL, 140 mg/kg, Centravet, France). They were transcardially perfused with sterile PBS and tissues were quickly dissected. Tissues were then fixed for 45 min in 4% paraformaldehyde solution (AntigenFix, Diapath, reference POO 14, France) at room temperature or directly snap-frozen in liquid nitrogen and stored at -80°C for H4C and molecular/biochemical analysis respectively.

For whole cornea imaging, mice were euthanized by cervical dislocation, enucleated with curved scissors by cutting the optic nerve. Collected eyes were then fixed for 20 min in 4% paraformaldehyde solution (AntigenFix, Diapath, reference P0014, France) at room temperature. After PBS washes, eyes were dehydrated during 2 h in 50% ethanol/PBS and then stored at 4°C in 70% ethanol/PBS. Tear volume, protein concentration and mNGF analyses in tears

The volume of tears per minute (pl / min) and the protein concentration in tears (mg / ml) using the BCA protein assay kit (Fisher Scientific, reference 10678484, France) were measured and expressed as the mean ± SD.

The mNGF level in tears upon a AAV2/9 CAG-mNGF injection in the LG was dosed using the mNGF ELISA kit (Sigma-Aldrich, reference RABI 119, France) following the manufacturer’s recommendations. Measures were performed on a CLARIOstar microplate reader (BMG Labtech, France), and analyzed using the CLARIOstar software (version 5.60 R2). Results are expressed as the mean ± SD.

IHC study

The following antibodies were used for IHC studies: chicken anti-GFP (Aves Labs, reference GFP-1020, 1/1000), mouse anti-Ecadh (BD Biosciences, reference 610182, 1/300), rabbit anti-Krtl9 (Abeam, reference ab52625, 1/200), rabbit anti-pill tubulin (Abeam, reference abl8207, 1/1000), goat anti-chicken Alexa Fluor 488 (Thermo Fisher Scientific, reference A-32931, 1/500), goat anti-mouse Alexa Fluor 568 (Thermo Fisher Scientific, reference A-l 1004, 1/500) and goat anti-rabbit Alexa Fluor 568 (Abeam, reference abl75471, 1/500), goat anti-rabbit Alexa Fluor 488 (Abeam, reference abl l008, 1/500) and goat antirabbit Alexa Fluor 568 (Abeam, reference abl75471, 1/500). Nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific, reference H3570, 1/2000) and BioTracker NIR694 (Merck, reference SCT118, 1/400).

Immunohistochemistry on LG frozen sections

Following fixation, LG were incubated 24 h in two successive baths of 6 % and 30 % sucrose and then embedded in Optimal Cutting Temperature (OCT tissue freezing medium, MM France, reference F/TFM-C) and stored at -80 °C. Longitudinal sections (10 pm of thickness) were cut using a cryostat apparatus (LEICA CM3050). For primary antibodies produced in rabbit and chicken, cryosections were blocked with a mixture of 5 % Goat Serum (GS, Thermo Fisher Scientific, reference 16210064), 5 % of fish skin gelatin (FSG, Sigma- Aldrich, reference G7765) and 0.1 % triton X-100 in PBS for 1 h at room temperature. For primary antibodies produced in mouse (mouse anti-Ecadh), the blocking step described above was followed by an incubation with goat anti-mouse immunoglobulins (Abeam, reference ab6668, 1/200) during 1 h at room temperature. Cryosections were then incubated overnight at 4 °C with primary antibodies diluted in GS/FSG/Triton/PBS mixture, washed three times with 0.1 % triton X-100 / PBS and subsequently incubated 1 h at room temperature with secondary antibodies diluted in GS / FSG / Triton / PBS mixture. After several PBS washes, cryosections were mounted in Fluoromount-G mounting medium (Invitrogen, reference 00-4958-02).

Immunohistochemistry on whole cornea

Eyes were rehydrated in 50 % ethanol/PBS during 2 h and washed twice in PBS for 15 minutes at room temperature. Corneas were dissected and permeabilized with 0.5% Triton-X- 100/PBS on a rocking agitator for 1 h and then blocked in 5 % GS (Thermo Fisher Scientific, reference 16210064) 2.5 % FSG (Sigma- Aldrich, reference G7765) in 0.1 % Triton X-100/PBS at room temperature. Corneas were incubated in primary antibody diluted in blocking solution overnight at 4°C on a rocking agitator and rinsed in 0.1 % Triton X-100/PBS at room temperature (three times 1 h). Next, samples were incubated with secondary antibodies as previously mentioned. After the washes, nuclei were stained 10 minutes with BioTracker NIR694 (Merck, reference SCT118) and washed in PBS. Corneas were cut at four comers and mounted in Fluoromount-G mounting medium (Invitrogen, reference 00-4958-02), epithelium facing the coverslip.

Imaging

LG images were acquired using Zen Black software (version 2.3 SP1, Zeiss, France) on a LSM 880 confocal microscope (Zeiss, France). Whole LG section images were obtained using a 20x / 0.8 objective while co-immunostaining of GFP with E-cadherin or Keratinl9 proteins were observed via 0.36 pm step size z-stacks using a 63x / 1.4 oil immersion objective. Images were then processed with Zen Black software (version 2.3 SP1, Zeiss, France) and Zen Blue lite software (version 3.2, Zeiss, France).

Whole cornea images were acquired using the navigator module on a Leica Thunder Imager Tissue microscope with Large Volume Computational Clearing (LVCC) process. Images were obtained using a 20x/0.55 objective with LAS X software (3.7.4) and processed with Imaris Bitplane software (version 9.8.0).

Western blot

Frozen LG were crushed with a pestle and mortar pre cooled at -80°C, solubilized in Pierce® RIPA lysis buffer (Thermo Fisher Scientific, reference 89900, France) supplemented with protease inhibitors (Halt™ Protease Inhibitor Cocktail, Thermo Fisher Scientific, reference 87786, France), homogenized on a rotating wheel at 4 °C overnight and then centrifuged at 16 900 g (Centrifuge 5418R, Eppendorf) for 30 min at 4 °C. Supernatants were recovered and protein concentration of LG lysates or collected tears were quantified using the BCA protein assay kit (Thermo Fisher Scientific, reference 10678484, France). Fourty and 8 pg of proteins from LG lysates and collected tears respectively were loaded on any kD precast polyacrylamide gels (Mini-Protean® TGX™ gels, Bio Rad, reference 4568124, France). Proteins were transferred to nitrocellulose membranes (Trans-Blot Turbo Mini 0.2 pm Nitrocellulose Transfer Pack, Bio Rad, reference 1704158, France) through semi-dry transfer process (Bio Rad Trans-Blot Turbo system). Membranes were incubated with the REVERT™ total protein stain solution (LLCOR Biosciences, reference 926-11015, France) to record the overall amount of protein per well. They were then blocked for 1 h at room temperature using Intercept® blocking buffer (LLCOR Biosciences, reference 927-60001, France). They were incubated with the following primary antibodies overnight at 4 °C in LLCOR blocking buffer: chicken anti-GFP (Aves Labs, reference GFP-1020, 1/2000) or rabbit mNGF (Abeam, reference Ab52918, 1/500). Following three washes with TBS containing 0.1% Tween (TBST) for 15 min, secondary antibodies were incubated at a 1/15000 dilution in LLCOR blocking buffer: donkey anti chicken IR Dye 800CW (LLCOR Biosciences, reference 926-32218) or donkey anti rabbit IR Dye 800CW (LLCOR Biosciences, reference 926-32213). After three washes in TBST for 15 min, images were acquired with an Odyssey CLX LLCOR Imaging System (LLCOR Biosciences, France) and the quantifications were performed with Image Studio lite software (version 5.2). The GFP and the mNGF protein levels were both normalized to the total amount of protein loaded per well. The results are expressed as the mean ± SD.

A A V2/9 biodistribution study

LG, liver and heart from AAV2/9 CAG-mNGF-injected mice (10 ¹¹ vg / LG, n = T) were collected one month post injection in DNA-free, RNAse/DNAse-free and PCR inhibitor-free certified microtubes. Tissue samples were collected immediately after sacrifice, snap-frozen in liquid nitrogen and stored at -80 °C in conditions that minimize cross-contamination and avoid qPCR inhibition. Extraction of genomic DNA (gDNA) from tissues using the Gentra Puregene kit (Qiagen, reference 158445, France) and Tissue Lyser II (Qiagen, reference 85300, France) was performed in accordance with the manufacturer’s recommendations. Vector genome copy number was determined using a primer/FAM-TAMRA probe combination designed to amplify a specific region of the BGH transgene. qPCR analyses were conducted on a StepOne Plus apparatus (Applied Biosystems®, Thermo Fisher Scientific, France) using 50 ng of gDNA in triplicates and the following cycling conditions: denaturation step (20 sec, 95°C) followed by a total of 45 cycles (1 sec, 95°C; 20 sec, 60°C). All reactions were performed in a final volume of 20 pl containing template DNA, Premix Ex Taq (Takara/Ozyme, reference RR390L, France), 0.4pl of ROX reference Dye (Takara/ozyme, reference RR390L, France), 0.2 pmol/L of each primer and 0.1 pmol/L of Taqman® probe (Dual-Labeled Probes, Sigma- Aldrich, France). Endogenous gDNA copy numbers were determined using the following primers/FAM- BHQ1 probe combination, designed to amplify a specific region of the murine Albumin sequence. qPCR analyses were conducted on a Cl 000 touch thermal cycler (Bio-Rad, France) using 50 ng of gDNA in triplicates and the following cycling conditions: denaturation step (20 sec, 95°C) followed by a total of 45 cycles (3 sec, 95°C; 30 sec, 60°C). All reactions were performed in a final volume of 20 pl containing template DNA, Premix Ex Taq (Takara/Ozyme, reference RR390L, France), 0.25 pmol/L of each primer and 0.2 pmol/L of Taqman® probe (Dual-Labeled Probes, Sigma- Aldrich, France). For each sample, threshold cycle (Ct) values were compared with those obtained with different dilutions of linearized standard plasmids (containing either the BGH expression cassette or the murine Albumin gene) using Bio-Rad CFX Maestro 2.2 software (version version 5.2.008.0222) or StepOne software (version 2.3) for the Cl 000 touch thermal cycler and the StepOne Plus apparatus respectively. The absence of qPCR inhibition in the presence of gDNA was checked by analyzing 50 ng of gDNA extracted from tissue samples from two AAV2/9 empty capsid-injected control mice. Results were expressed in vector genome copies per diploid genome (vg / dg) as the mean ± SD. The lowest limit of quantification (LLOQ) was determined as 0.001 vg/dg. Only values of vg / dg above the LLOQ were presented.

Von Frey test

Corneal sensitivity was evaluated using von Frey filaments (Bioseb, reference bio-VF- M) as previously described ⁵¹ . Filaments with define forces from 0.008 to 0.6 g were applied on the cornea of an immobilized mouse until an eye-blink reflex was observed. Mice were habituated every day for five days. Von Frey test was performed on each cornea for two consecutive days (contralateral side and injected side). The values obtained from these two days were averaged for each cornea. As the values were represented in g, we displayed them as 1/g to reflect the sensitivity. Results are expressed as the mean ± SD. Behavioural experiments and analysis were performed by the same experimenter in single-blinded conditions throughout the study.

Statistical analysis

Data were analyzed with GraphPad Prism software (version 9.1.2, Prism, CA, USA) and expressed as the mean ± SD as indicated in the Figure legends. Statistical differences between mean values were tested using Brown-Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test, repeated measures two-way ANOVA test followed by Sidak’s or Tukey’s multiple comparisons test, repeated measure one-way ANOVA test followed by Dunnett’s comparisons test, Friedman’s one-way ANOVA test followed by Dunn’s multiple comparisons test or simple linear regression as indicated in the Figure legends. Differences between values were considered significant with: *p < 0.05, ** p < 0.01, *** p < 0 001, **** p < 0.0001.

Results

AAV2/9 andAAV2/5 mediate an efficient gene transfer in the lacrimal gland

To investigate the use of the LG as bioreactor for protein secretion in the tear film, we first established an injection protocol that allows an efficient AAV-mediated gene transfer after a single injection into murine LG. We used AAV2/9-CAG-GFP and AAV2/5-CAG-GFP to monitor the extent of vector diffusion within LG. The immunostaining of GFP demonstrated that these AAV serotypes induced GFP expression in all territories of the LG (Figure 1). To rule-out a possible tropism of AAV serotypes for a specific-cell type, we looked at GFP expression, one month after injection, in two different LG epithelial cell populations, namely the E-Cadherin (E-Cadh) positive cells, located in the acinar compartment, and the Keratinl9 (Krtl9) positive subpopulation, specifically localized in the ducts ²⁵ . The GFP / E-Cadh colabelling showed that almost all acinar cells were GFP positive regardless of the used serotype (data not shown). Similarly, the GFP / Krtl9 co-labelling demonstrated that most of the ductal cells were GFP positive after AAV2/9-CAG-GFP and AAV2/5-CAG-GFP injection (data not shown).

To evaluate a possible discrepancy between AAV serotypes 2/5 and 2/9 in the transduction efficiency, we analyzed by Western-blot the levels of GFP protein expressed in LG after injection of AAV2/9- or AAV2/5-CAG-GFP (Figure 1). As expected, while in the LG injected with AAV2/9 or AAV2/5 empty capsids, no GFP protein was detected (data not shown), whereas high levels of GFP were detected in the LG injected with AAV2/9 or AAV2/5-CAG- GFP respectively. Interestingly, when comparing the two AAV serotypes, GFP levels were found significantly higher after AAV2/9-CAG-GFP injection than after AAV2/5-CAG-GFP injection (Figure 1).

Taken together, our results demonstrate that LG can be widely transduced by AAV2/9 and AAV2/5, and that serotype 2/9 exhibits a higher transduction efficiency.

The transgene expression leads to the secretion of the resulting protein in the tear film

To evaluate if AAV2/9 and AAV2/5 serotypes have a different impact on the amount of protein secreted in the tear film, we chose to express the murine nerve growth factor (mNGF) in the LG, using either AAV2/9 (AAV2/9-CAG-mNGF) or AAV2/5 (AAV2/5-CAG-mNGF). Following AAV injection into LG, the transgene expression should lead to the secretion of mNGF in the tear film. To assess this secretion, we measured mNGF level in the tear film, using ELISA and Western-blot analysis (Figure 2). Tear film contains a basal level of mNGF ²⁶ , which can have two forms ^27,28 . The pro-mNGF, of higher molecular weight is cleaved to generate the mature mNGF, the active form. As expected from the GFP results, injection of AAV2/9 and AAV2/5 CAG-mNGF induced a significant increase of the mNGF levels in the tear film, whereas only endogeneous basal expression levels of mNGF were detected with the empty AAV vectors. Moreover, consistant with the GFP expression data in LG, mNGF amount in the tear film, was more than 3 -fold higher after AAV2/9 injection than after AAV2/5 injection (Figure 2). Moreover, Western blot analysis confirmed that injection of AAV2/9 and AAV2/5 CAG-mNGF led to a significant increase of the total mNGF level in the tear film (Figure 2). Importantly, this analysis revealed that mNGF was consistently found in the tear film in its pro- mNGF form (data not shown). However, the analysis of the mature mNGF showed a maturation process (data not shown). Indeed, the mature mNGF form was close to absent from the tear film of mice injected in LG with empty AAV2/9 or AAV2/5, whereas a large amount of mature mNGF was detected after AAV-mNGF gene transfer. Notably, AAV2/9-CAG-mNGF gave rise to 4-times more of the mature form than with AAV2/5-CAG-mNGF injection (data not shown).

AAV2/9-mediated gene transfer induces a dose-dependent and long-term protein secretion

Consequently to our results indicating a better protein secretion using AAV2/9 compared to AAV2/5, we chose to focus on the serotype 2/9. To establish the optimal amount of vector genomes (vg) to be injected in LG, we tested three doses and checked the amount of secreted mNGF one month after injection (Figure 3 A). While injection of 10 ⁹ vg did not lead to a drastic increase of the mNGF secretion, a single injection of 10 ¹¹ vg induced the secretion of 75 ng of mNGF per mL of tear film. This high concentration represents a 10-time increase in comparison to physiological mNGF secretion during cornea wound healing process, where a concentration of 7.5 ng / mL was found seven days after abrasion (data not shown). We concluded that 10 ¹¹ vg / LG was suitable to have a significantly high increase of the protein secretion in the tear film.

Subsequently, we evaluated the dynamics of the mNGF secretion over a 6-month period (Figure 3B). The injection of 10 ¹¹ vg into the LG induced a significant increase of the mNGF level found in the tear film already after a week, before reaching a peak at 30 days post injection. The mNGF secretion then decreased to reach a plateau, 120 days after injection with stable expression during the 60 following days. However, although injection of 10 ¹⁰ vg/LG of AAV2/9-CAG-mNGF or AAV2/5-CAG-mNG induced a peak 30 days after injection, only the serotype 2/9 induced a significant increase of the mNGF level in the tear film (data not shown). Nonetheless, the secreted mNGF level for both serotypes decreased and were no more different than the basal level. Taken together, these results show the long-lasting protein secretion when injecting 10 ¹¹ vg/LG, compared to 10 ⁹ or IO ¹⁰ vg, and confirm that AAV2/9 is more efficient than AAV2/5.

A A V2/9 vector genome copies in the murine LG correlates with the amount of protein secreted in the tear film

To further analyze further the use of 10 ¹¹ vg / LG, we investigated the biodistribution of the AAV2/9-CAG-mNGF vector after injection into LG. We measured the amount of vector genomes (vg) per diploid genome (dg) in LG, liver and heart, one month after vector injection (Figure 4 A). All injected mice showed vector genome copies in LG, indicating that the injection technique is reliable and reproducible. Interestingly, we found around 0.25 vg / dg in the LG, meaning that an average of 1 out of 4 cells was transduced with 1 copy of the AAV2/9-CAG- mNGF vector, which reflects an efficient gene transfer. Despite the detection of the AAV2/9- CAG-mNGF vector genome in liver and heart of injected mice, the levels were very low to negligeable, with 14 and 80-times less vg / dg in liver and heart respectively than in LG.

Then, we compared the vg / dg values to the concentration of mNGF in the tear film of the same injected mice (Figure 4B). Remarkably, we found a nice correlation between the vg / dg values detected in the LG and the amounts of secreted mNGF in the tears. The increase of vg / dg value corresponded to a higher mNGF secretion.

Taken together, these results confirmed the high transduction efficiency displayed by AAV2/9, which is additionally correlated with the amount of protein secreted in the tear film. Moreover, they showed a very limited distribution of AAV2/9 vector to peripheral organs after a single injection into murine LG.

AAV2/9-mediated mNGF secretion does not affect LG physiology nor corneal integrity

After resolving the parameters for an efficient AAV2/9-mediated mNGF gene transfer, its biodistribution, and induced mNGF secretion, we investigated the impact of the mNGF over secretion on LG physiology and corneal integrity. We injected 10 ¹¹ vg/LG of AAV2/9-CAG- mNGF and measured the impact on tear volume and protein concentration in tears over a period of 6 months. (Figure 5A-B). Simultaneously to the increase of mNGF secretion we detected a significant increase of the tear volume from 7 to 120 days post-injection, when compared to the injection of AAV2/9 empty capsid (Figure 5A). Interestingly, this increase of the tear volume was matched with a stability of the protein concentration in tears (Figure 5B). Moreover, to rule out any impact of the injection procedure on LG physiology, we analyzed the tears before and after the AAV2/9 empty capsid injection (data not shown). Importantly, after injection of the AAV2/9 empty capsid and during the whole 6-month-period, no modification of tear volume nor of total protein concentration was observed (data not shown). This rule out any impact of the injection procedure itself on LG physiology.

In addition, we used fluorescein staining to monitor the corneal epithelium integrity and visualize any adverse effect that the injection procedure and the tear film modification could have on the cornea. Fluorescein stains areas where the corneal epithelial barrier is defected ¹ , as after corneal abrasion (data not shown). During the 180 days after AAV2/9 injection, we never observed any fluorescein staining, whether we injected an empty capsid, or induced mNGF over secretion. We concluded that AAV2/9 injection does not impact the corneal epithelium integrity.

To investigate further the effect of the mNGF over secretion on cornea, we visualized corneal innervation with pill tubulin immunolabelling, which is a pan neuronal marker. We showed that the gross morphology of corneal fibers was not affected by the over secretion of mNGF in the tear film (data not shown). Furthermore, we performed von Frey tests on the corneas of injected mice. We demonstrated that the constant over secretion of mNGF did not modify corneal sensitivity, i.e does not have deleterious effect (Figure 6). All together, these results demonstrate that the injection procedure and mNGF over secretion respect the LG physiology and the corneal integrity.

Discussion

Among the sight threatening diseases, corneal defects are the fourth cause for blindness globally. Except for physical harm, corneal blindness often results from a combination of intrinsic and extrinsic causes. In the case of genetic diseases, such as aniridia ^29,30 , or systemic diseases, such as diabetes ¹³ , corneal defect is often associated with corneal microenvironment dysregulation ³¹ .

The aim of this study was to establish an innovative and attractive strategy to tackle corneal defects by sustainably modulating the tear film composition. For this purpose, we chose an AAV-mediated gene transfer to use LG as a bioreactor producing specific transgenes. Nevertheless, several major variables are crucial to achieve an efficient transduction of the target cells, and thus require to be well-designed ^17,32 . First, the route of administration determines both the efficacy and the biosafety pattern of an AAV-based gene transfer. In this study, we used a local injection into LG as this organ is easily accessible by surgery. Moreover, we showed that a local injection allows concentration and time residence of AAV vectors in vicinity to the target cells, limits the biodistribution of the vector to non-targeted tissues and thus the risk of toxicity. For instance, local injection is well-established for ocular diseases, particularly in retinal disorders, as several anatomical sites are commonly targeted: subretinal, intravitreal, intracameral, suprachoroidal and topical ^33,34 . Furthermore, the injection method is a key parameter to reach a large diffusion in the target organ. While a systemic injection leads to large diffusion in all the organism including the target organ ³⁵ , when using local injection, the development of specific injection procedures are necessary. For example, several injections at precise coordinates are required for a large diffusion in the brain ³⁶ . Interestingly, a pneumatic picopump system applying multiple short-time pressure pulses has been reported to transduce the whole sciatic nerve of rodents ³⁷ . In this study, we used 34-gauge beveled needle linked to a 10-pL Hamilton syringe to perform injections into LG. We injected 3 pL of AAV vectors by following the path of the LG main duct. This procedure of injection led to a large diffusion in the gland, and showed an efficient gene transfer in all epithelial cell types in the murine LG. The promoter used to drive the expression of the transgene is a second parameter determining the transduction efficiency and the biosafety pattern of the injection ^17,32 . Indeed, the promoter controls the transgene expression level. Commonly, AAV-mediated gene transfer uses an ubiquitous and strong promoter, such as CAG and CMV, to achieve high transgene expression ^38,39 . Moreover, researchers usually employ such promoters when trying to establish a proof of concept for AAV-mediated gene transfer into a specific organ. In this study, we utilized a CAG promoter to provide a proof of concept of the tear fluid composition modulation after an AAV-mediated gene transfer into murine LG. On the other hand, a high transgene expression level is not always desired. For instance, transgene over expression above physiological levels has been reported to be toxic ⁴⁰ . This toxicity must be correlated to the serotype of the AAV vector. Indeed, the serotype represents another parameter influencing both the transduction efficiency and the biosafety of an injection as each AAV serotypes exhibit different cell and tissue tropisms ⁴¹ . Therefore, the combination of the route of administration, the AAV serotype, the promoter driving the transgene expression and the dose of vector have to be carefully designed as it may induce transgene expression in off-target tissues and thus lead to dramatic toxicities ⁴² . In this study, we compared the AAV serotypes 2/5 and 2/9 to first transduce the murine LG efficiently and then to modulate the tear film composition. Notably, while both serotypes led to an efficient gene transfer in the LG, AAV2/9 gave a better yield of GFP production and mNGF secretion compared to AAV2/5. Interestingly, although the difference of transduction efficiency was reported previously ²⁴ , the 2 to 4 time fold in mNGF secretion is startling. Indeed, until now, no study has reported a transgene secretion in the tear fluid after an AAV-mediated gene transfer into LG. Furthermore, we tested increasing dose of AAV2/9 to evaluate the mNGF secretion in the tear film. Interestingly, while 10 ¹¹ vg / LG dose led to the highest mNGF secretion, IO ¹⁰ vg / LG was sufficient to induce a significant secretion. Given that we used the serotype 2/9, known to show high heart and liver tropism in rodent ⁴³ , and a strong CAG promoter, we performed a biodistribution study for AAV2/9. This study was paramount to determine the potential off-targets of our strategy, which may lead to both unwanted toxicity and immunogenicity. At the highest injected dose of 10 ¹¹ vg / LG, we showed an average of 0.25 vg / dg in LG, only 0.017 vg / dg in liver and 0.003 vg /dg in heart of injected mice, which corresponded to 14- and 80-times less vg / dg than in LG respectively. Importantly, these levels of AAV2/9 found in liver and heart were very low to negligible, from 100 to 10 000 times lower than those obtained after intravenous ³⁹ or intrathecal ⁴⁴ injections. Different strategies exist to limit the transgene expression in off-targets and its related adverse effects, including the use of cell-type specific promoters ³⁹ or the incorporation of miRNA binding sites in the AAV gene expression cassette ⁴⁵ . We can speculate that the use of a 10-time lower dose of AAV2/9 vector, providing a high secretion of mNGF, could bring a lower off- target transduction. Injection of 10 ¹⁰ vg / LG of AAV led to a faster secretion of mNGF with AAV2/9 compared to AAV2/5. After a peak 30-day post-injection, the mNGF secretion decreased to the physiological level with both serotypes. However, injection of 10 ¹¹ vg / LG results in over secretion of mNGF during several months. This is an important aspect for the subsequent use of this method. While a lower dose could be used for transitory pathologies, such as corneal graft, or recurrent corneal abscesses, a higher dose could be of interest for chronical pathologies, such as neurotrophic keratitis, or dry eye diseases.

The long-term secretion of mNGF is a remarkable discovery, which must be linked to the LG physiology. As all epithelial organs, such as skin, cornea or mammary glands, the epithelial compartment contains stem cells regenerating continuously the organ, and healing it if necessary ⁴⁶ . The AAV vector is a non-integrative vector that can be lost when cell division occurs ⁴⁷ . Therefore, only non-proliferating cells will keep the transgene and lead to protein secretion. Our results demonstrate that gene therapy is successful on this epithelial organ and highlighting the low turn-over of epithelial cells in the LG. Knowing that 30 days after injection, there was around one AAV2/9 vector genome in every 4 cells, it would be of great interest to monitor over a long period of time to see which cells retain the AAV2/9 genome. This method would give a deeper understanding of LG fundamental biology. Of course, this long term follow-up could only be done at the cellular level, as with age, the LG physiology is randomly affected in individuals ⁸ . Therefore, measuring tear volume and tear protein content could be misleading because of aging impact more than AAV2/9 injection. Nevertheless, we demonstrated that up to 6 months after injection into the murine LG, our approach has no detrimental impact on the LG physiology.

Despite the large amount of mNGF and its constant presence on the cornea, we did not detect any impact on corneal innervation, nor on corneal sensitivity. We hypothesize that the robustness of corneal innervation maintains the system under control in physiological conditions, and mNGF alone is not sufficient to disturb this system. Most likely, under pathological conditions, i.e., physical harm, or neurotrophic keratopathy, the system would be sufficiently perturbed to visualize the effect of mNGF on corneal innervation.

Collectively, our results show that LG gene therapy could be established to modify specifically the tear film to support corneal physiology. The main challenge in using AAV vectors for epithelial organ gene therapy is the high renewal rate of epithelial cells. Here, we demonstrate that there is a non-renewing cell population in the LG that retains the secretory capabilities to produce a large amount of mNGF for over 6 months. The long term over secretion of mNGF could replace the use of NGF supplemented eyedrops, used to treat neurotrophic keratitis, such as observed in diabetes ¹² or neurodegenerative diseases ⁴⁸ . Notably, by substituting mNGF with another gene, other corneal defects could be treated. Indeed numerous growth factor are suitable for promote healing of corneal epithelial defect (and to promote reinnervation of corneal epithelial after abrasion). Indeed local treatment of cenergermin, a recombinant human nerve growth factor, has shown higher rates of corneal healing in neurotrophic keratopathy ⁵⁷ . Topical VEGF-A application further promoted corneal nerve regeneration without inducing pathological neovascularization ⁵⁸ . PEDF has also been demonstrated to have an essential role in corneal structure and function ⁵⁹ .

EXAMPLE!: Injection mRNA into murine lacrimal gland

Tears were collected 24 and 48hrs after injection of NGF mRNA in murine lacrimal gland. The tear fluid was then subjected to ELISA immunodosage. Physiologically a murine tear film contains about 0.5ng/ml of NGF, the injection of 0.85pg of mRNA coding for NGF leads to a transitory peak of NGF secretion in the tear, with around 9ng/ml 24hrs after injection, and 4ng/ml at 48hrs post-injection.

The Injection of mRNA in the murine lacrimal gland can lead to a transitory expression of the protein coded by the injected mRNA.

The transitory secretion of the chosen protein make this approach perfect to treat transitory defects of corneal surface. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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