DNA constructs for the treatment of ocular conditions

Abstract

The present invention relates primarily to a DNA construct for use in the treatment of ocular conditions and for the non-viral transfer of nucleic acids into ocular muscle cells of a patient suffering from an ocular condition; it is particularly characterized in that it comprises a first sequence encoding a first therapeutic protein and a second sequence encoding a second therapeutic protein different from the first therapeutic protein, the DNA construct being administered to the patient by injection into the ciliary muscle followed by electrotransfer into the ciliary muscle cells.

Description

[Technical Field]

[0001] The present invention relates to a novel DNA construct and its application in the treatment of ocular conditions by non-viral gene therapy. The method of the present invention more particularly relates to the DNA construct, which allows for the targeted intraocular production of two therapeutic proteins for a period that can last up to several months. The DNA construct of the present invention and its use are more particularly suitable for the treatment of retinal conditions using injection of the DNA construct into the ciliary muscle followed by electrotransfer for long-lasting intraocular production of the therapeutic protein of interest.

[0002] prior art Blindness caused by metabolic and inflammatory diseases or aging is increasing significantly and poses an increasingly serious social problem from a public health perspective in Europe and around the world. The main causes of blindness are related to retinal pathologies, including age-related macular degeneration (ARMD), diabetic retinopathy (DR), uveitis, glaucoma, retinitis pigmentosa, bleeding after ocular injury, and retinal detachment. Age-related macular degeneration (ARMD), which leads to blindness, has a prevalence of 8.7% and affects approximately 26% of the population aged 50 and over. Due to the aging of the population, age-related macular degeneration is becoming a major public health and social welfare problem. The sustained and significant increase in the incidence of diabetes, the primary cause of diabetic retinopathy (DR), is, in turn, becoming a priority for public health and scientific research. Statistics indicate that type 2 diabetes will affect more than 4.5% of the population between now and 2030, and nearly 30% of these will suffer from diabetic retinopathy. Ultimately, uveitis represents a group of inflammatory eye diseases, with an estimated prevalence of 1 / 1000 and incidence of 0.5 / 1000. Uveitis accounts for 10% of cases of blindness and, therefore, is rarer than the diseases mentioned above, but has a significant social and economic impact in young, working-age patients. Glaucoma is the second leading cause of irreversible blindness worldwide. The number of people with glaucoma worldwide is predicted to increase from 76 million in 2020 to 111 million in 2040. Glaucoma is characterized by abnormal intraocular pressure, which induces progressive optic neuropathy characterized by retinal ganglion cell degeneration and visual field loss. Intraocular pressure is currently the only risk factor, for which treatments exist. However, glaucomatous damage persists in approximately 50% of patients despite reduction of intraocular pressure. Retinitis pigmentosa represents a clinically and genetically heterogeneous group of inherited disorders of the retina characterized by the progressive loss of photoreceptors in the periphery of the retina and then progressing to the macula. Visual impairment is typically manifested by night blindness and progressive visual field loss. Its prevalence is 1 / 3000 to 1 / 5000. More than 50 genes responsible for retinitis pigmentosa have been identified to date.

[0003] Intraocular or even intravitreal injection of therapeutic agents has been developed to treat these specific pathologies. In 2006, the first anti-VEGF (VEGF: vascular endothelial growth factor) therapeutic protein was administered intraocularly for the treatment of choroidal neovascularization in age-related macular degeneration. The inventors may particularly mention Lucentis® (ranibizumab) as an example of an anti-VEGF protein, which has been approved for the treatment of choroidal neovascularization in age-related macular degeneration and diabetic macular edema. Intraocular injection of therapeutic proteins, particularly recombinant proteins, has since become common for the treatment of age-related macular degeneration, diabetic retinopathy, and macular edema in vein occlusion.

[0004] To ensure sustained efficacy against ocular conditions, these anti-VEGF antibodies are administered monthly, or at most every two months, depending on the patient. Therefore, monitoring of each patient is necessary to determine the frequency of anti-VEGF antibody administration to ensure full effectiveness of the treatment. This monitoring creates considerable stress for patients, caregivers, and nursing staff, often resulting in suboptimal and ineffective treatment over the long term (Ciulla 2020, Ophthalmology Retina 2020;4:19-30). Furthermore, this treatment method induces changes in the level of the therapeutic protein in the patient's eye, i.e., a high concentration (peak) at the time of injection and a gradually decreasing concentration that tends to zero by the next injection. Therefore, the concentration of the therapeutic protein is inconsistent and not optimal throughout the entire treatment period. Furthermore, the risk of side effects associated with intravitreal administration increases with each repeated administration (Schargus 2020, Clinical Ophthalmology 2020: 14 897-904).

[0005] Among ocular conditions, uveitis is defined as an inflammatory process affecting the iris, ciliary body, or choroid of a patient's eye; these three components form the uvea. It is a general term encompassing several different conditions, the causes of which remain unknown, but which generally fall into two categories: infectious and noninfectious uveitis. A distinction is made between anterior uveitis, which affects the iris or ciliary body and is the most common type of uveitis in Western countries; intermediate uveitis, which affects the anterior vitreous; and posterior uveitis, which affects the choroid and retina. Noninfectious uveitis often has an autoimmune component. Acute anterior uveitis associated with the HLA-B27 antigen represents the major cause of uveitis (Rothova et al., Br J Ophthalmol. 1992; 76:137-41). Additionally, anterior uveitis is seen with many rheumatic diseases, such as sarcoidosis. Non-infectious posterior uveitis is often associated with Behçet's disease, Vogt-Koyanagi-Harada disease, and shotcretic chorioretinopathy.

[0006] Oral and / or topical treatment with corticosteroids is widely used to treat non-infectious posterior uveitis. Furthermore, in the most refractory cases, immunosuppressants may be added to the treatment to enhance the anti-inflammatory effects of corticosteroids. This applies particularly, but not exclusively, to cyclosporine, methotrexate, azathioprine, mycophenolate mofetil, tacrolimus, and chlorambucil. For the past decade or so, treatment with anti-TNFα antibodies has also been available, including Humira® (adalimumab), which was recently approved for the treatment of non-infectious uveitis with inflammation of the ocular fundus.

[0007] Clinical trials have been conducted on the use of the following therapeutic compounds (cyclosporin A, rapamycin, tacrolimus, and anti-TNFα antibodies) to evaluate the efficacy and safety of these compounds, thereby treating autoimmune ocular conditions such as Behçet's disease. However, systemic administration of these therapeutic compounds has many long-term side effects, and for some of them, their topical administration has been shown to provide less efficacy or poor patient tolerance.

[0008] Therefore, the above treatment methods have several drawbacks. Therapeutic compounds administered by systemic and local routes cause numerous side effects. Recombinant proteins administered by intravitreal injection must be administered frequently, with large fluctuations in concentration between each administration. These repeated injections are still very tedious and stressful for patients and may cause side effects (e.g., increased intraocular pressure, intraocular inflammation, endophthalmitis, cataracts, etc.). Therefore, many patients eventually discontinue their treatment.

[0009] To overcome this problem, the inventors have previously proposed, as shown in particular in application FR3031112, to reduce the number and / or frequency of surgical interventions and therefore the invasive aspect of intraocular injections while ensuring a stable and constant production of therapeutic proteins for several months, through the application of a DNA construct aimed at the non-viral transfer of nucleic acids into the muscle cells of the patient's eye. This construct comprises an origin of replication, a promoter allowing the expression of the DNA in the patient's eye, one or more sequences promoting the expression of the DNA in the patient's eye, and a polynucleotide encoding a therapeutic protein selected for its activity in the treatment of an ocular condition, and the construct is delivered to the eye by direct injection into the ciliary muscle followed by electrotransfer.

[0010] However, treatment of the above ocular conditions may require the application of two active therapeutic ingredients, a second compound to enhance the effectiveness of the active therapeutic ingredient, or a compound consisting of two peptide subunits.

[0011] In the context of the above method, it may be envisioned to administer to a patient a composition containing two types of DNA constructs (the first type allows the expression of a first molecule of interest, while the second type allows the expression of a second molecule of interest). However, such a method would not be ideal in that (i) it would require the development of two products, which would increase development and production costs and require the evaluation of the activity and safety of each product taken separately and in combination; (ii) it would impose significant constraints in terms of dosage, halving the maximum dose of each of the two active therapeutic components that can be administered; and finally (iii) it would not be possible to completely control the amount of each of these constructs that penetrates into the targeted cells, resulting in uncertainty regarding the ratio of these molecules of interest expressed at the ocular level. Such constraints and uncertainty are undesirable in the context of treating a pathological condition.

[0012] The inventors consequently propose to employ in the context of the present invention a DNA construct comprising sequences encoding two proteins of interest.

[0013] Summary of the Invention The present invention therefore relates firstly to a DNA construct for its use in the treatment of an ocular condition, said DNA construct being intended for the non-viral transfer of nucleic acid into ocular muscle cells of a patient having said ocular condition; The DNA construct is (a) a bacterial or prokaryotic origin of replication, in particular a bacterial origin of replication; (b) one or more sequences that promote expression of DNA in the patient's eye; (c) a first therapeutic protein, and - a signal peptide that allows the secretion of this first therapeutic protein a first nucleotide sequence encoding (wherein the signal peptide is contiguous with the sequence of the first therapeutic protein at the N-terminus of the first therapeutic protein), (d) a promoter that allows expression of the first therapeutic protein in the patient's eye; (e) a polyadenylation sequence 3' of the first nucleotide sequence; (f) a second therapeutic protein, different from the first therapeutic protein, and - a signal peptide that allows secretion of this second therapeutic protein a second nucleotide sequence encoding (the signal peptide is contiguous with the sequence of the second therapeutic protein at the N-terminus of the second therapeutic protein); (g) a promoter that allows expression of the second therapeutic protein in the patient's eye; and (h) a polyadenylation sequence 3' of the second nucleotide sequence; characterized in that it comprises; The DNA construct is administered to the patient by injection into the ciliary muscle followed by electrical transduction into the ciliary muscle cells.

[0014] In fact, contrary to all expectations, the significant increase in size of the DNA construct, resulting from the introduction of not one but two sequences encoding the molecule of interest, does not have a negative effect on its ability to penetrate targeted cells in the context of the method as shown above, which includes not only direct injection of the construct into the ciliary muscle but also a step of electrical introduction.

[0015] As a result, the methods and constructs of the present invention, contrary to all expectations, - as well as increasing the possibilities for treating ocular conditions by allowing the in situ production of two active ingredients or one active ingredient and a compound suitable for enhancing the activity / effectiveness of said active ingredient; Advantageously, it makes it possible to maintain, without diminishing, the advantages of the methods set out above in terms of reducing the number and / or frequency of surgical interventions and, consequently, the invasive aspects of intraocular injections, while ensuring a stable and sustained production of therapeutic proteins over several months.

[0016] According to one embodiment, the first therapeutic protein of the DNA construct of the invention is an anti-VEGF type protein, in particular an anti-VEGF type protein selected from the group consisting of S-Flt1, aflibercept, conbercept, brolucizumab, in particular a protein having at least 85% sequence identity to the peptide sequence of SEQ ID NO: 3, and more particularly aflibercept.

[0017] According to one embodiment, the first therapeutic protein is encoded by a nucleotide sequence having at least 75% sequence identity to the sequence of SEQ ID NO:1, more particularly the nucleotide sequence of SEQ ID NO:2.

[0018] According to one embodiment, the second therapeutic protein of the DNA construct of the invention is a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 8, which protein is more particularly decorin.

[0019] According to one embodiment, the second therapeutic protein is encoded by a nucleotide sequence having at least 70% sequence identity to the sequence of SEQ ID NO:6, in particular consisting of the nucleotide sequences of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, more particularly a sequence selected from the group consisting of the sequence of SEQ ID NO:7 and the sequence of SEQ ID NO:1, in particular the sequence of SEQ ID NO:11.

[0020] According to one embodiment, the DNA construct for use in the present invention comprises: (c) the first nucleotide sequence is - a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 3 (this protein is more particularly aflibercept); and - a signal peptide of the peptide sequence SEQ ID NO: 4 code; and (f) the second nucleotide sequence: a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 8 (this protein is more particularly decorin); and - a signal peptide of the peptide sequence SEQ ID NO: 13 It's like coding:

[0021] According to one embodiment, the origin of replication of such a DNA construct is a bacterial origin of replication, in particular an origin of replication derived from the native Escherichia coli plasmid R6K, in particular the R6K gamma origin of replication of the native Escherichia coli plasmid R6k, in particular the sequence of SEQ ID NO: 31.

[0022] The DNA construct of the present invention may be in linear or circular form, in particular in circular form. In a particular embodiment, the DNA construct of the present invention is a circular plasmid.

[0023] In certain embodiments, the DNA construct is a naked DNA construct.

[0024] According to one embodiment, the DNA construct for such use as described above is characterized in that the ocular pathology is a retinal degeneration selected from the group consisting of retinal degeneration, in particular exudative or atrophic age-related macular degeneration (ARMD); diabetic retinopathy (DR); retinal vein occlusion, in particular central retinal vein occlusion (CRVO) or branch retinal vein occlusion (BRVO); myopic choroidal neovascularization (CNV); uveitis, in particular non-infectious uveitis; retinitis pigmentosa and glaucoma, more particularly the retinal degeneration is characterized in that it is selected from the group consisting of age-related macular degeneration (ARMD), in particular the (exudative) neovascular type of ARMD; loss of vision due to diabetic macular edema (DME); retinal vein occlusion, in particular central retinal vein occlusion (CRVO) or branch retinal vein occlusion (BRVO); and myopic choroidal neovascularization (CNV).

[0025] The present invention further relates to a DNA construct for the non-viral transfer of nucleic acids into ocular muscle cells of a patient for the treatment of ocular conditions, which (a) a bacterial or prokaryotic origin of replication, in particular a bacterial origin of replication; (b) one or more sequences that promote expression of DNA in the patient's eye; (c) - a first therapeutic protein, said first therapeutic protein being a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 3, and more particularly aflibercept; and a signal peptide allowing the secretion of this first therapeutic protein, in particular a signal peptide of the peptide sequence SEQ ID NO: 4 (This signal peptide is contiguous with the sequence of the first therapeutic protein at the N-terminus of the first therapeutic protein.) a first nucleotide sequence encoding the (d) a promoter that allows expression of the first therapeutic protein in the patient's eye; (e) a polyadenylation sequence 3' of the first nucleotide sequence; (f) - a second therapeutic protein, said second therapeutic protein being a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 8, and more particularly, said protein being decorin, and a signal peptide allowing the secretion of the first therapeutic protein, in particular the signal peptide of the peptide sequence SEQ ID NO: 13 (This signal peptide is contiguous with the second therapeutic protein sequence at the N-terminus of the second therapeutic protein.) a second nucleotide sequence encoding (g) a promoter that enables expression of the second therapeutic protein in the patient's eye; and (h) a polyadenylation sequence 3' of the second nucleotide sequence; The present invention is characterized by comprising:

[0026] In particular, the DNA construct of the present invention is c) the first nucleotide sequence is a nucleotide sequence encoding aflibercept, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2; and - the nucleotide sequence of SEQ ID NO: 5 encoding the signal peptide and (f) the second nucleotide sequence: a nucleotide sequence encoding decorin, more particularly a nucleotide sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 6, more particularly a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, in particular the sequence of SEQ ID NO: 7 or the sequence of SEQ ID NO: 11; and - the nucleotide sequence of SEQ ID NO: 14 encoding the signal peptide It is something that includes. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows a DNA construct (plasmid A) of the present invention. [Figure 2] FIG. 2 shows a DNA construct that is not according to the invention, since it only encodes a single therapeutic protein (transferrin - the same sequence as used in Plasmid A), the latter being under the control of a CMV-type promoter, just like Plasmid A (Plasmid a'). [Figure 3] 3 shows the variation of the transferrin concentration (vertical axis: pg / mL) of the sequence of SEQ ID NO: 17 in the ocular fluid of rats from 3 to 30 days (horizontal axis: days after electrotransfer (day 0)) after administration into the ciliary muscle of both eyes of rats of a construct according to the invention (plasmid A) or a construct not according to the invention (plasmid a') encoding only transferrin (the latter under the control of the same promoter as plasmid A). Thus, each group of rats received a specific construct from the two described. [Figure 4]Figure 4 shows the changes in the concentration (vertical axis: pg / mL) of the anti-TNFα fusion protein of sequence number 22 in the ocular fluid of rats over 3 to 30 days (horizontal axis: number of days after electrotransfer (day 0)) after administration of the construct of the present invention (plasmid A) to the ciliary muscles of both eyes of the rats. [Figure 5] FIG. 5 shows a DNA construct (plasmid B) of the present invention. [Figure 6] FIG. 6 shows a DNA construct that is not according to the invention, since it only encodes a single therapeutic protein (aflibercept - the same sequence as used in Plasmid B, i.e., sequence SEQ ID NO: 2), the latter being under the control of a CAG-type promoter, just like Plasmid B (Plasmid b'). [Figure 7] 7 shows the variation of aflibercept concentrations (vertical axis: pg / mL) of the sequence of SEQ ID NO: 3 in the ocular fluid of rats from 3 to 21 days (horizontal axis: days after electrotransfer (day 0)) after administration into the ciliary muscle of both eyes of the rats of a construct of the present invention (plasmid B) or a construct not according to the present invention (plasmid b') encoding only aflibercept (the latter under the control of the same promoter as plasmid B). Thus, each group of rats received a specific construct from the two described. [Figure 8] Figure 8 shows the fluctuations in the concentration of decorin (vertical axis: pg / mL) of sequence number 8 in the ocular fluid of rats from 3 to 21 days after administration of the construct of the present invention (plasmid B) into the ciliary muscles of both eyes of the rats (horizontal axis: number of days after electrical induction (day 0)). [Figure 9] FIG. 9 shows a DNA construct (plasmid C) of the present invention. [Figure 10] Figure 10 shows the proportion of effects causing severe leakage (grade 3) (vertical axis: % of grade 3 choroidal neovascular lesions) as a function of treatment (horizontal axis). Left: Vehicle (control). Right: Plasmid C.

[0028] Detailed Description definition In the context of the present document, the terms "treat" and "treatment" in relation to an ocular condition refer to the reduction, or even cessation, of said condition.

[0029] The term "patient" as used in the present document preferably refers to a mammal (including non-human mammals), more particularly a human.

[0030] The terms "first nucleotide sequence" and "second nucleotide sequence" are used in the present document to allow a clear distinction between these two sequences and the proteins they encode while reading the latter.

[0031] The above nucleotide sequences represent expression cassettes, each of which is as defined hereinafter.

[0032] However, the terms "first nucleotide sequence" and "second nucleotide sequence" are not intended to indicate the order in which these sequences / expression cassettes are present in the construct of the present invention. Thus, according to one embodiment, the first nucleotide sequence may precede the second nucleotide sequence in terms of reading the construct of the present invention. In another embodiment, the second nucleotide sequence may precede the first nucleotide sequence in terms of reading the construct of the present invention.

[0033] As set forth above, a "first nucleotide sequence" comprises a sequence encoding a first therapeutic protein and a sequence encoding a signal peptide, which sequences are present in the "first nucleotide sequence" in the order specifically set forth relative to each other, i.e., the sequence encoding the signal peptide is at the N-terminus of the first therapeutic protein, i.e., the sequence encoding the signal peptide is 5' to the sequence encoding the first therapeutic protein.

[0034] Further, as set forth above, the "second nucleotide sequence" comprises a sequence encoding a second therapeutic protein and a sequence encoding a signal peptide, which sequences are present in the "second nucleotide sequence" in the order specifically set forth relative to each other, i.e., the sequence encoding the signal peptide is at the N-terminus of the second therapeutic protein, i.e., the sequence encoding the signal peptide is 5' to the sequence encoding the second therapeutic protein.

[0035] The "percent identity" between two amino acid sequences or two nucleic acid sequences, in the sense of the present invention, is determined by comparing two optimally aligned sequences over a comparison window.

[0036] Thus, some of the nucleotide sequences in the comparison window may contain additions or deletions (e.g., "gaps") compared to the reference sequence (which does not contain these additions or these deletions) to obtain optimal alignment between the two sequences.

[0037] The percent identity is calculated by determining the number of positions where identical amino acids (or identical nucleic acid bases) are observed for the two sequences being compared, then dividing the number of positions that are identical between the two amino acids (or between the two nucleic acid bases) by the total number of positions in the comparison window, and then multiplying the result to obtain a percentage, thereby obtaining the percentage amino acid identity (or nucleotide identity) of the two sequences between them.

[0038] Optimal alignment of sequences for comparison can be performed by computer using known algorithms.

[0039] Overall, percent sequence identity is preferably determined using CLUSTAL W software (version 1.82), with the following parameters set: (1) CPU MODE=ClustalWmp; (2) alignment="complete"; (3) output format="aln" w / numbers"; (4) output order = "aligned"; (5) color alignment = "no"; (6) KTUP (word size) = "default"; (7) window length = "default"; (8) score type = "percent"; (9) TOPDIAG = "default"; (10) pair gap = "default"; (11) dendrogram / tree type = "none"; (12) matrix = "default"; (13) gap open = "default"; (14) end gap = "default"; (15) gap extension = "default"; (16) gap distance = "default"; (17) tree type = "cladogram" and (18) tree gap distance = "hide".

[0040] In the sense of the present invention, an amino acid sequence having at least 80% amino acid identity to a reference amino acid sequence includes an amino acid sequence having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% amino acid sequence identity to the reference sequence.

[0041] In the sense of the present invention, a nucleotide sequence having, for example, at least 80% nucleotide identity to a reference nucleotide sequence includes a nucleotide sequence having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% nucleotide identity to the reference sequence.

[0042] DNA constructs As noted above, the present invention primarily relates to DNA constructs for use therein in the treatment of ocular conditions.

[0043] This construct is intended for the non-viral transfer of nucleic acid into ocular muscle cells of patients with ocular conditions.

[0044] Furthermore, the DNA construct of the present invention is characterized in that it (a) contains a bacterial or prokaryotic origin of replication.

[0045] According to a particular embodiment, said origin of replication is in particular a bacterial origin of replication, and may for example be an origin of replication of the E. coli type, more particularly selected from the group consisting of origins of replication derived from the native E. coli plasmid R6K, in particular the R6K gamma origin of replication of the native E. coli plasmid R6k; and the origin of replication of pUC OriC.

[0046] The origin of replication from the natural plasmid R6K of E. coli is notably defined in patent EP 1366176 B2.

[0047] Furthermore, the DNA construct of the present invention is characterized in that it (b) contains one or more sequences that enhance the expression of the DNA in the patient's eye. Such sequences that enhance the expression of the DNA are well known to those skilled in the art and are, for example, enhancer-type sequences, also called amplification sequences or activation sequences. We may, for example, mention enhancer sequences derived from cytomegalovirus (CMV) and / or tumor viruses carrying simian DNA SV40.

[0048] Furthermore, the DNA construct of the present invention is also characterized in that it comprises (c) a first nucleotide sequence encoding, in particular, a first therapeutic protein, and (f) a second nucleotide sequence encoding, in particular, a second therapeutic protein different from the first therapeutic protein.

[0049] According to a particular embodiment, the DNA construct of the present invention comprises only two coding sequences for a Therapeutic protein, i.e., only two expression cassettes, each of which comprises one of the two coding sequences for a Therapeutic protein.

[0050] These first and second therapeutic proteins may be selected from proteins that are known for their effects on ocular conditions in particular.

[0051] The effects of these two proteins may be additive or complementary, and one of these two proteins may have a potentiating effect on the therapeutic activity of the other protein produced starting from the DNA construct of the present invention.

[0052] In particular, the first and second therapeutic proteins, which are different from each other, can be, e.g., (i) a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 17 (this protein is more particularly transferrin); (ii) proteins with anti-fibrotic properties, such as BMP7 protein (bone morphogenetic protein 7), anti-TGF (tumor growth factor) beta-type proteins, anti-FGF (fibroblast growth factor) type 2 proteins, anti-CTGF-type proteins (connective tissue growth factors), in particular proteins having at least 85% sequence identity with the sequence of SEQ ID NO: 8 (this protein is more particularly decorin); (iii) proteins with anti-inflammatory properties, in particular anti-TNF type proteins, such as human TNFR (tumor necrosis factor receptor)-Is, human TNFR-Is / mouse IgG1, Lenercept, or fusion proteins comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to a constant fragment of human immunoglobulin IgG1, more particularly anti-TNFα type proteins, in particular fusion proteins comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to a constant fragment of human immunoglobulin IgG1 (Peppel et al., J Exp Med, 174: 1483-1489 - Murphy et al., Arch Ophthalmol, 22: 845-851), more particularly proteins having at least 85% sequence identity to the sequence of SEQ ID NO: 22; (iv) anti-VEGF (vascular endothelial growth factor) type proteins, in particular proteins selected from the group consisting of S-Flt1, aflibercept, conbercept, brolucizumab, in particular proteins having at least 85% sequence identity to the peptide sequence of SEQ ID NO: 3 (this protein is more particularly aflibercept); (v) proteins with anti-angiogenic properties, such as angiostatin, endostatin, thrombospondin, anti-angiopoietin-2 type proteins, anti-FGF2 type proteins, anti-PLGF (placental growth factor) type proteins, anti-PDGF type proteins; and (vi) Proteins that regulate the activation of complement, such as complement factor I (CFI) and proteins having at least 85% sequence identity to the sequence of SEQ ID NO: 26 (more particularly, this protein is complement factor H). may be selected from the group consisting of:

[0053] In particular, (i) a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 17 includes proteins having at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% sequence identity to the sequence of SEQ ID NO: 17. In particular, the protein is more particularly transferrin (i.e., a protein having 100% sequence identity to the sequence of sequence identity 17).

[0054] In particular, (ii) a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 8 includes proteins having at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% sequence identity to the sequence of SEQ ID NO: 8. In particular, the protein is more particularly decorin (i.e., a protein having 100% sequence identity to the sequence of SEQ ID NO: 8).

[0055] In particular, (iii) a protein having at least 85% sequence identity to the fusion protein of sequence SEQ ID NO: 22 includes proteins having at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% sequence identity to the sequence SEQ ID NO: 22. In particular, this protein is more particularly a fusion protein comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 of sequence SEQ ID NO: 22.

[0056] In particular, (iv) a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 3 includes proteins having at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% sequence identity to the sequence of SEQ ID NO: 3. In particular, the protein is more particularly aflibercept (i.e., a protein having 100% sequence identity to the sequence of SEQ ID NO: 3).

[0057] In particular, (vi) a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 26 includes proteins having at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% sequence identity to the sequence of SEQ ID NO: 26. In particular, the protein is more particularly complement factor H (i.e., a protein having 100% sequence identity to the sequence of SEQ ID NO: 26).

[0058] In particular, the coding sequences for the first and second therapeutic proteins, which differ from each other, can be, e.g., (i) a nucleotide sequence encoding transferrin, in particular a nucleotide sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 15, in particular the sequence of SEQ ID NO: 16; (ii) nucleotide sequences encoding proteins with anti-fibrotic properties, such as BMP7 proteins (bone morphogenetic protein 7), anti-TGFβ-type proteins, anti-FGF2-type proteins, anti-CTGF-type proteins (CTGF: connective tissue growth factor), in particular proteins encoding decorin, more particularly nucleotide sequences having at least 70% sequence identity to the sequence SEQ ID NO: 6, more particularly a sequence selected from the group consisting of the sequences SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, in particular the sequence SEQ ID NO: 7 or the sequence SEQ ID NO: 11, more particularly the sequence SEQ ID NO: 11; (iii) nucleotide sequences encoding proteins with anti-inflammatory properties, in particular anti-TNF type proteins, such as human TNFR-Is, human TNFR-Is / mouse IgG1, Lenercept or fusion proteins comprising the extracellular domain of the human receptor p55 for TNF alpha linked via a hinge to a constant fragment of human immunoglobulin IgG1, more particularly anti-TNF alpha type proteins, in particular fusion proteins comprising the extracellular domain of the human receptor p55 for TNF alpha linked via a hinge to a constant fragment of human immunoglobulin IgG1, more particularly sequences having at least 85% sequence identity to the sequence of SEQ ID NO: 21; (iv) nucleotide sequences encoding anti-VEGF proteins, such as S-Flt1, aflibercept, conbercept, brolucizumab, in particular sequences encoding aflibercept, more particularly sequences having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2; (v) sequences encoding proteins with anti-angiogenic properties, such as angiostatin, endostatin, thrombospondin, anti-angiopoietin-2 type proteins, anti-FGF2 type proteins, anti-PLGF type proteins, anti-PDGF type proteins; and (vi) a sequence encoding a protein that regulates the activation of complement, such as complement factor I (CFI) and complement factor H, particularly complement factor H, more particularly a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 25 may be selected from the group consisting of:

[0059] "Sequences encoding a first and second therapeutic protein" are not to be understood as "first nucleotide sequence" and "second nucleotide sequence" as previously indicated, but rather sequences that are present within the first and second nucleotide sequences of the invention and that specifically encode the first and second therapeutic proteins.

[0060] According to one embodiment, the first or second therapeutic protein of the DNA construct of the invention is a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 17, which protein is more particularly transferrin.

[0061] In particular, the DNA construct of the present invention is a DNA construct in which the first nucleotide sequence or the second nucleotide sequence is a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 17 (this protein is more particularly transferrin); and - a signal peptide of the peptide sequence SEQ ID NO: 18 The nucleic acid sequence may be characterized in that it encodes:

[0062] Thus, one of the sequences encoding the first and second therapeutic proteins of the DNA construct of the invention may in particular be a sequence having at least 75% sequence identity to a nucleotide sequence encoding transferrin, in particular the sequence of SEQ ID NO: 15, in particular the sequence of SEQ ID NO: 16.

[0063] In particular, the DNA construct of the present invention is a DNA construct in which the first nucleotide sequence or the second nucleotide sequence is - a nucleotide sequence encoding transferrin, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 15, in particular the sequence of SEQ ID NO: 16; and - the nucleotide sequence of SEQ ID NO: 20 encoding the signal peptide The method may be characterized in that it comprises:

[0064] According to one embodiment, the first and second therapeutic proteins encoded by the DNA construct of the present invention are, respectively: a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 17 (this protein is more particularly transferrin); and - proteins of the anti-TNFα type, in particular proteins with at least 85% sequence identity to the sequence of SEQ ID NO: 22, more particularly fusion proteins comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 of sequence SEQ ID NO: 22 is.

[0065] Thus, according to one embodiment, the sequences encoding the first and second therapeutic proteins of the DNA construct of the invention are respectively: - a nucleotide sequence encoding transferrin, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 15, in particular the sequence of SEQ ID NO: 16; and a sequence encoding an anti-TNFα type protein, in particular a sequence encoding a fusion protein comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 of the peptide sequence SEQ ID NO: 22, in particular a nucleotide sequence having at least 85% sequence identity to the sequence SEQ ID NO: 21 is.

[0066] Inflammation and oxidative stress are important components of retinal degeneration, such as age-related macular degeneration (AMD) or glaucomatous neuropathy, following increased intraocular pressure caused by glaucoma. In particular, elevated intraocular concentrations of TNFα have been observed in glaucomatous eyes (Tezel et al., 2001, Invest Ophthalmol Vis Sci. 2001 Jul; 42(8): 1787-94), and injection of TNFα into rodent eyes induces optic nerve axon degeneration and programmed death of retinal ganglion cells (Kitaoka 2006, Invest Ophthalmol Vis Sci. 2006; 47: 1448-1457). Increased expression of genes regulating iron levels has also been observed in glaucomatous eyes, suggesting that iron-induced oxidative stress may play a role in the pathogenesis of glaucoma (Farkas et al., 2004). Administration of anti-TNF antibodies and iron chelators, such as transferrin, can advantageously reduce both iron-mediated inflammation and oxidative stress.

[0067] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is - a nucleotide sequence encoding transferrin, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 15, in particular the sequence of SEQ ID NO: 16; and - the nucleotide sequence of SEQ ID NO: 20 encoding the signal peptide and (f) the second nucleotide sequence: a nucleotide sequence encoding an anti-TNFα type protein, in particular a sequence encoding a fusion protein comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 of the peptide sequence SEQ ID NO: 22, in particular a nucleotide sequence having at least 85% sequence identity with the sequence SEQ ID NO: 21, in particular the sequence SEQ ID NO: 21; and - the nucleotide sequence of SEQ ID NO: 23 encoding the signal peptide The method may be characterized in that it comprises:

[0068] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 17 (this protein is more particularly transferrin); and - a signal peptide of the peptide sequence SEQ ID NO: 18 and (f) the second nucleotide sequence: a protein with at least 85% sequence identity to the sequence of SEQ ID NO: 22 (this protein is more particularly a fusion protein comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 of sequence SEQ ID NO: 22); and - a signal peptide of the peptide sequence SEQ ID NO: 23 The nucleic acid sequence may be characterized in that it encodes:

[0069] According to another embodiment, the first and second therapeutic proteins encoded by the DNA construct of the present invention are each a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 17 (this protein is more particularly transferrin); and a protein with antifibrotic properties, in particular a protein with at least 85% sequence identity to the sequence of SEQ ID NO: 8 (this protein is more particularly decorin); is.

[0070] Thus, according to one embodiment, the sequences encoding the first and second therapeutic proteins of the DNA construct of the invention are respectively: - a nucleotide sequence encoding transferrin, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 15, in particular the sequence of SEQ ID NO: 16; and a protein encoding a protein with antifibrotic properties, such as a BMP7 protein (bone morphogenetic protein 7), an anti-TGFβ-type protein, an anti-FGF2-type protein, an anti-CTGF-type protein (connective tissue growth factor), in particular a protein encoding decorin, more particularly a nucleotide sequence having at least 70% sequence identity to the sequence SEQ ID NO: 6, more particularly a sequence selected from the group consisting of the sequences SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, in particular the sequence SEQ ID NO: 7 or the sequence SEQ ID NO: 11, more particularly the sequence SEQ ID NO: 11 is.

[0071] Glaucoma, especially open-angle glaucoma, is characterized by the increase in intraocular pressure after trabecular meshwork fibrosis, as well as the loss of retinal ganglion cells and optic nerve degeneration.Current treatments for glaucoma reduce intraocular pressure, but cannot stop the progression of neurodegeneration.Administering neuroprotective agents, such as anti-TNF antibodies or transferrin, can advantageously enhance the anti-fibrotic effects of decorin and the like, thereby reducing intraocular pressure and preventing the degeneration of the retina and optic nerve.

[0072] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is a nucleotide sequence encoding transferrin, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 15, in particular the sequence of SEQ ID NO: 16; and - the nucleotide sequence of SEQ ID NO: 20 encoding the signal peptide and (f) the second nucleotide sequence: a nucleotide sequence encoding decorin, more particularly a nucleotide sequence having at least 70% sequence identity to the sequence SEQ ID NO: 6, more particularly a sequence selected from the group consisting of the sequences SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, in particular the sequence SEQ ID NO: 7 or the sequence SEQ ID NO: 11, more particularly the sequence SEQ ID NO: 11; and - the nucleotide sequence of SEQ ID NO: 14 encoding the signal peptide The method may be characterized in that it comprises:

[0073] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 17 (this protein is more particularly transferrin); and - a signal peptide of the peptide sequence SEQ ID NO: 18 code; and (f) the second nucleotide sequence: a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 8 (this protein is more particularly decorin); and - a signal peptide of the peptide sequence SEQ ID NO: 13 The nucleic acid sequence may be characterized in that it encodes:

[0074] According to one embodiment, the first or second therapeutic protein of the DNA construct of the invention is an anti-VEGF type protein, in particular a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 3 (this protein is more particularly aflibercept).

[0075] In particular, the DNA construct of the present invention is a DNA construct in which the first nucleotide sequence or the second nucleotide sequence is - a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 3 (this protein is more particularly aflibercept); and - a signal peptide of the peptide sequence SEQ ID NO: 4 The nucleic acid sequence may be characterized in that it encodes:

[0076] Thus, one of the sequences encoding the first and second therapeutic proteins of the DNA construct of the present invention may in particular be a nucleotide sequence encoding an anti-VEGF type antibody protein, in particular S-Flt1, aflibercept, conbercept, brolucizumab, in particular a sequence encoding aflibercept, more particularly a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2.

[0077] In particular, the DNA construct of the present invention is a DNA construct in which the first nucleotide sequence or the second nucleotide sequence is - a nucleotide sequence encoding aflibercept, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2; and - the nucleotide sequence of SEQ ID NO: 5 encoding the signal peptide Contains The present invention may be characterized by the above.

[0078] According to another embodiment, the first and second therapeutic proteins of the DNA construct of the invention are respectively: a protein having antifibrotic properties, in particular a protein having at least 85% sequence identity with the sequence of SEQ ID NO: 8 (this protein is more particularly decorin); and - an anti-VEGF type protein, in particular a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 3 (this protein is more particularly aflibercept) is.

[0079] Thus, according to one embodiment, the sequences encoding the first and second therapeutic proteins of the DNA construct of the invention are respectively: a nucleotide sequence encoding a protein having antifibrotic properties, such as a BMP7 protein (bone morphogenetic protein 7), an anti-TGFβ-type protein, an anti-FGF2-type protein, an anti-CTGF-type protein (connective tissue growth factor), in particular a protein encoding decorin, more particularly a nucleotide sequence having at least 70% sequence identity to the sequence SEQ ID NO: 6, more particularly a sequence selected from the group consisting of the sequences SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, in particular the sequence SEQ ID NO: 7 or the sequence SEQ ID NO: 11; and - a nucleotide sequence encoding a protein of an anti-VEGF type antibody, in particular S-Flt1, aflibercept, conbercept, brolucizumab, in particular a sequence encoding aflibercept, more particularly a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2 is.

[0080] The presence of an anti-fibrotic active ingredient can advantageously enhance the action of anti-VEGF antibodies, thereby improving the efficacy of these compounds in treating the target ocular condition. In particular, even in patients receiving optimally spaced injections of anti-VEGF antibodies, the development of subretinal fibrosis appears over time in more than half of patients, and the efficacy of anti-VEGF antibodies has been observed to decrease over time (Daniel et al. 2014, Ophthalmology 121, 656-666). Furthermore, the development of subretinal fibrosis in patients with age-related macular degeneration who do not respond to anti-VEGF antibodies has been identified as a cause of poor therapeutic response to anti-VEGF antibodies (Cohen et al. 2012, Retina 32, 1480-1485).

[0081] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is - a nucleotide sequence encoding aflibercept, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2; and - the nucleotide sequence of SEQ ID NO: 5 encoding the signal peptide and (f) the second nucleotide sequence: a nucleotide sequence encoding decorin, more particularly a nucleotide sequence having at least 70% sequence identity to the sequence of SEQ ID NO: 6, more particularly a sequence selected from the group consisting of the sequences of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, in particular the sequence of SEQ ID NO: 7 or the sequence of SEQ ID NO: 11; and - the nucleotide sequence of SEQ ID NO: 14 encoding the signal peptide The method may be characterized in that it comprises:

[0082] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is - a nucleotide sequence encoding aflibercept, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2; and - the nucleotide sequence of SEQ ID NO: 5 encoding the signal peptide and (f) the second nucleotide sequence: - a nucleotide sequence encoding decorin, more particularly the sequence SEQ ID NO: 7; and It may be characterized in that it contains the nucleotide sequence of SEQ ID NO: 14 which codes for a signal peptide.

[0083] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is - a nucleotide sequence encoding aflibercept, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2; and - the nucleotide sequence of SEQ ID NO: 5 encoding the signal peptide and (f) the second nucleotide sequence: - a nucleotide sequence encoding decorin, more particularly the sequence SEQ ID NO: 11; and - the nucleotide sequence of SEQ ID NO: 14 encoding the signal peptide The method may be characterized in that it comprises:

[0084] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is - a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 3 (this protein is more particularly aflibercept); and - a signal peptide of the peptide sequence SEQ ID NO: 4 code; and (f) the second nucleotide sequence: a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 8 (this protein is more particularly decorin); and - a signal peptide of the peptide sequence SEQ ID NO: 13 The nucleic acid sequence may be characterized in that it encodes:

[0085] According to another embodiment, the first and second therapeutic proteins of the DNA construct of the invention are respectively: - proteins of the anti-TNFα type, in particular proteins with at least 85% sequence identity to the sequence of SEQ ID NO: 22, more particularly fusion proteins comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 of sequence SEQ ID NO: 22; - a protein of the anti-VEGF type, in particular a protein having at least 85% sequence identity with the sequence of SEQ ID NO: 3 (this protein is more particularly aflibercept) is.

[0086] Thus, according to one embodiment, the sequences encoding the first and second therapeutic proteins of the DNA construct of the invention are respectively: a sequence encoding a protein of the anti-TNFα type, in particular a sequence encoding a fusion protein comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 of the peptide sequence SEQ ID NO: 22, in particular a nucleotide sequence having at least 85% sequence identity with the sequence SEQ ID NO: 21; and nucleotide sequences encoding anti-VEGF type proteins, such as S-Flt1, aflibercept, conbercept, brolucizumab, in particular sequences encoding aflibercept, more particularly sequences having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2 is.

[0087] The presence of the active ingredient of anti-TNFα antibody can advantageously enhance the effect of anti-VEGF antibody, thereby improving the effectiveness of this compound in treating the target ocular pathology.In particular, VEGF induces retinal permeability, but it is well known that inflammatory substances such as TNFα can also cause vascular permeability, especially in patients who do not respond to treatment with anti-VEGF antibody, as may be observed in patients with diabetic retinopathy (Arias L. et al.; Retina 2010, 30: 1601-1608 and Sfikakis et al.; Diabetes Care 2010, 33: 1523-1528).Recent studies suggest that VEGF and TNFα induce permeability through different mechanisms.

[0088] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is a nucleotide sequence encoding a protein of the anti-TNFα type, in particular a sequence encoding a fusion protein comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 of the peptide sequence SEQ ID NO: 22, in particular a nucleotide sequence having at least 85% sequence identity with the sequence SEQ ID NO: 21, in particular the sequence SEQ ID NO: 21; and - comprising the nucleotide sequence of SEQ ID NO: 23 encoding the signal peptide; and (f) the second nucleotide sequence: a nucleotide sequence encoding aflibercept, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2; and - the nucleotide sequence of SEQ ID NO: 5 encoding the signal peptide The method may be characterized in that it comprises:

[0089] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 22 (this protein is more particularly a fusion protein comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 of sequence SEQ ID NO: 22); and - a signal peptide of the peptide sequence SEQ ID NO: 23 code; and (f) the second nucleotide sequence: - a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 3 (this protein is more particularly aflibercept); and - a signal peptide of the peptide sequence SEQ ID NO: 4 The nucleic acid sequence may be characterized in that it encodes:

[0090] According to another embodiment, the first and second therapeutic proteins of the DNA construct of the invention are respectively: - proteins that regulate the activation of complement, in particular a protein having at least 85% sequence identity with the sequence of SEQ ID NO: 26 (this amino acid sequence is more particularly complement factor H); - an anti-VEGF type protein, in particular a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 3 (this protein is more particularly aflibercept) is.

[0091] Thus, according to one embodiment, the sequences encoding the first and second therapeutic proteins of the DNA construct of the invention are respectively: - a nucleotide sequence encoding a protein that regulates the activation of complement, such as complement factor I (CFI) and complement factor H, in particular a sequence encoding complement factor H, more particularly a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 25; and - a nucleotide sequence encoding an anti-VEGF type protein, such as S-Flt1, aflibercept, conbercept, brolucizumab, in particular a sequence encoding aflibercept, more particularly a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2 is.

[0092] Activation of the alternative complement pathway is a key component of age-related macular degeneration (AMD). This activation leads to the formation of a membrane attack complex, recruitment of macrophages, and inflammation associated with inflammasome-mediated cytokine production. Complement factor H is involved in regulating complement autoactivation. Several polymorphic variants in the gene encoding complement factor H, which affect protein function, confer a strong susceptibility to developing two forms of AMD: wet and dry. Conversely, inhibition of the alternative pathway by intraocular injection of complement factor H reduces neovascularization in animal models of choroidal neovascularization. Therefore, administration of an active ingredient that modulates complement activation and an anti-VEGF antibody, such as aflibercept, can beneficially reduce both neovascularization and inflammation associated with AMD.

[0093] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is - a nucleotide sequence encoding a protein that regulates the activation of complement, such as complement factor I (CFI) and complement factor H, in particular complement factor H, more particularly a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 25; and - the nucleotide sequence of SEQ ID NO: 28 encoding the signal peptide and (f) the second nucleotide sequence: - a nucleotide sequence encoding aflibercept, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2; and - the nucleotide sequence of SEQ ID NO: 5 encoding the signal peptide The method may be characterized in that it comprises:

[0094] In particular, the DNA construct of the present invention is (c) the first nucleotide sequence is - a protein that regulates the activation of complement, in particular a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 26 (this protein is more particularly complement factor H); and - a signal peptide of the peptide sequence SEQ ID NO: 27 code; and (f) the second nucleotide sequence: - a protein having at least 85% sequence identity to the sequence of SEQ ID NO: 3 (this protein is more particularly aflibercept); and - a signal peptide of the peptide sequence SEQ ID NO: 4 The nucleic acid sequence may be characterized in that it encodes:

[0095] Furthermore, the DNA construct of the present invention is characterized in that the first nucleotide sequence also encodes a signal peptide allowing the secretion of the first therapeutic protein.

[0096] Signal peptides of this type are well known to those skilled in the art. This signal peptide can be, for example, the signal peptide of human tissue plasminogen activator (tPA) of the peptide sequence SEQ ID NO: 4 (which can be encoded, for example, by the nucleotide sequence SEQ ID NO: 5) or the signal peptide of the HTLV-1 envelope of the peptide sequence SEQ ID NO: 29 (which can be encoded, for example, by the nucleotide sequence SEQ ID NO: 30). It is also possible that the natural peptide signal of the therapeutic protein in question can be, for example, the native signal peptide of decorin, of the peptide sequence SEQ ID NO: 13 (which can be encoded, for example, by the nucleotide sequence SEQ ID NO: 14); - the signal peptide of transferrin, of the peptide sequence SEQ ID NO: 18 (which can be encoded, for example, by the nucleotide sequence SEQ ID NO: 19 or by the nucleotide sequence SEQ ID NO: 20); a natural peptide signal fusion protein comprising the signal peptide of a protein of the anti-TNFα type, in particular the extracellular domain of the human receptor p55 for TNFα, linked via a hinge to the constant fragment of human immunoglobulin IgG1, this signal peptide having the peptide sequence of SEQ ID NO: 23 (which can, for example, be encoded by the nucleotide sequence of SEQ ID NO: 24); or the native signal peptide of factor H, of the peptide sequence SEQ ID NO: 27 (which can be encoded, for example, by the nucleotide sequence SEQ ID NO: 28); It could be.

[0097] As noted above, this signal peptide is contiguous with the first therapeutic protein, i.e., it is fused directly at the N-terminus of the first therapeutic protein, and therefore the sequence encoding the signal peptide is 5' to the sequence encoding the first therapeutic protein.

[0098] Furthermore, the DNA construct of the present invention is characterized in that the second nucleotide sequence also encodes a signal peptide allowing the secretion of the second therapeutic protein.

[0099] This signal peptide can be, for example, the signal peptide of human tissue plasminogen activator (tPA) of the peptide sequence SEQ ID NO: 4 (which can be encoded, for example, by the nucleotide sequence SEQ ID NO: 5) or the signal peptide of the HTLV-1 envelope of the peptide sequence SEQ ID NO: 29 (which can be encoded, for example, by the nucleotide sequence SEQ ID NO: 30). It can also be the natural signal peptide of the therapeutic protein in question, for example the native signal peptide of decorin, of the peptide sequence SEQ ID NO: 13 (which can, for example, be encoded by the nucleotide sequence SEQ ID NO: 14); the natural signal peptide of transferrin, of the peptide sequence SEQ ID NO: 18 (which can be encoded, for example, by the nucleotide sequence SEQ ID NO: 19 or by the nucleotide sequence SEQ ID NO: 20); a natural signal peptide fusion protein comprising the extracellular domain of the human receptor for TNFα, p55, linked via a hinge to the signal peptide of a protein of the anti-TNFα type, in particular the constant fragment of human immunoglobulin IgG1, this signal peptide having the peptide sequence of SEQ ID NO: 23 (which can, for example, be encoded by the nucleotide sequence of SEQ ID NO: 24); or the native signal peptide of factor H, of the peptide sequence SEQ ID NO: 27 (which can be encoded, for example, by the nucleotide sequence SEQ ID NO: 28); But it's possible.

[0100] This signal peptide may be the same as or different from the signal peptide encoded by the first nucleotide sequence, and is preferably different from the signal peptide encoded by the first nucleotide sequence.

[0101] As noted above, this signal peptide is contiguous with the second therapeutic protein, i.e., it is fused directly to the N-terminus of the second therapeutic protein, and therefore the sequence encoding the signal peptide is 5' to the sequence encoding the second therapeutic protein.

[0102] According to a particular embodiment, the signal peptide encoded by the first nucleotide sequence and the signal peptide encoded by the second nucleotide sequence are independently selected from the group consisting of the peptide sequences SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 27 and SEQ ID NO: 29.

[0103] According to one embodiment, the signal peptide-encoding sequence encoded by the first nucleotide sequence and the signal peptide-encoding sequence encoded by the second nucleotide sequence are independently selected from the group consisting of the nucleotide sequences of SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28 and SEQ ID NO:30.

[0104] Furthermore, the DNA construct of the present invention also (d) a promoter that enables expression of a first therapeutic protein of the construct of the present invention; and (g) a promoter that enables expression of a second therapeutic protein of the construct of the present invention.

[0105] These promoters may be the same or different. According to a particular embodiment, these two promoters are different from each other.

[0106] The promoter may be, for example, a CAG-type promoter or a CMV-type promoter.

[0107] Furthermore, the DNA construct of the present invention is characterized in that it comprises (e) a polyadenylation sequence 3' of the first nucleotide sequence, and (h) a polyadenylation sequence 3' of the second nucleotide sequence.

[0108] The polyadenylation sequence contains in particular the conserved motif of the sequence AATAAA, which is well known to those skilled in the art.

[0109] These two polyadenylation sequences may be identical to each other or different. According to one embodiment, they are different from each other.

[0110] These polyadenylation sequences can be, for example, polyadenylation sequences of the RBG type (rabbit beta globin) or BGH (bovine growth hormone).

[0111] Finally, as described above, the DNA constructs of the present invention are administered to the patient's ciliary muscle by injection and then electrotransferred into the cells of the ciliary muscle.

[0112] In a particular embodiment, the DNA construct of the present invention is in a circular form.

[0113] In one embodiment of the present invention, the DNA construct is a naked DNA construct.

[0114] According to one embodiment of the present invention, the DNA construct of the present invention is a naked DNA construct in circular form.

[0115] In one embodiment of the present invention, the DNA construct of the present invention is a naked DNA construct in circular form, in which the sequences encoding the first and second therapeutic proteins of the DNA construct of the present invention are - a nucleotide sequence encoding transferrin, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 15, in particular the sequence of SEQ ID NO: 16; and a sequence encoding a protein of the anti-TNFα type, in particular a sequence encoding a fusion protein comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 of the peptide sequence SEQ ID NO: 22, in particular a nucleotide sequence having at least 85% sequence identity with the sequence SEQ ID NO: 21 is.

[0116] In one embodiment of the present invention, the DNA construct of the present invention is a naked DNA construct in circular form, in which the sequences encoding the first and second therapeutic proteins of the DNA construct of the present invention are a nucleotide sequence encoding a protein having antifibrotic properties, such as a BMP7 protein (bone morphogenetic protein 7), an anti-TGFβ-type protein, an anti-FGF2-type protein, an anti-CTGF-type protein (connective tissue growth factor), in particular a protein encoding decorin, more particularly a nucleotide sequence having at least 70% sequence identity to the sequence SEQ ID NO: 6, more particularly a sequence selected from the group consisting of the sequences SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, in particular the sequence SEQ ID NO: 7 or the sequence SEQ ID NO: 11, more particularly the sequence SEQ ID NO: 11; and - a nucleotide sequence encoding an anti-VEGF type protein, such as S-Flt1, aflibercept, conbercept, brolucizumab, in particular a sequence encoding aflibercept, more particularly a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2 is.

[0117] In one embodiment, the DNA construct of the invention is a naked DNA construct in circular form, in which the sequences encoding the first and second therapeutic proteins of the DNA construct of the invention are, respectively: - a nucleotide sequence encoding transferrin, in particular a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 15, in particular a sequence which is the sequence of SEQ ID NO: 16; a nucleotide sequence encoding a protein having antifibrotic properties, such as a BMP7 protein (bone morphogenetic protein 7), an anti-TGFβ type protein, an anti-FGF2 type protein, an anti-CTGF type protein (connective tissue growth factor), in particular a protein encoding decorin, more particularly a nucleotide sequence having at least 70% sequence identity to the sequence SEQ ID NO: 6, more particularly a sequence selected from the group consisting of the sequences SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, in particular the sequence SEQ ID NO: 7 or the sequence SEQ ID NO: 11, more particularly the sequence SEQ ID NO: 11 is.

[0118] In one embodiment of the present invention, the DNA construct of the present invention is a naked DNA construct in circular form, in which the sequences encoding the first and second therapeutic proteins of the DNA construct of the present invention are respectively a sequence encoding an anti-TNFα type protein, in particular a sequence encoding a fusion protein comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 of the protein sequence of SEQ ID NO: 22, in particular a nucleotide sequence having at least 85% sequence identity to the sequence of SEQ ID NO: 21; and - a nucleotide sequence encoding an anti-VEGF type protein, such as S-Flt1, aflibercept, conbercept, brolucizumab, in particular a sequence encoding aflibercept, more particularly a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular the sequence of SEQ ID NO: 2 is.

[0119] In one embodiment of the present invention, the DNA construct of the present invention is a naked DNA construct in circular form, in which the sequences encoding the first and second therapeutic proteins of the DNA construct of the present invention are respectively - a nucleotide sequence encoding a protein that regulates the activation of complement, such as complement factor I (CFI) and complement factor H, in particular a sequence encoding complement factor H, more particularly a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 25; and a nucleotide sequence encoding an anti-VEGF type protein, such as S-Flt1, aflibercept, conbercept, brolucizumab, in particular a sequence encoding aflibercept, more particularly a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 1, in particular a sequence that is SEQ ID NO: 2 is.

[0120] The present invention also relates to a DNA construct for the non-viral transfer of nucleic acids into ocular muscle cells of a patient for the treatment of an ocular condition, comprising: (a) a bacterial or prokaryotic origin of replication, in particular a bacterial origin of replication; (b) one or more sequences that promote expression of DNA in the patient's eye; (c) a first therapeutic protein, wherein said first therapeutic protein is aflibercept; and a signal peptide allowing secretion of said first therapeutic protein, said signal peptide being contiguous with the sequence of said first therapeutic protein at the N-terminus of said first therapeutic protein; a first nucleotide sequence encoding the (d) a promoter that allows expression of the first therapeutic protein in the patient's eye; (e) a polyadenylation sequence 3' of the first nucleotide sequence; (f) a second therapeutic protein, wherein the second therapeutic protein is decorin, and a signal peptide allowing secretion of this second therapeutic protein, said signal peptide being contiguous with the sequence of the second therapeutic protein at the N-terminus of said second therapeutic protein; a second nucleotide sequence encoding (g) a promoter that enables expression of the second therapeutic protein in the patient's eye; and (h) a polyadenylation sequence 3' of the second nucleotide sequence; The DNA construct, characterized in that it comprises It is characterized by:

[0121] Application of the above DNA constructs The DNA constructs as defined above are particularly intended for the treatment of ocular conditions.

[0122] The ocular condition of the present invention is retinal degeneration.

[0123] The retinal degeneration may in particular be selected from the group consisting of wet or dry age-related macular degeneration (ARMD); diabetic retinopathy (DR); retinal vein occlusion, in particular central retinal vein occlusion (CRVO) or branch retinal vein occlusion (BRVO); myopic choroidal neovascularization (CNV); uveitis, in particular non-infectious uveitis; retinitis pigmentosa and glaucoma.

[0124] Diabetic retinopathy is particularly intended to refer to vision loss resulting from diabetic macular edema (DME), intravitreal hemorrhage, retinal detachment, or neovascular glaucoma.

[0125] According to a particular embodiment, when the construct of the invention, which is used in particular for the treatment of an ocular condition, comprises aflibercept and decorin as defined above as the first and second therapeutic proteins, the ocular condition may more particularly be selected from the group consisting of age-related macular degeneration (ARMD), in particular the exudative type; diabetic retinopathy (DR); retinal vein occlusion, in particular central retinal vein occlusion (CRVO) or branch retinal vein occlusion (BRVO); and myopic choroidal neovascularization (CNV), and more particularly retinal degeneration, which may be selected from the group consisting of age-related macular degeneration (ARMD), in particular the (exudative) neovascular type of ARMD; vision loss due to diabetic macular edema (DME); retinal vein occlusion, in particular central retinal vein occlusion (CRVO) or branch retinal vein occlusion (BRVO); and myopic choroidal neovascularization (CNV).

[0126] Diabetic retinopathy is particularly intended to refer to the loss of vision resulting from diabetic macular edema (DME) and the formation of new blood vessels observed in proliferative diabetic retinopathy.

[0127] As described above, the DNA constructs of the present invention are injected into the ciliary muscle, an eye muscle, and then electrotransferred there. A known non-viral gene therapy technique used in the present invention involves the injection of a DNA construct into the eye muscle followed by electrotransfer, thereby inducing transient permeabilization of the ciliary muscle cells and DNA migration, optimizing transfection of the DNA construct. This DNA electrotransfer technique (also called electroporation or electropermeabilization) is easy to apply, reliable, and safe for patients. In contrast to viral vectors, DNA electrotransfer does not induce an immune response, thus allowing long-term expression of the introduced gene. Furthermore, studies performed with lentiviruses and retroviruses have shown that the latter are prone to inducing insertional mutagenesis during their integration into the host genome. The DNA constructs described herein are free of these drawbacks, are easy to produce and manipulate, do not induce an immune response, and are therefore perfectly suitable for gene therapy in patients, particularly human patients.

[0128] According to the present invention, DNA constructs are injected into the ciliary muscle, because its location allows for uniform and sustained protein production and facilitates the diffusion of these proteins throughout the eye (Blocquel et al. "Plasmid electrotransfer of eye ciliary muscle: principles and therapeutic efficacy using hTNF-alpha soluble receptor in uveitis", FASEB J. 2006 Feb; 20(2): 389-91). Smooth muscle cells exhibit a low regeneration rate and are well distributed on either side of the lens. The amount of protein to be produced is proportional to the surface area of ​​the transfected muscle (Touchard "The ciliary smooth muscle electrotransfer: basic principles and potential for sustained intraocular production of therapeutic proteins", J Gene Med. 2010 Nov; 12(11): 904-19). Thus, the production of the protein of interest of the present invention, in particular a therapeutic protein as described above, will be uniform and constant within the entire eyeball and will be limited to this eyeball. By way of example, the inventors may refer to the electrotransfer method described in patent application EP 2266656, which relates to the injection of a composition that may contain DNA at the level of the ciliary body tissue and / or extraocular muscle tissue.

[0129] The ciliary muscle forms part of the ciliary body near the limbus and just behind the sclera, and therefore injection of the DNA constructs of the present invention into the latter is much less invasive as opposed to subretinal injection, and therefore represents an advantageous injection site for the DNA constructs of the present invention.

[0130] According to another aspect, the present invention also relates to the use of a DNA construct for the treatment of an ocular condition, said DNA construct being intended for the non-viral transfer of nucleic acids into ocular muscle cells of a patient having said ocular condition; said DNA construct being (a) a bacterial or prokaryotic origin of replication, in particular a bacterial origin of replication; (b) one or more sequences that promote expression of DNA in the patient's eye; (c) a first therapeutic protein, and - a signal peptide that allows the secretion of this first therapeutic protein a first nucleotide sequence encoding (wherein the signal peptide is contiguous with the sequence of the first therapeutic protein at the N-terminus of the first therapeutic protein), (d) a promoter that allows expression of the first therapeutic protein in the patient's eye; (e) a polyadenylation sequence 3' of the first nucleotide sequence; (f) a second therapeutic protein, different from the first therapeutic protein, and - a signal peptide that allows secretion of this second therapeutic protein a second nucleotide sequence encoding (the signal peptide is contiguous with the sequence of the second therapeutic protein at the N-terminus of the second therapeutic protein); (g) a promoter that allows expression of the second therapeutic protein in the patient's eye; and (h) a polyadenylation sequence 3' of the second nucleotide sequence; characterized in that it comprises; The DNA construct is administered to the patient by injection into the ciliary muscle followed by electrical transduction into the cells of the ciliary muscle.

[0131] According to another aspect, the present invention also relates to a method for treating an ocular condition comprising administering to a patient a DNA construct by injection into the ciliary muscle followed by electrotransfer into ciliary muscle cells, The DNA construct is intended for the non-viral transfer of nucleic acid into ocular muscle cells of a patient having the ocular condition; (a) a bacterial or prokaryotic origin of replication, in particular a bacterial origin of replication; (b) one or more sequences that promote expression of DNA in the patient's eye; (c) a first therapeutic protein, and - a signal peptide that allows the secretion of this first therapeutic protein a first nucleotide sequence encoding (wherein the signal peptide is contiguous with the sequence of the first therapeutic protein at the N-terminus of the first therapeutic protein), (d) a promoter that allows expression of the first therapeutic protein in the patient's eye; (e) a polyadenylation sequence 3' of the first nucleotide sequence; (f) a second therapeutic protein, different from the first therapeutic protein, and - a signal peptide that allows secretion of this second therapeutic protein a second nucleotide sequence encoding (the signal peptide is contiguous with the sequence of the second therapeutic protein at the N-terminus of the second therapeutic protein); (g) a promoter that allows expression of the second therapeutic protein in the patient's eye; and (h) a polyadenylation sequence 3' of the second nucleotide sequence; The present invention is characterized by comprising:

[0132] The invention will now be described in more detail below using the following examples, which are presented for illustrative purposes only.

[0133] Example 1 The present inventors have demonstrated the expression of two therapeutic proteins encoded by the DNA construct (plasmid) of the present invention in the vitreous of rat eyes after electrotransfer of this DNA construct into the ciliary muscle.

[0134] Plasmid - sequence SEQ ID NO: 16, which encodes human transferrin of sequence SEQ ID NO: 17 (the sequence encoding its signal peptide is sequence SEQ ID NO: 20); - as well as sequence SEQ ID NO: 21, which encodes a fusion protein comprising the extracellular domain of the human receptor p55 for TNFα linked via a hinge to the constant fragment of human immunoglobulin IgG1 (human TNFR-Is / human IgG1) of sequence SEQ ID NO: 22, the sequence encoding its signal peptide being sequence SEQ ID NO: 24; -These two sequences are under the control of a CMV-type promoter A DNA construct of the present invention, designated Plasmid A, containing the following sequence was first prepared by conventional methods and is shown in Figure 1A. A comparative construct, designated Simple because it encodes only one of the proteins of interest (i.e., the sequence of SEQ ID NO: 16, which encodes human transferrin of the sequence of SEQ ID NO: 17), was also prepared by similar methods using a similar plasmid backbone to the Plasmid A construct, only the sequence encoding the protein of interest is also under the control of a CMV-type promoter. This comparative plasmid (designated Plasmid A') is shown in Figure 2.

[0135] animal Seven-week-old Long-Evan rats were used according to the ARVO (Association for Research in Vision and Ophthalmology) protocol. Rats were anesthetized with intramuscular injections of ketamine (40 mg / kg) and xylazine (4 mg / kg) prior to bilateral injection of plasmid (30 μg per eye) and electrotransfer on day 0 (DO). Six rats were used for each analysis time point (days 3, 7, 14, 21, and 30).

[0136] At each analysis time point, rats are euthanized by administration of a lethal dose of pentobarbital (400 mg / kg), after which the eyes are removed from the animals and the ocular fluids (vitreous and aqueous humor) are collected and stored at -80°C until analysis.

[0137] Electrotransfer at the level of the ciliary muscle in rats Electrotransfer is performed as described in Blocquel et al. "Plasmid electrotransfer of eye ciliary muscle: principles and therapeutic efficacy using hTNF-alpha soluble receptor in uveitis" (FASEB J 2006; 20:389-391), with the injection route modified by a transscleral approach (Touchard, "The ciliary smooth muscle electrotransfer: basic principles and potential for sustained intraocular production of therapeutic proteins," J Gene Med. 2010 Nov; 12(11): 904-19). Plasmids are injected into the ciliary muscle of animals at a ratio of 30 μg in 10 μL of Tris-EDTA NaCl solution using an appropriate syringe.

[0138] Electrical impulses are administered using a specialized iridium / platinum electrode with a diameter of 250 μm. This internal electrode is introduced through an existing transscleral tunnel. The external electrode is a thin sheet of stainless steel curved to fit the shape of the eye and is placed at the level of the limbus, opposite the internal electrode. Electrotransfer is performed at a rate of eight unipolar square-wave electrical impulses (200 V / cm, 10 ms, 5 Hz) generated by an electroporation device similar to that described by Touchard et al. (J Gene Med. 2010 Nov; 12(11): 904-19).

[0139] Test results Ocular fluid samples are collected on days 3 (D+3), 7 (D+7), 14 (D+14), 21 (D+21), and 30 (D+30) after injection of the plasmid followed by electrotransfer (day 0). For each of these samples, an ELISA assay is performed to measure the amount of human transferrin and / or anti-TNFα fusion protein of sequence SEQ ID NO: 22 present in the sample.

[0140] In this way, mean concentrations are calculated for each group over time.

[0141] The results obtained are: - in Figure 3, which shows the concentration (pg / mL) of human transferrin produced in ocular fluid collected from day 3 to day 30 by a construct according to the invention compared to a construct not according to the invention; and In Figure 4, which shows the concentration (pg / mL) of the anti-TNFα fusion protein of sequence SEQ ID NO: 22 produced in ocular fluid collected from day -3 to day 30, is shown.

[0142] Inspection of these figures shows that the concentrations of anti-TNFα fusion protein and human transferrin remain constant over time in rats receiving the constructs of the invention.

[0143] These experiments therefore demonstrate the fact that application of the construct of the invention, which is of very large size due to the presence of not one but two coding sequences for the protein of interest, allows it to effectively penetrate the target cell and to express the two proteins encoded by said construct at the level of the site of interest.

[0144] Furthermore, they also demonstrate, quite surprisingly, the ability of the constructs of the present invention to produce the proteins they encode more stably over time than constructs encoding only one of these proteins (Figure 3).

[0145] Example 2 The inventors confirmed the observations made in the second experimental protocol of the example using a different plasmid of the invention than that used in example 1.

[0146] Plasmid - sequence SEQ ID NO: 7, which codes for decorin of sequence SEQ ID NO: 8; as well as the sequence SEQ ID NO: 2 which codes for aflibercept of sequence SEQ ID NO: 3; - these two sequences under the control of a CMV-type and a CAG-type promoter, respectively First, a DNA construct of the present invention, designated as plasmid B, comprising: is prepared by conventional methods, as shown in Figure 5. A comparative construct, designated as simple, is also prepared by similar methods, using a similar plasmid backbone to the plasmid B construct, since it encodes only one protein of interest (i.e., the sequence of SEQ ID NO: 2, which encodes aflibercept of the sequence of SEQ ID NO: 3). The single sequence encoding the protein of interest is also under the control of a CAG-type promoter. The expression cassette of this protein of interest is therefore identical in the two constructs. This comparative plasmid (designated as plasmid b') is shown in Figure 6.

[0147] The animals used in this protocol are as described in Example 1. Six rats are used at each analysis time point (days 3, 7, 14 and 21).

[0148] At each analysis time point, rats are euthanized by administration of a lethal dose of pentobarbital (400 mg / kg), after which the animals' eyes are removed and the ocular fluids (vitreous and aqueous humor) are collected and stored at -80°C until analysis.

[0149] Electrotransfer is performed as described in Example 1.

[0150] Test results Ocular fluid samples are collected on days 3 (D+3), 7 (D+7), 14 (D+14), and 21 (D+21) following injection of the plasmid followed by electrotransfer (Day 0). For each of these samples, an ELISA assay is performed to measure the amount of decorin and / or aflibercept present in the sample.

[0151] In this way, mean concentrations are calculated for each group over time.

[0152] The results obtained are: - in Figure 7, which shows the aflibercept concentrations (pg / mL) produced in ocular fluid collected from day 3 to day 21 by constructs according to the invention compared to constructs not according to the invention; and In Figure 8, the concentration (pg / mL) of decorin produced in ocular fluid collected from day -3 to day 21 is shown. is shown.

[0153] Inspection of these figures shows that the plasmid of the invention allows the expression of two therapeutic proteins of interest.

[0154] These experiments therefore demonstrate that application of the construct of the invention, which is of very large size due to the presence of not one but two coding sequences for the protein of interest, allows it to effectively penetrate the target cell and to express the two proteins encoded by the construct at the level of the site of interest.

[0155] Furthermore, as shown above in Example 1, they also demonstrate, quite surprisingly, the ability of the constructs of the invention to produce the proteins they encode more stably over time compared to constructs encoding only one of these proteins.

[0156] Example 3 Furthermore, the inventors have also confirmed the observations made above in experimental protocols using plasmids of the invention different from those used in Examples 1 and 2.

[0157] Plasmid - sequence SEQ ID NO: 11, which encodes decorin of sequence SEQ ID NO: 8, under the control of a CMV-type promoter; and the sequence SEQ ID NO: 2, which encodes aflibercept of the sequence SEQ ID NO: 3, under the control of a CAG-type promoter. A DNA construct of the present invention, designated Plasmid C, containing:

[0158] animal Seven- to eight-week-old Brown Norway rats were used according to the ARVO (Association for Research in Vision and Ophthalmology) protocol. Rats were anesthetized with intramuscular injections of ketamine (40 mg / kg) and xylazine (4 mg / kg) prior to bilateral injection of plasmid (30 μg per eye) or vehicle (10 μL) and electrotransfer on day 0 (DO). Six rats were used for each treatment. Electrotransfer was performed as described in Example 1.

[0159] On day 3, choroidal neovascularization is induced in all animals by laser photocoagulation in several locations of the retina (4-5 laser shocks per eye).

[0160] Test results 14 days after the lesion (D17), the vascular leakage of the neovessels is assessed by fluorescein angiography and the assignment of a score as a function of the severity of the vascular leakage according to the table below.

[0161] Observation Grade Hyperfluorescence 0 Slight hyperfluorescence without increase in intensity or size1 Hyperfluorescence (moderate leakage) with late-stage increase in intensity without size increase2 Hyperfluorescence (severe leakage) with early leakage, with increased size and intensity in the later stages3

[0162] The results obtained are shown in FIG. 10, which in particular shows the proportion of effects with severe leakage (grade 3) as a function of treatment.

[0163] Inspection of this figure shows that the plasmid of the invention appears to result in a 38% reduction in the number of events that exhibited severe neovascular leakage compared to animals receiving vehicle.

[0164] Sequence Listing SEQ ID NO: 1: Nucleotide sequence encoding aflibercept [Table 1]

[0165] SEQ ID NO: 2: Nucleotide sequence encoding aflibercept [Table 2]

[0166] SEQ ID NO: 3: Peptide sequence of aflibercept [Table 3]

[0167] SEQ ID NO: 4: Peptide sequence of the TPA signal peptide [Table 4]

[0168] SEQ ID NO: 5: Nucleotide sequence encoding the TPA signal peptide [Table 5]

[0169] SEQ ID NO: 6: Nucleotide sequence encoding decorin [Table 6]

[0170] SEQ ID NO: 7: Nucleotide sequence encoding decorin [Table 7]

[0171] SEQ ID NO: 8: Peptide sequence of decorin [Table 8]

[0172] SEQ ID NO: 9: Nucleotide sequence encoding decorin [Table 9]

[0173] SEQ ID NO: 10: Nucleotide sequence encoding decorin [Table 10]

[0174] SEQ ID NO: 11: Nucleotide sequence encoding decorin [Table 11]

[0175] SEQ ID NO: 12: Nucleotide sequence encoding decorin [Table 12]

[0176] SEQ ID NO: 13: Peptide sequence of the native signal peptide of decorin [Table 13]

[0177] SEQ ID NO: 14: Nucleotide sequence encoding the native signal peptide of decorin [Table 14]

[0178] SEQ ID NO: 15: Nucleotide sequence encoding transferrin [Table 15]

[0179] SEQ ID NO: 16: Nucleotide sequence encoding transferrin [Table 16]

[0180] SEQ ID NO: 17: Peptide sequence of human transferrin [Table 17]

[0181] SEQ ID NO: 18: Peptide sequence of the native signal peptide of human transferrin [Table 18]

[0182] SEQ ID NO: 19: Nucleotide sequence encoding the native signal peptide of transferrin [Table 19]

[0183] SEQ ID NO: 20: Nucleotide sequence encoding the signal peptide of transferrin [Table 20]

[0184] SEQ ID NO: 21: Nucleotide sequence encoding a fusion protein comprising the extracellular domain of the human receptor for TNFα, p55, connected via a hinge to the constant fragment of human immunoglobulin IgG1 (Peppel et al., J Exp Med, 174: 1483-1489 - Murphy et al., Arch Ophthalmol, 22: 845-851). [Table 21]

[0185] SEQ ID NO: 22: Peptide sequence of a fusion protein comprising the extracellular domain of the human receptor for TNFα, p55, connected via a hinge to the constant fragment of human immunoglobulin IgG1 (Peppel et al., J Exp Med, 174: 1483-1489 - Murphy et al., Arch Ophthalmol, 22: 845-851). [Table 22]

[0186] SEQ ID NO: 23: Peptide sequence of the native signal peptide of the protein of sequence SEQ ID NO: 22 [Table 23]

[0187] SEQ ID NO: 24: Nucleotide sequence encoding the signal peptide of the sequence SEQ ID NO: 23 [Table 24]

[0188] SEQ ID NO: 25: Nucleotide sequence encoding complement factor H [Table 25] TIFF0007766054000026.tif250165 TIFF0007766054000027.tif23165

[0189] SEQ ID NO: 26: Peptide sequence of complement factor H [Table 26]

[0190] SEQ ID NO: 27: Peptide sequence of the native signal peptide of Factor H [Table 27]

[0191] SEQ ID NO: 28: Nucleotide sequence encoding the native signal peptide of Factor H [Table 28]

[0192] SEQ ID NO: 29: Peptide sequence of the signal peptide of the HTLV-1 envelope [Table 29]

[0193] SEQ ID NO: 30: Nucleotide sequence encoding the signal peptide of the HTLV-1 envelope [Table 30]

[0194] SEQ ID NO: 31: Sequence of the replication origin of E. coli R6K plasmid gamma [Table 31]

Claims

1. 1. A DNA construct for use in treating an ocular condition, comprising: the DNA construct is intended for non-viral transfer of nucleic acid into ocular muscle cells of a patient having the ocular condition; The DNA construct is (a) a bacterial or prokaryotic origin of replication; (b) one or more sequences that promote expression of the DNA in the patient's eye; (c) a first nucleotide sequence encoding: a first therapeutic protein, and a signal peptide allowing secretion of said first therapeutic protein, said signal peptide being contiguous with the sequence of the first therapeutic protein at the N-terminus of the first therapeutic protein, (d) a promoter that allows expression of the first therapeutic protein in the patient's eye; (e) a polyadenylation sequence 3' to the first nucleotide sequence; (f) a second nucleotide sequence encoding: a second therapeutic protein that is different from the first therapeutic protein, and a signal peptide allowing secretion of said second therapeutic protein, said signal peptide being contiguous with the sequence of the second therapeutic protein at the N-terminus of the second therapeutic protein, (g) a promoter that allows expression of the second therapeutic protein in the patient's eye; and (h) a polyadenylation sequence 3' to the second nucleotide sequence. characterized in that it comprises: The DNA construct is administered to the patient by injection into the ciliary muscle followed by electrotransfer into the ciliary muscle cells. DNA constructs.

2. 2. The DNA construct of claim 1, wherein the first therapeutic protein is an anti-VEGF protein.

3. The DNA construct described in claim 2, wherein the anti-VEGF protein is selected from the group consisting of S-Flt1, aflibercept, conbercept, and brolucizumab.

4. A DNA construct as described in claim 2, wherein the anti-VEGF type protein is a protein having at least 95% sequence identity to the peptide sequence of SEQ ID NO:

3.

5. The DNA construct described in claim 2, wherein the anti-VEGF protein is aflibercept.

6. The DNA construct of any one of claims 1 to 5, wherein the first therapeutic protein is encoded by a nucleotide sequence having at least 95% sequence identity to the sequence of SEQ ID NO:

1.

7. The DNA construct described in claim 6, wherein the first therapeutic protein is encoded by the nucleotide sequence of SEQ ID NO:

2.

8. (c) the first nucleotide sequence is - a protein having at least 95% sequence identity to the sequence of SEQ ID NO: 3, and - a signal peptide of the peptide sequence SEQ ID NO: 4 The DNA construct according to any one of claims 1 to 7, which encodes

9. The DNA construct of any one of claims 1 to 8, wherein the second therapeutic protein is a protein having at least 95% sequence identity to the sequence of SEQ ID NO:

8.

10. The DNA construct described in claim 9, wherein the second therapeutic protein is decorin.

11. The DNA construct of any one of claims 1 to 10, wherein the second therapeutic protein is encoded by a nucleotide sequence having at least 95% sequence identity to the sequence of SEQ ID NO:

6.

12. The DNA construct described in claim 11, wherein the second therapeutic protein is encoded by a sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:

12.

13. (c) the first nucleotide sequence is - a protein having at least 95% sequence identity to the sequence of SEQ ID NO: 3, and - a signal peptide of the peptide sequence SEQ ID NO: 4 and (f) the second nucleotide sequence is - a protein having at least 95% sequence identity to the sequence of SEQ ID NO: 8, and - a signal peptide of the peptide sequence SEQ ID NO: 13 Encode A DNA construct according to any one of claims 1 to 12.

14. The DNA construct according to any one of claims 1 to 13, wherein the origin of replication is a bacterial origin of replication.

15. A DNA construct according to any one of claims 1 to 14, characterized in that it is in circular form.

16. A DNA construct according to any one of claims 1 to 14, characterized in that the construct is naked DNA.

17. The DNA construct according to any one of claims 1 to 16, wherein the ocular pathology is retinal degeneration.

18. The DNA construct described in claim 17, characterized in that the ocular pathology is a retinal degeneration selected from the group consisting of exudative or atrophic age-related macular degeneration (ARMD); diabetic retinopathy (DR); retinal vein occlusion; myopic choroidal neovascularization (CNV); uveitis; retinitis pigmentosa and glaucoma.

19. A DNA construct for the non-viral introduction of nucleic acid into ocular muscle cells of a patient for the treatment of an ocular condition, (a) a bacterial or prokaryotic origin of replication; (b) one or more sequences that promote expression of the DNA in the patient's eye; (c) a first nucleotide sequence encoding: a first therapeutic protein, which is a protein having at least 95% sequence identity to the sequence of SEQ ID NO: 3, and a signal peptide allowing secretion of said first therapeutic protein, said signal peptide being contiguous with the sequence of the first therapeutic protein at the N-terminus of the first therapeutic protein, (d) a promoter that allows expression of the first therapeutic protein in the patient's eye; (e) a polyadenylation sequence 3' to the first nucleotide sequence; (f) a second nucleotide sequence encoding: a second therapeutic protein, which is a protein having at least 95% sequence identity to the sequence of SEQ ID NO: 8, and a signal peptide allowing secretion of said second therapeutic protein, said signal peptide being contiguous with the sequence of the second therapeutic protein at the N-terminus of the second therapeutic protein, (g) a promoter that allows expression of the second therapeutic protein in the patient's eye; and (h) a polyadenylation sequence 3' to the second nucleotide sequence. characterized in that it comprises DNA constructs. (c) the first nucleotide sequence is - a nucleotide sequence encoding aflibercept, and - the nucleotide sequence of SEQ ID NO: 5 encoding the signal peptide and (f) the second nucleotide sequence is - a nucleotide sequence encoding decorin; and - the nucleotide sequence of SEQ ID NO: 14 encoding the signal peptide Including, 20. The DNA construct of claim 19.

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