Patent Application
20240189448
The present invention relates to a new DNA construct as well as the application thereof in the treatment of ocular pathologies by nonviral gene therapy. A method according to the invention relates more particularly to said DNA construct making possible the targeted intraocular production of two therapeutic proteins during a period that may last for up to several months. The DNA construct and its use according to the invention are more particularly suitable for treating pathologies of the retina by means of an injection of the DNA construct into the ciliary muscle followed by electrotransfer for long-lasting intraocular production of the therapeutic proteins of interest.
Blindness related to metabolic and inflammatory diseases or to age is increasing considerably and poses a more and more significant social problem in Europe and throughout the world in terms of public health. The main causes of blindness are related to pathologies of the retina, including age-related macular degeneration (ARMD), diabetic retinopathies (DR), uveitis, glaucoma, retinitis pigmentosa, hemorrhages following eye injury and retinal detachment. Age-related macular degeneration (ARMD) leading to blindness has a prevalence of the order of 8.7%, and affects nearly 26% of the population aged 50 years and over. ARMD is becoming a major problem of public and social health on account of the increasing age of the population. The continual major increase in the incidence of diabetes is the major cause of diabetic retinopathies (DR), which in their turn are becoming a priority of public health and scientific research. The statistics show that type 2 diabetes will affect, from now to 2030, more than 4.5% of the population, nearly 30% of whom will suffer diabetic retinopathy. Finally, uveitis represents a group of inflammatory ocular diseases whose prevalence is estimated at 1/1000 and incidence at 0.5/1000. Uveitis is responsible for 10% of cases of blindness and therefore, although rarer than the diseases mentioned above, has a major social and economic impact in young patients of working age. Glaucoma is the second cause of irreversible blindness in the world. The number of people with glaucoma in the world is expected to increase from 76 million in 2020 to 111 million in 2040. Glaucoma is characterized by abnormal intraocular pressure inducing a progressive optical neuropathy characterized by degeneration of the retinal ganglion cells and a loss of visual field. The intraocular pressure is currently the only risk factor for which there are treatments. However, the glaucomatous damage persists in nearly 50% of patients, despite a decrease in intraocular pressure. Retinitis pigmentosa represents a clinically and genetically heterogeneous group of hereditary disorders of the retina characterized by a progressive loss of photoreceptors at the periphery of the retina, which then progresses to the macula. The visual deficiency is generally manifested by night blindness and a progressive loss of visual field. Its prevalence is from 1/3000 to 1/5000. More than 50 genes responsible for retinitis pigmentosa have been identified to date.
For treating certain of these pathologies, intraocular, or even intravitreal, injections of therapeutic agents have been developed. In 2006, the first therapeutic proteins of the anti-VEGF type (VEGF: Vascular Endothelial Growth Factor) were administered by the intraocular route for treating choroid neovascularization in ARMD. We may mention in particular, as an example of a protein of the anti-VEGF type, Lucentis® (ranibizumab), which has been given marketing authorization for treating choroid neovascularization in ARMD and diabetic macular edema. Intraocular injections of therapeutic proteins, and in particular of recombinant proteins, have since become common for treating macular edemas in ARMD, diabetic retinopathy and venous occlusions.
In order to ensure a continuing effect on the ocular pathologies, the anti-VEGFs described above are administered once a month or at best once every two months depending on the patients. It is therefore necessary to monitor each patient in order to determine the frequency of administration of the anti-VEGFs so that the treatment is fully effective. This monitoring generates considerable stress for the patients, careers and nursing staff, which most often results in treatment that is suboptimal and ineffective in the long term (Ciulla 2020, Ophthalmology Retina 2020; 4: 19-30). Moreover, this method of treatment induces changes in the level of therapeutic protein in the patient's ocular sphere, namely a high concentration at the time of injection (a peak) and a concentration that gradually diminishes and tends toward zero until the next injection. Therefore the concentration of therapeutic protein is irregular and is not optimal for the entire duration of the treatment. Moreover, the risk of side effects associated with intravitreal administration increases with repeated administration (Schargus 2020, Clinical Ophthalmology 2020: 14 897-904).
Among the ocular pathologies, uveitis is defined as an inflammatory process that affects the iris, ciliary body or the choroid of the patient's eye, these three elements forming the uvea. It is a general term that covers several different pathologies whose causes remain unknown but are generally of two types: infectious uveitides and noninfectious uveitides. A distinction is made between anterior uveitis, which affects the iris or the ciliary body and is the commonest of the uveitides in countries in the west; intermediate uveitis, which affects the anterior vitreous body; and posterior uveitis, which affects the choroid and the retina. Noninfectious uveitis often has an autoimmune component. Acute anterior uveitis associated with the HLA-B27 antigen represents the primary cause of uveitis (Rothova et al., Br J Ophthalmol. 1992; 76:137-41). Moreover, anterior uveitis is found associated with many rheumatologic diseases such as sarcoidosis. Posterior uveitis of noninfectious cause is most often associated with Behçet disease, Vogt Koyanagi Harada disease, birdshot chorioretinopathy, etc.
Treatment with corticoids by the oral and/or topical route is widely used in the treatment of noninfectious posterior uveitis. Moreover, for the most refractory pathologies, immunosuppressants may be added to the treatment to increase the anti-inflammatory effects of the corticosteroids. This relates in particular but not exclusively to ciclosporin, methotrexate, azathioprine, mycophenolate mofetil, tacrolimus and chlorambucil. For about the last ten years, treatments using anti-TNF alpha antibodies could also be used, including Humira® (Adalimumab), recently approved for treating noninfectious uveitis with inflammation of the back of the eye.
Clinical trials into the use of the following therapeutic compounds: ciclosporin A, rapamycin, tacrolimus, and anti-TNF alpha, have been conducted to evaluate the efficacy and safety of these compounds and thus be able to treat autoimmune ocular pathologies such as Behçet disease. However, it has been shown that systemic administration of these therapeutic compounds leads in the long term to many side effects and that for some of them, their local administration provides little efficacy or poor tolerance by the patient.
The methods of treatment described above therefore have several drawbacks. The therapeutic compounds administered by the systemic and topical route lead to numerous side effects. Recombinant proteins administered by intravitreal injections must be administered frequently, with large fluctuations in concentrations between each administration. Repetition of these injections is still extremely onerous and stressful for the patients and may also cause effects (increase in intraocular pressure, intraocular inflammation, endophthalmitis, cataract, etc.). Thus, in the end many patients abandon their treatment.
In order to overcome this problem, the inventors have previously proposed, as illustrated in particular in application FR3031112, to reduce the number and/or frequency of surgical interventions, and therefore reduce the invasive aspect of the intraocular injections, while ensuring a stable and constant production of a therapeutic protein for several months through application of a DNA construct intended for nonviral transfer of nucleic acids into the muscle cells of a patient's ocular sphere. This construct comprises an origin of replication, a promoter allowing expression of the DNA in the patient's ocular sphere, one or more sequences promoting the expression of DNA in the patient's ocular sphere, and a polynucleotide coding for a therapeutic protein selected for its activity in the treatment of ocular pathologies, said construct being delivered into the ocular sphere by direct injection into the ciliary muscle followed by electrotransfer.
Treatment of the ocular pathologies mentioned above may, however, require the application of two active therapeutic ingredients, of a second compound for potentiating the efficacy of an active therapeutic ingredient, or of a compound consisting of 2 peptide subunits.
In the context of the method mentioned above, it could thus be envisaged to administer to the patient a composition comprising two types of DNA constructs, differing in that a first type allows expression of the first molecule of interest while a second type allows expression of the second molecule of interest. Such a method would not, however, be ideal, (i) in that it would require the development of two products, which would lead to an increase in the costs of development and production, and to the need to evaluate the activity and safety of each of the products taken separately and in combination, (ii) in that it would impose a considerable constraint in terms of dosage, halving the maximum dose of each of the two active therapeutic ingredients that may be administered, and finally (iii) in that it would not allow complete control of the quantity of each of these constructs penetrating the targeted cells, which would lead in consequence to uncertainty regarding the proportions of these molecules of interest expressed at the level of the eye. Such constraints and uncertainties are undesirable in the context of the treatment of a pathology.
The inventors consequently propose, in the context of the present invention, to employ a DNA construct comprising the sequences coding for the two proteins of interest.
The present invention therefore relates firstly to a DNA construct for use thereof in the treatment of an ocular pathology,
In fact, against all expectations, the significant increase in the size of the DNA construct, a consequence of introducing not one but two sequences coding for the molecules of interest, does not have a negative effect on its capacity to penetrate the targeted cells in the context of a method such as indicated above, comprising not only direct injection of said construct into the ciliary muscle, but also a step of electrotransfer.
Consequently, a method and a construct according to the invention allow advantageously, and against all expectations:
According to one embodiment, the first therapeutic protein of a DNA construct according to the invention is a protein of the anti-VEGF type, in particular selected from the group consisting of S-Flt1, aflibercept, conbercept, brolucizumab, and in particular of a protein having at least 85% sequence identity with the peptide sequence SEQ ID NO: 3, this protein more particularly being aflibercept.
According to one embodiment, the first therapeutic protein is encoded by a nucleotide sequence having at least 75% sequence identity with the sequence SEQ ID NO: 1, and is more particularly encoded by the nucleotide sequence SEQ ID NO: 2.
According to one embodiment, the second therapeutic protein of a DNA construct according to the invention is a protein having at least 85% sequence identity with the sequence SEQ ID NO: 8, this protein more particularly being decorin.
According to one embodiment, the second therapeutic protein is encoded by a nucleotide sequence having at least 70% sequence identity with the sequence SEQ ID NO: 6, and in particular by a sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, more particularly consisting of the sequence SEQ ID NO: 7 and the sequence SEQ ID NO: 1, and in particular the sequence SEQ ID NO: 11.
According to one embodiment, a DNA construct for use thereof according to the invention is such that:
According to one embodiment, the origin of replication of a DNA construct such as mentioned above is bacterial, and is in particular an origin of replication derived from the natural plasmid R6K of Escherichia coli, in particular the origin of replication R6K gamma of the natural plasmid R6k of Escherichia coli, in particular of sequence SEQ ID NO: 31.
The DNA construct according to the invention may be of linear or circular shape, in particular of circular shape. In a particular embodiment, the DNA construct according to the invention is a circular plasmid.
In a particular embodiment, the DNA construct is a naked DNA construct.
According to one embodiment, a DNA construct for use thereof as mentioned above is characterized in that the ocular pathology is a retinal degeneration, in particular a retinal degeneration selected from the group consisting of wet or dry age-related macular degeneration (ARMD); diabetic retinopathies (DR); a retinal venous occlusion, in particular a central retinal vein occlusion (CRVO) or a branch retinal vein occlusion (BRVO); a myopic choroid neovascularization (CNV); a uveitis, in particular a noninfectious uveitis; a retinitis pigmentosa and a glaucoma, and more particularly in that the retinal degeneration is selected from the group consisting of age-related macular degeneration (ARMD), in particular the (wet) neovascular form of ARMD; a decline of visual acuity due to diabetic macular edema (DME); a retinal venous occlusion, in particular a central retinal vein occlusion (CRVO) or a branch retinal vein occlusion (BRVO); and a myopic choroid neovascularization (CNV).
The present invention further relates to a DNA construct intended for the nonviral transfer of nucleic acids into the muscle cells of a patient's ocular sphere for treating ocular pathologies, characterized in that it comprises:
In particular, the DNA construct according to the invention is such that:
In the context of the present text, the terms “treat” and “treatment” associated with an ocular pathology denote a decrease, or even an interruption of said pathology.
The term “patient” as used in the present text preferably denotes a mammal, including a nonhuman mammal, and more particularly a human being.
The terms “first nucleotide sequence” and “second nucleotide sequence” are used in the present text in order to allow, during reading of the latter, to make a clear distinction between these two sequences and the proteins that they encode.
Said nucleotide sequences correspond to expression cassettes, each of these cassettes being as defined hereunder.
These terms “first nucleotide sequence” and “second nucleotide sequence” do not, however, aim to indicate the order in which these sequences/expression cassettes are present in a construct according to the invention. Thus, according to one embodiment, in the sense of reading a construct according to the invention, the first nucleotide sequence may be present before the second nucleotide sequence. In another embodiment, in the sense of reading a construct according to the invention, the second nucleotide sequence may be present before the first nucleotide sequence.
In accordance with what is indicated above, the “first nucleotide sequence” comprises a sequence coding for a first therapeutic protein and a sequence coding for a signal peptide, these sequences being, within the “first nucleotide sequence”, in the order as specifically indicated relative to one another, namely that the sequence coding for the signal peptide is such that the signal peptide is at N-terminal of the first therapeutic protein, i.e. the sequence coding for the signal peptide is at 5′ of the sequence coding for the first therapeutic protein. Moreover, in accordance with what is indicated above, the “second nucleotide sequence” comprises a sequence coding for a second therapeutic protein and a sequence coding for a signal peptide, these sequences being, within the “second nucleotide sequence”, in the order as specifically indicated relative to one another, namely that the sequence coding for the signal peptide is such that the signal peptide is at N-terminal of the second therapeutic protein, i.e. the sequence coding for the signal peptide is at 5′ of the sequence coding for the second therapeutic protein.
The “percentage identity” between two amino acid or nucleic acid sequences, in the sense of the present invention, is determined by comparing the two optimally aligned sequences, through a comparison window.
The part of the nucleotide sequence in the comparison window may thus comprise additions or deletions (for example “gaps”) relative to the reference sequence (which does not comprise these additions or these deletions) so as to obtain an optimal alignment between the two sequences.
The percentage identity is calculated by determining the number of positions at which an identical amino acid (or an identical nucleic base) is observed for the two sequences compared, then dividing the number of positions at which there is identity between the two amino acids (or between the two nucleic bases) by the total number of positions in the comparison window, and then multiplying the percentage result in order to obtain the percentage of amino acid identity (or nucleotide identity) of the two sequences between them.
The optimal alignment of the sequences for comparison may be carried out by computer using known algorithms.
Totally preferably, the percentage sequence identity is determined using the CLUSTAL W software (version 1.82), the parameters being fixed as follows: (1) CPU MODE=ClustalW mp; (2) ALIGNMENT=“full”; (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) PAIRGAP=“default”; (11) PHYLOGENETIC TREE/TREE TYPE=“none”; (12) MATRIX=“default”; (13) GAP OPEN=“default”; (14) END GAPS=“default”; (15) GAP EXTENSION=“default”; (16) GAP DISTANCES=“default”; (17) TREE TYPE=“cladogram” and (18) TREE GAP DISTANCES=“hide”.
In the sense of the invention, an amino acid sequence having for example at least 80% amino acid identity with a reference amino acid sequence includes the amino acid sequences 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 identity with said reference sequence.
In the sense of the invention, a nucleotide sequence having for example at least 80% nucleotide identity with a reference nucleotide sequence includes the nucleotide sequences 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 with said reference sequence.
As mentioned above, the present invention relates firstly to a DNA construct for use thereof in the treatment of an ocular pathology.
This construct is intended for the nonviral transfer of nucleic acids into the muscle cells of the ocular sphere of a patient with said ocular pathology.
Moreover, a DNA construct according to the present invention is characterized in that it comprises (a) a bacterial or prokaryotic origin of replication.
According to a particular embodiment, said origin of replication is in particular bacterial and may for example be an origin of replication of the Escherichia coli type, more particularly selected from the group consisting of an origin of replication derived from the natural plasmid R6K of Escherichia coli, in particular the origin of replication R6K gamma of the natural plasmid R6k of Escherichia coli; and the origin of replication pUC OriC.
The origins of replication derived from the natural plasmid R6K of Escherichia coli are in particular defined in patent EP1366176B2.
Moreover, a DNA construct according to the invention is characterized in that it comprises (b) one or more sequences promoting expression of the DNA in the patient's ocular sphere. Such sequences promoting the expression of DNA are familiar to a person skilled in the art, such as for example the sequences of the enhancer type, also called amplifier or activator sequences. We may mention for example the enhancer sequences derived from cytomegalovirus (CMV) and/or the tumoral virus with simian DNA SV40.
Moreover, a DNA construct according to the invention is also characterized in that it comprises (c) a first nucleotide sequence coding in particular for a first therapeutic protein as well as (f) a second nucleotide sequence coding in particular for a second therapeutic protein, different than the first therapeutic protein.
According to a particular embodiment, a DNA construct according to the invention only comprises two coding sequences for therapeutic proteins, i.e. only comprises two expression cassettes, each of these expression cassettes comprising one of the two coding sequences for a therapeutic protein.
These first and second therapeutic proteins may in particular be selected from proteins known for their effect on ocular pathologies.
The effects of these two proteins may be additional or complementary. Moreover, one of these two proteins may have a potentiating effect on the therapeutic activity of the other protein produced starting from the DNA construct according to the invention.
In particular, the first and the second therapeutic proteins, different than one another, may for example be selected from the group consisting of:
In particular, (i) a protein having at least 85% sequence identity with the sequence SEQ ID NO: 17 comprises a protein 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 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and 100% sequence identity with the sequence SEQ ID NO: 17. In particular, this protein is more particularly transferrin (i.e. a protein having 100% sequence identity with the sequence SEQ ID NO: 17).
In particular, (ii) a protein having at least 85% sequence identity with the sequence SEQ ID NO: 8 comprises a protein 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 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and 100% sequence identity with the sequence SEQ ID NO: 8. In particular, this protein is more particularly decorin (i.e. a protein having 100% sequence identity with the sequence SEQ ID NO: 8).
In particular, (iii) a protein having at least 85% sequence identity with the fusion protein of sequence SEQ ID NO: 22 comprises a protein 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 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and 100% sequence identity with the sequence SEQ ID NO: 22. In particular, this protein is more particularly the fusion protein comprising the extracellular domain of the human receptor p55 to TNF alpha coupled via a hinge to the constant fragment of human immunoglobulin IgG1 of sequence SEQ ID NO: 22.
In particular, (iv) a protein having at least 85% sequence identity with the sequence SEQ ID NO: 3 comprises a protein 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 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and 100% sequence identity with the sequence SEQ ID NO: 3. In particular, this protein is more particularly aflibercept (i.e. a protein having 100% sequence identity with the sequence SEQ ID NO: 3).
In particular, (vi) a protein having at least 85% sequence identity with the sequence SEQ ID NO: 26 comprises a protein 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 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and 100% sequence identity with the sequence SEQ ID NO: 26. In particular, this protein is more particularly complement factor H (i.e. a protein having 100% sequence identity with the sequence SEQ ID NO: 26).
In particular, the coding sequences for the first and second therapeutic proteins, different than one another, may for example be selected from the group consisting of:
“Sequences coding for the first and the second therapeutic proteins” is not to be understood as “first nucleotide sequence” and “second nucleotide sequence” as indicated previously, but rather the sequences that are present within the first nucleotide sequence and within the second nucleotide sequence according to the invention, and which code specifically for the first and the second therapeutic proteins.
According to one embodiment, the first or the second therapeutic protein of a DNA construct according to the invention is a protein having at least 85% sequence identity with the sequence SEQ ID NO: 17, this protein more particularly being transferrin.
In particular, a DNA construct according to the invention may be characterized in that the first nucleotide sequence or the second nucleotide sequence codes for:
Thus, one of the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention may in particular be a nucleotide sequence coding for transferrin, and in particular a sequence having at least 75% sequence identity with the sequence SEQ ID NO: 15, and is in particular the sequence SEQ ID NO: 16.
In particular, a DNA construct according to the invention may be characterized in that the first nucleotide sequence or the second nucleotide sequence comprises:
According to one embodiment, the first and the second therapeutic proteins encoded by a DNA construct according to the invention are respectively:
Thus, according to one embodiment, the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
Inflammation and oxidative stress are important components of retinal degeneration such as ARMD) or glaucomatous neuropathy following an increase in intraocular pressure caused by glaucoma. In particular, an increase in the intraocular concentrations of TNF alpha is observed in glaucomatous eyes (Tezel et al., 2001, Invest Ophthalmol Vis Sci. 2001 July; 42(8): 1787-94) and injection of TNF-alpha in the eye of a rodent induces an axonal degeneration of the optic nerve and programmed death of the ganglion cells of the retina (Kitaoka 2006, Invest Ophthalmol Vis Sci. 2006; 47: 1448-1457). An increase in expression of the genes regulating the level of iron has also been observed in glaucomatous eyes suggesting that the oxidative stress induced by iron may play a role in the pathogenesis of glaucoma (Farkas et al., 2004). The administration of an anti-TNF and an iron chelating agent, such as transferrin, makes it possible advantageously to reduce both the inflammation and the oxidative stress mediated by iron.
In particular, a DNA construct according to the invention may be characterized in that:
In particular, a DNA construct according to the invention may be characterized in that:
According to another embodiment, the first and the second therapeutic proteins encoded by a DNA construct according to the invention are respectively:
Thus, according to one embodiment, the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
Glaucoma, in particular primary open-angle glaucoma, is characterized by an increase in intraocular pressure following fibrosis of the trabecular network, and by loss of ganglion cells of the retina and degeneration of the optic nerve. The current treatments for glaucoma reduce the intraocular pressure but do not allow the evolution of neurodegeneration to be halted. The administration of an agent with neuroprotective action, such as an anti-TNF or transferrin, advantageously makes it possible to potentiate the effects of an antifibrotic such as decorin, and both reduce the intraocular pressure and protect the retina and the optic nerve from degeneration.
In particular, a DNA construct according to the invention may be characterized in that:
In particular, a DNA construct according to the invention may be characterized in that:
According to one embodiment, the first or the second therapeutic protein of a DNA construct according to the invention is a protein of the anti-VEGF type, in particular a protein having at least 85% sequence identity with the sequence SEQ ID NO: 3, this protein more particularly being aflibercept.
In particular, a DNA construct according to the invention may be characterized in that the first nucleotide sequence or the second nucleotide sequence codes for:
Thus, one of the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention may in particular be a nucleotide sequence coding for a protein of the anti-VEGF type, such as S-Flt1, aflibercept, conbercept, brolucizumab, and in particular a sequence coding for aflibercept, more particularly a sequence having at least 75% sequence identity with the sequence SEQ ID NO: 1, and is in particular the sequence SEQ ID NO: 2.
In particular, a DNA construct according to the invention may be characterized in that the first nucleotide sequence or the second nucleotide sequence comprises:
According to another embodiment, the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
Thus, according to one embodiment, the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
The presence of the antifibrotic active ingredient advantageously makes it possible to potentiate the effects of the anti-VEGF, thus improving the efficacy of this compound in the treatment of the target ocular pathologies. In particular, it was observed that even in patients receiving injections of anti-VEGF at optimal intervals, development of a subretinal fibrosis appears with time in more than half of the patients, reducing the efficacy of the anti-VEGFs with the passage of time (Daniel et al. 2014, Ophthalmology 121, 656-666). Moreover, development of subretinal fibrosis was identified as a cause of poor therapeutic response to anti-VEGFs in ARMD patients not responding to anti-VEGFs (Cohen et al. 2012, Retina 32, 1480-1485).
In particular, a DNA construct according to the invention may be characterized in that:
In particular, a DNA construct according to the invention may be characterized in that:
In particular, a DNA construct according to the invention may be characterized in that:
In particular, a DNA construct according to the invention may be characterized in that:
According to another embodiment, the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
Thus, according to one embodiment, the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
The presence of the anti-INF-alpha active ingredient makes it possible advantageously to potentiate the effects of the anti-VEGF, thus improving the efficacy of this compound in the treatment of the target ocular pathologies. In particular, it is well known that VEGF induces retinal permeability but inflammatory agents, such as TNF-alpha, may also lead to vascular permeability, in particular in patients who do not respond to anti-VEGF treatment, such as may be observed in patients with diabetic retinopathy (Arias L. et al.; Retina 2010, 30: 1601e1608 and Sfikakis et al.; Diabetes Care 2010, 33: 1523e1528). Recent tests suggest that VEGF and TNF-alpha induce permeability by different mechanisms.
In particular, a DNA construct according to the invention may be characterized in that:
In particular, a DNA construct according to the invention may be characterized in that:
According to another embodiment, the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
Thus, according to one embodiment, the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
Activation of the complement alternative pathway is an important component of ARMD. This activation leads to formation of the membrane attack complex, recruitment of macrophages, and induction of inflammation with production of cytokines involved in the inflammasome. Complement factor H is involved in regulating the autoactivation of complement. Several polymorphic variants in the gene coding for CFH, affecting the function of the protein, confer strong susceptibility for developing the two forms of ARMD, wet and dry. Conversely, inhibition of the alternative pathway by intraocular injection of CFH reduces neovascularization in animal models of choroid neovascularization. Thus, administration of an active ingredient regulating the activation of complement and of an anti-VEGF, such as aflibercept, advantageously makes it possible to reduce both the neovascularization and the inflammation associated with ARMD.
In particular, a DNA construct according to the invention may be characterized in that:
In particular, a DNA construct according to the invention may be characterized in that:
Moreover, a DNA construct according to the invention is characterized in that the first nucleotide sequence also codes for a signal peptide allowing secretion of the first therapeutic protein.
A signal peptide of this kind is familiar to a person skilled in the art. This signal peptide may for example be human tissue plasminogen activator (tPA) of peptide sequence SEQ ID NO: 4 (and which may for example be encoded by the nucleotide sequence SEQ ID NO: 5) or the signal peptide of HTLV-1 Env of peptide sequence SEQ ID NO: 29 (and which may for example be encoded by the nucleotide sequence SEQ ID NO: 30). It may also be the native peptide signal of the therapeutic protein in question, such as for example:
As mentioned above, this signal peptide is contiguous with the first therapeutic protein, i.e. it is fused directly at the N-terminal of the first therapeutic protein, and therefore the sequence coding for the signal peptide is at 5′ of the sequence coding for the first therapeutic protein.
Moreover, a DNA construct according to the invention is characterized in that the second nucleotide sequence also codes for a signal peptide allowing secretion of the second therapeutic protein.
This signal peptide may for example be human tissue plasminogen activator (tPA) of peptide sequence SEQ ID NO: 4 (and which may for example be encoded by the nucleotide sequence SEQ ID NO: 5) or the signal peptide of HTLV-1 Env of peptide sequence SEQ ID NO: 29 (and which may for example be encoded by the nucleotide sequence SEQ ID NO: 30). It may also be the native peptide signal of the therapeutic protein in question, such as for example:
This signal peptide may be identical to or different than the signal peptide encoded by the first nucleotide sequence, and is preferably different than the signal peptide encoded by the first nucleotide sequence.
As mentioned above, this signal peptide is contiguous with the second therapeutic protein, i.e. it is fused directly at the N-terminal of the second therapeutic protein, and therefore the sequence coding for the signal peptide is at 5′ of the sequence coding for the second therapeutic protein.
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 of one another, 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.
According to one embodiment, the sequence coding for the signal peptide encoded by the first nucleotide sequence and the sequence coding for the signal peptide encoded by the second nucleotide sequence are, independently of one another, selected from the group consisting of the nucleotide sequences 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.
Moreover, a DNA construct according to the invention is also characterized in that it comprises (d) a promoter allowing expression of the first therapeutic protein of a construct according to the invention as well as (g) a promoter allowing expression of the second therapeutic protein of a construct according to the invention.
These promoters may be identical or different. According to a particular embodiment, these two promoters are different than one another.
Said promoters may for example be promoters of the CAG or CMV type.
Moreover, a DNA construct according to the invention is characterized in that it comprises (e) a polyadenylation sequence at 3′ of the first nucleotide sequence and (h) a polyadenylation sequence at 3′ of the second nucleotide sequence.
A polyadenylation sequence contains in particular a conserved motif of sequence AATAAA, familiar to a person skilled in the art.
These two polyadenylation sequences may be identical to or different than one another.
According to one embodiment, they are different than one another.
These polyadenylation sequences may for example be polyadenylation sequences of the type RBG (Rabbit Beta Globin), or BGH (Bovine Growth Hormone).
Finally, as mentioned above, a DNA construct according to the invention is administered to the patient by injection into a ciliary muscle and then electrotransfer into the cells of the ciliary muscle.
In a particular embodiment, the DNA construct according to the invention is of circular shape.
In one embodiment of the invention, the DNA construct is a naked DNA construct.
According to one embodiment of the invention, the DNA construct according to the invention is a naked DNA construct of circular shape.
In one embodiment of the invention, the DNA construct according to the invention is a naked DNA construct of circular shape in which the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
In one embodiment of the invention, the DNA construct according to the invention is a naked DNA construct of circular shape in which the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
In one embodiment, the DNA construct according to the invention is a naked DNA construct of circular shape in which the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
In one embodiment according to the invention, the DNA construct according to the invention is a naked DNA construct of circular shape in which the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
In one embodiment according to the invention, the DNA construct according to the invention is a naked DNA construct of circular shape in which the sequences coding for the first and the second therapeutic proteins of a DNA construct according to the invention are respectively:
The present invention also relates to a DNA construct intended for the nonviral transfer of nucleic acids into the muscle cells of a patient's ocular sphere for treating ocular pathologies, characterized in that it comprises:
A DNA construct as defined above is in particular intended for the treatment of ocular pathologies.
An ocular pathology according to the present invention is a retinal degeneration.
This retinal degeneration may in particular be selected from the group consisting of wet or dry age-related macular degeneration (ARMD); diabetic retinopathies (DR); a retinal venous occlusion, in particular a central retinal vein occlusion (CRVO) or a branch retinal vein occlusion (BRVO); a myopic choroid neovascularization (CNV); a uveitis, in particular a noninfectious uveitis; a retinitis pigmentosa and a glaucoma.
Diabetic retinopathy is intended in particular to denote a decline of visual acuity due to diabetic macular edema (DME), an intravitreal hemorrhage, a retinal detachment, or a neovascular glaucoma.
According to a particular embodiment, and especially when a construct according to the invention used for treating an ocular pathology comprises, as first and second therapeutic proteins, aflibercept and decorin as defined above, said ocular pathology is a retinal degeneration that may more particularly be selected from the group consisting of age-related macular degeneration (ARMD), in particular the wet form; diabetic retinopathies (DR); a retinal venous occlusion, in particular a central retinal vein occlusion (CRVO) or a branch retinal vein occlusion (BRVO); and a myopic choroid neovascularization (CNV);
and more particularly may be selected from the group consisting of age-related macular degeneration (ARMD), in particular the (wet) neovascular form of ARMD; a decline of visual acuity due to diabetic macular edema (DME); a retinal venous occlusion, in particular a central retinal vein occlusion (CRVO) or a branch retinal vein occlusion (BRVO); and a myopic choroid neovascularization (CNV).
Diabetic retinopathy is intended in particular to denote a decline of visual acuity due to diabetic macular edema (DME) and the formation of neovasculature observed in the proliferative form of diabetic retinopathy.
As stated above, a DNA construct according to the invention is injected into an ocular muscle, the ciliary muscle, where it is submitted to electrotransfer. The known technique of nonviral gene therapy used in the present invention is injection of the DNA construct into an ocular muscle and then electrotransfer to induce a transient permeabilization of the cells of the ciliary muscle and migration of the DNA to optimize transfection of the DNA construct. This technique of electrotransfer of DNA (also called electroporation or electropermeabilization) is easy to apply, reliable and safe for the patient. In contrast to viral vectors, electrotransfer of DNA does not induce an immune response and allows long-term expression of the genes thus introduced. Moreover, studies conducted on lentiviruses and retroviruses show that the latter are liable to induce insertion mutations during their integration in the host genome. The DNA constructs described here do not have drawbacks of this type, are easy to produce and manipulate and do not induce an immune response, thus making them perfectly suitable for gene therapy of patients, especially human patients.
According to the present invention, a DNA construct is injected into the ciliary muscle as the latter is capable of producing the proteins homogenously and continually and, owing to its position, promotes diffusion of these proteins in the whole ocular sphere (Blocquel et al. “Plasmid electrotransfer of eye ciliary muscle: principles and therapeutic efficacy using hTNF-alpha soluble receptor in uveitis”, FASEB J. 2006 February; 20(2): 389-91). Smooth muscle cells have a low renewal rate and are well distributed on either side of the lens. The quantity of protein to be produced is proportional to the surface area of the muscle transfected (Touchard “The ciliary smooth muscle electrotransfer: basic principles and potential for sustained intraocular production of therapeutic proteins”, J Gene Med. 2010 November; 12(11): 904-19). Thus, production of the proteins of interest according to the present invention, and in particular of therapeutic proteins as described above, will be homogeneous and constant in the whole ocular sphere and will be limited to this ocular sphere. We may mention, as an example, the method of electrotransfer described in patent application EP2266656, which relates to a method of injection of a composition that may contain DNA at the level of the tissues of the ciliary body and/or extraocular muscle tissues.
The ciliary muscle forms part of the ciliary body, near the limbus and just behind the sclera. Injection of a DNA construct according to the invention into the latter is therefore very slightly invasive in contrast to subretinal injections and therefore advantageously constitutes the injection site of the DNA construct according to the invention.
According to another aspect, the present invention also relates to the use of a DNA construct for treating an ocular pathology, said DNA construct being intended for the nonviral transfer of nucleic acids into the muscle cells of the ocular sphere of a patient with said ocular pathology; said DNA construct being characterized in that it comprises:
According to another aspect, the present invention also relates to a method of treating an ocular pathology, comprising the administration of a DNA construct to a patient by injection into a ciliary muscle and then electrotransfer into the cells of the ciliary muscle, said DNA construct being intended for the nonviral transfer of nucleic acids into the muscle cells of the ocular sphere of a patient with said ocular pathology;
said DNA construct being characterized in that it comprises:
The invention is described below in more detail by means of the following examples, which are presented only for purposes of illustration.
The inventors have demonstrated the expression of two therapeutic proteins encoded in a DNA construct according to the invention (plasmid) in the vitreous body of the eye of rats after electrotransfer of this DNA construct in the ciliary muscle.
A DNA construct according to the invention, designated plasmid A, comprising:
Long Evans rats aged 7 weeks are used in accordance with the provisions of the ARVO protocol (Association for Research in Vision and Ophthalmology). The rats are anesthetized by intramuscular injection of a dose of ketamine (40 mg/kg) and xylazine (4 mg/kg) before bilateral injection of the plasmid (30 μg/eye) and electrotransfer on day 0 (D0). Six rats are used for each of the analysis times (D3, D7, D14, D21, and D30).
At each analysis time, the rats are euthanized by administration of a lethal dose of pentobarbital (400 mg/kg) and then the animals are enucleated and the ocular fluids (vitreous humor and aqueous humor) are taken and stored at −80° C. until analysis.
Electrotransfer is carried out 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), modifying the route of injection 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 November; 12(11): 904-19). The plasmids are injected at a rate of 30 μg in 10 μL of Tris-EDTA NaCl solution, in the ciliary muscle of the animals using a suitable syringe.
The electrical impulses are administered by means of a special iridium/platinum electrode of 250 μm diameter. This internal electrode is introduced into the existing transscleral tunnel. The external electrode is a thin sheet of stainless steel curved to match the shape of the eye and placed at the level of the limbus opposite the internal electrode. Electrotransfer is carried out at a rate of 8 unipolar square electrical impulses (200V/cm, 10 ms, 5 Hz) generated by an electroporator similar to that described in Touchard et al. (J Gene Med. 2010 November; 12(11): 904-19).
Samples of ocular fluids are taken at 3 days (D+3), 7 days (D+7), 14 days (D+14), 21 days (D+21) and 30 days (D+30) after injection of plasmid followed by electrotransfer (D0). For each of these samples, an ELISA assay is carried out in order to measure the amount of human transferrin and/or of the anti-TNF-alpha fusion protein of sequence SEQ ID NO: 22 present in the samples.
A mean concentration is thus calculated for each of the groups with the passage of time.
The results obtained are shown:
Examination of these figures shows that the concentration of anti-TNF-alpha fusion protein and of human transferrin is constant over time in the rats that received the construct according to the invention.
Thus, these experiments demonstrate the fact that the application of a construct according to the invention, of very large size owing to the presence of not one but two coding sequences for proteins of interest, penetrates the targeted cells effectively and allows expression of the two proteins encoded by said construct at the level of the site of interest.
Moreover, they also illustrate, quite unexpectedly, the capacity of a construct according to the invention to produce, more stably over time, the proteins that it encodes compared to constructs only coding for a single one of these proteins (
The inventors confirmed the observations made in example 1 in a second experimental protocol using a plasmid according to the invention different than that used in example 1.
A DNA construct according to the invention, designated plasmid B, comprising:
The animals used in this protocol are as described in example 1. Six rats are used for each of the analysis times (D3, D7, D14 and D21).
At each analysis time, the rats are euthanized by administration of a lethal dose of pentobarbital (400 mg/kg) and then the animals are enucleated and the ocular fluids (vitreous humor and aqueous humor) are taken and stored at −80° C. until analysis.
Electrotransfer is carried out as described in example 1.
Samples of ocular fluids are taken at 3 days (D+3), 7 days (D+7), 14 days (D+14) and 21 days (D+21) after injection of plasmid followed by electrotransfer (D0). For each of these samples, an ELISA assay is carried out in order to measure the amount of decorin and/or of aflibercept present in the samples.
A mean concentration is thus calculated for each of the groups with the passage of time.
The results obtained are shown:
Examination of these figures shows that the plasmid according to the invention allows expression of the two therapeutic proteins of interest.
Thus, these experiments demonstrate the fact that the application of a construct according to the invention, of very large size owing to the presence of not one but two coding sequences for proteins of interest, penetrates the targeted cells effectively and allows expression of the two proteins encoded by said construct at the level of the site of interest.
Moreover, as had been shown above in example 1, they also illustrate, quite unexpectedly, the capacity of a construct according to the invention to produce, more stably over time, the proteins that it encodes compared to constructs only coding for a single one of these proteins (
Moreover, the inventors also confirmed the observations made above in an experimental protocol using a plasmid according to the invention different than that used in examples 1 and 2.
A DNA construct according to the invention, designated plasmid C, comprising:
Brown Norway rats aged from 7 to 8 weeks are used according to the provisions of the ARVO protocol (Association for Research in Vision and Ophthalmology). The rats are anesthetized by intramuscular injection of a dose of ketamine (40 mg/kg) and xylazine (4 mg/kg) before bilateral injection of the plasmid (30 μg/eye) or vehicle (10 μL) and electrotransfer on day 0 (D0). Six rats are used for each of the treatments. Electrotransfer is carried out as described in example 1.
On D3, choroid neovascularization is induced in all the animals by laser photocoagulation in several places of the retina (4 to 5 laser impacts per eye).
Fourteen days after lesion (D17), the vascular leakage of the neovascularization is evaluated by fluorescent angiography and attribution of a score as a function of the severity of the vascular leakage according to the following table.
The results obtained are shown in
On examining this figure, it appears that the plasmid according to the invention gives a 38% reduction in the number of impacts showing severe neovascular leakage compared to the animals that received the vehicle.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR2006898 | Jun 2020 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/068085 | 6/30/2021 | WO |
