WO2024100176A1

WO2024100176A1 - Controlled gene therapy of ocular diseases

Info

Publication number: WO2024100176A1
Application number: PCT/EP2023/081264
Authority: WO
Inventors: Pierre Fafournoux; Alain BRUHAT; Cyrielle VITURET
Original assignee: Nutritheragene; Institut National De Recherche Pour L'agriculture, L'alimentation Et L'environnement; Université Clermont Auvergne; Centre National De La Recherche Scientifique
Priority date: 2022-11-10
Filing date: 2023-11-09
Publication date: 2024-05-16

Abstract

The present invention concerns a controlled gene therapy of ocular diseases. This technology can be used in gene therapy protocols and avoids the side effects resulting from a continuous and uncontrolled expression of a transgene.

Description

CONTROLLED GENE THERAPY OF OCULAR DISEASES

Many ophthalmological diseases are treated, or could potentially be treated, by gene therapy. The eye lends itself well to this type of treatment because it is a closed organ. Viral vectors, which are generally used for transgene delivery, do not usually travel significantly to other areas of the body, so there is limited risk of an immune response elsewhere. In addition, treatment with a viral vector will require a relatively low dose of virus, thereby reducing treatment costs. If the treatment is aimed at the retina, sophisticated diagnostic technologies are available to monitor its structure and function without traumatic intervention.

Several treatments using gene therapy have been or are being tested (Gordon et al. Nature Reviews Drug Discovery 18, 415-416 (2019)). In addition to many genetic diseases that affect vision, several Phase 1 clinical trials have been conducted in the treatment of age-related macular degeneration (AMD). In this case, the aim was to express anti- angiogenic peptides (e.g. sFLTOI , RGX-314, CD59). However, the long-term use of anti- VEGF could lead to adverse effects and is not safe (Saint-Geniez M, et al. (2008) PLoS ONE 3(11 ): e3554).

Hence, improved methods of gene therapy are needed in order to be able to control the level of transgene expression in the eye.

The inventors have previously described that the amino-acid response pathway (elF2a-ATF4 pathway) can be harnessed as a regulatory system for gene therapy (Chaveroux et aL, Nat Biotechnol, 34, 746-751 (2016); international patent applications published as WO 2013/068096 A1 and WO 2017/207744 A1 ). Using a bioluminescent transgenic mouse model, the induction of transgene was found functional in the brain, liver, pancreas, duodenum, brown and white adipose tissue, kidney and skeletal muscle, while the transgene expression was not activated in spleen, lung and heart (Chaveroux et aL, Sci. Signal. 8, rs5 (2015))

It is now shown that this amino-acid response pathway is adapted to control transgene expression in the eye of a subject.

SUMMARY OF THE INVENTION

The invention relates to an isolated nucleic acid comprising: i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, said regulatory polynucleotide being activated in an individual upon consumption of a diet deficient in at least one essential amino acid; and ii) a transgene for gene therapy of an ocular disease, which is placed under the control of the said regulatory polynucleotide.

The invention further relates to a vector comprising a nucleic acid that comprises: i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, said regulatory polynucleotide being activated in an individual upon consumption of a diet deficient in at least one essential amino acid; and ii) a transgene for gene therapy of an ocular disease, which is placed under the control of the said regulatory polynucleotide.

The invention further relates to a pharmaceutical composition comprising the nucleic acid or a vector according to the invention, and a pharmaceutically acceptable carrier.

The invention also encompasses the nucleic acid, the vector, or the pharmaceutical composition according to the invention, for use for gene therapy of an ocular disease.

FIGURES

Figure 1 shows the result of bioluminetry measurement of Luciferase expression in the left eye injected with the AARE-TK Luc lentivirus (LV-Luc) of mice fed with control diet (Ctrl) or with the isoleucine deficient diet for 8 hours (-lie 8h), and in the right eye injected with PBS (Nl) of mice fed with control diet (Ctrl) or with the isoleucine deficient diet for 8 hours (-lie 8h}.

Figure 2 shows the result of bioluminetry measurement of Luciferase expression in the extracted eye of representative transgenic mice having the AARE-TK-Luc construct integrated into their genome, and that were fasted overnight and fed either with a control diet (Ctrl) or with a lie deficient diet (-lie), for 6 hours.

Figure 3 shows immunohistochemical analysis of Luciferase protein expression in sections of the extracted eye of transgenic mice having the AARE-TK-Luc construct integrated into their genome fasted overnight and fed either with a control diet (Ctrl) or with a lie deficient diet (-lie), for 8 hours.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a technology for controlling the expression of a transgene encoding a peptide with a therapeutic action in the eye. This technology can be used in gene therapy protocols and will avoid the side-effects resulting from a continuous and uncontrolled expression of a transgene. The technology is based on a construction comprising a promoter inducible by a deficiency in an amino acid that controls the expression of a cDNA encoding a peptide or protein with a therapeutic function. This construction can be delivered to a targeted area of the eye, and will allow the expression of the transgene upon induction by a deficiency in at least one amino acid. The results obtained by the inventors show that transgene expression can be induced in the eye of an individual by the consumption of a meal deficient in an essential amino acid. In particular, the inventors demonstrated that transgene expression can be induced in the retina, and more specifically in the layers of the photoreceptors and ganglion cells which are of particular interest for gene therapy of ocular diseases. By contrast, no transgene expression could be detected in the Inner Plexiform Layer (IPL), Inner Nuclear Layer (INL), Outer Plexiform Layer (OPL), and Outer Nuclear Layer (ONL).A single delivery into the eye, e.g. by injection of a viral vector containing the construct, should allow the drug peptide or protein to be expressed on demand when needed. This should avoid frequent injections into the eye (every 3-4 weeks) which are traumatic for the patient and at risk for the integrity of the retina.

Regulatory polynucleotide

The regulatory polynucleotide comprises a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence.

As used herein, a "minimal promoter" is intended to mean a promoter including all the required elements to properly initiate transcription of a gene of interest positioned downstream. The expressions "minimal promoter" and "core promoter" are considered as equivalent expressions. A skilled in the art understands that the "minimal promoter" includes or consists in at least a transcription start site (TSS), and a TATA box (the TATA box consists is a consensus sequence consisting of TAT AAA or TAT ATA, in which the spacing between the first T of the TATA box and the +1 of the TSS is generally 30 bp). The "minimal promoter" or "core promoter" enables recruitment of a RNA polymerase, which transcribes the DNA into RNA, and of general transcription factors.

Suitable minimal promoters are known for a skilled artisan.

In some embodiments, a minimal promoter is selected from the group consisting of the promoter of the thymidine kinase (TK), the promoter of the p-globin, the promoter for cytomegalovirus (CMV), the SV40 promoter and the like. As used herein, an “AARE” or “amino acid response element” denotes a nucleic acid sequence which is bound by the activating transcription factor 4 (ATF4), after activation of the GCN2-elF2a-ATF4 pathway by deficiency in an essential amino acid (EAA), and thereby induce expression of a target gene driven by the AARE.

In mammals, after consumption of a diet deficient in one EAA, the blood concentration of the limiting EAA decreases rapidly and greatly, triggering an ubiquitous adaptive process referred to as the amino-acid response pathway. The initial step of this pathway is the activation of the mammalian GCN2 protein kinase by uncharged tRNAs. GCN2 then phosphorylates the a subunit of eukaryotic initiation factor 2 (elF2a) on serine 51 , leading to upregulation of the translation of the ATF4. Once induced, ATF4 activates transcription of specific target genes through binding to the AARE. The GCN2-elF2a-ATF4 pathway can be rapidly turned off by the administration of the missing EAA.

According to some embodiments, the amino acid response element (AARE) nucleic acid sequence is selected in the group consisting of the nucleic acid sequences SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

The regulatory polynucleotide which comprises at least one AARE may include at least 2, at least 3, at least 4 or at least 5 AARE nucleic acid sequences. The expression "at least one AARE nucleic acid sequence" thus includes e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 AARE nucleic acid sequences.

In certain embodiments, the regulatory polynucleotide comprises at least two AARE nucleic acid sequences. In some other embodiments, the regulatory polynucleotide comprises from 1 to 20 AARE nucleic acid sequences, preferably from 1 to 10 AARE nucleic acid sequences. In certain embodiments, the regulatory polynucleotide comprises from 2 to 6 AARE nucleic acid sequences.

In some embodiments, the regulatory polynucleotide comprises 2 AARE nucleic acid sequences selected from the group consisting of the nucleic acid sequences SEQ ID NO: 2 and SEQ ID NO: 4. In some embodiments, the regulatory polynucleotide comprises 6 AARE nucleic acid sequences of sequence SEQ ID NO: 1 .

In certain embodiments, the at least two AARE nucleic acid sequences may be identical or distinct.

According to an embodiment, the regulatory polypeptide comprises the Thymidine kinase (Tk) minimal promoter and six copies of the AARE nucleic acid sequence from the TRIB3 gene, and comprises or consists of a sequence as shown in SEQ ID NO: 6.

The regulatory polynucleotide construct thus comprises the at least one AARE nucleic acid sequence placed immediately upstream of the minimal promoter which controls the expression of the transgene placed downstream. The regulatory polynucleotide is activated in an individual upon consumption of a diet deficient in at least one essential amino acid.

The individual is a human or a non-human mammal, preferably a human. In some embodiments, the non-human mammal is selected from the group consisting of a pet such as a dog, a cat, a domesticated pig, a rabbit, a ferret, a hamster, a mouse, a rat and the like; a primate such as a chimp, a monkey, and the like; an economically important animal such as cattle, a pig, a horse, a sheep, or a goat.

In mammals, nine EAAs must be supplied in the diet, and a lack of any one of them can induce the AARE-driven expression system.

As used herein, an "essential amino acid" includes histidine (His, H), isoleucine (lie, I), leucine (Leu, L), Lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), threonine (Thr, T), tryptophan (Trp, W) and valine (Vai, V).

As used herein, a “diet deficient in at least one essential amino acid” is intended to mean a diet deficient in 1 , 2, 3, 4, 5, 6, 7, 8 or 9 essential amino acid(s).

Activation of the regulatory polynucleotide

For activation of the regulatory peptide, a diet deficient in at least one essential amino acid is administered to the individual.

According to preferred embodiments, the individual has been starved before the diet deficient in at least one essential amino acid is administered.

The blood-retinal barrier regulates the micro-environment of the retina, the extra cellular fluid and the vitreous. Blood-aqueous barrier and -retinal barrier play important roles not only in restricting molecular movement into the anterior and posterior compartments but also in their elimination from the ocular compartments. Ocular barriers effectively protect the eye also from pharmaceuticals.

The results disclosed herein demonstrate that administering a diet deficient in at least one essential amino acid makes it possible to activate a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence and induce expression of a transgene under its control even when the nucleic acid comprising said regulatory polynucleotide and transgene has been administered to the eye, and in particular the retina, of an individual. Transqene for gene therapy of an ocular disease

The regulatory polynucleotide controls the expression of a transgene for gene therapy of an ocular disease.

As used herein, “controls the expression" is intended to mean that the expression is induced or turned "on", and shut down or turned "off', in a precise manner, with respect to the moment of induction and/or the duration of induction.

The expression of the transgene may be measured by any suitable method available in the state of the art, including the measure of the mRNA expression resulting from the transcription of the transgene, and/or the measure of the expression of the protein encoded by the transgene.

An induced expression may be expressed as a time fold expression as compared to the basal, non-induced expression.

In some embodiments, the induced expression may vary from 2 fold to 10,000 fold, preferably from 4 fold to 500 fold, more preferably from 8 fold to 250 fold, most preferably from 10 fold to 100 fold, as compared to the basal expression. In particular, the induced expression may vary 3 fold, 4 5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 75 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 350 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1 ,000 fold, 2,000 fold, 3,000 fold, 4,000 fold, 5,000 fold, 6,000 fold, 7,000 fold, 8,000 fold and 9,000 fold.

The ocular disease is a retinal disease (i.e. it affects retinal cells such as RPE, rod and/or cone photoreceptors, or ganglion cells). According to an embodiment, the ocular disease affects rod and/or cone photoreceptors, and/or ganglion cells.

The ocular disease may be an inherited ocular disease or a sporadic ocular disease.

In some embodiments, the transgene for gene therapy of an ocular disease does not encode a pro-apoptotic protein, or the transgene is for gene therapy of an inherited ocular disease. According to certain embodiments, the transgene does not encode a pro-apoptotic protein when the ocular disease is a cancer or when the ocular disease affects a retinal cell.

Preferably, the ocular disease is an inherited ocular disease. In an inherited ocular disease, the ocular disease is caused by a mutated or defective gene. According to certain embodiments, the ocular disease is an inherited ocular disease that affects retinal cells.

In an embodiment, the mutated or defective gene may encode a protein having defective or partial function in retinal cells. In this embodiment, the transgene may encode a functional version of the protein having defective or partial function. In this embodiment, the transgene may alternatively encode a protein useful for optogenetics. Optogenetics is a method that uses light to modulate molecular events in a targeted manner in living cells or in an individual. It relies on the use of genetically-encoded proteins that change conformation in the presence of light to alter cell behavior, for example, by changing the membrane voltage potential of excitable cells. Optogenetic methods are currently developed to restore visual function in individuals having neurodegenerative eye disease, such as retinitis pigmentosa (RP). Accordingly, the transgene may encode an optogenetic actuator such as a light-sensitive membrane protein, for instance a microbial or mammalian opsin or a light-activatable G-protein coupled receptor (GPCR).

In another embodiment, the ocular disease is caused by a gain-of-function mutation in a gene that encodes a protein having function in retinal cells. In this embodiment, the transgene is transcribed into a RNA nucleic acid that inhibits or reduces expression of the protein having function in retinal cells and which is encoded by the gain-of-function mutated gene. For instance, the transgene is transcribed into a ribozyme, an antisense RNA, an iRNA, a shRNA.

In some embodiments, the ocular disease is selected from the group consisting of color blindness, blue cone monochromacy, lysosomal storage disease IV or VII, ocular or oculocutaneous albinism, maculopathies, glaucoma, early onset severe retinal dystrophy, age-related macular degeneration (AMD), retinoschisis (juvenile or X-linked retinoschisis), Leber hereditary optic neuropathy (LHON), retinal dystrophy (such as retinitis pigmentosa (RP), Congenital stationary night blindness, Leber's congenital amaurosis, progressive cone and cone-rod dystrophies, achromatopsia, choroideremia and Usher syndrome), and macular dystrophy (such as Stargardt's disease, Vitelliform macular dystrophy, or North Carolina macular dystrophy).

In an embodiment, the ocular disease is a retinal dystrophy (RD), in particular retinitis pigmentosa (RP), achromatopsia, Leber's congenital amaurosis (LCA), or Usher syndrome. Retinal dystrophies are degenerative diseases of the retina that have marked clinical and genetic heterogeneity. Common presentations among these disorders include night or colour blindness, tunnel vision and subsequent progression to complete blindness. The known causative disease genes have a variety of developmental and functional roles with mutations in more than 120 genes shown to be responsible for the phenotypes (Nash et aL, Transl. Pediatr., 2015 Apr; 4(2): 139-163).

Examples of possible transgenes for therapy of retinal dystrophies are listed in Table

1. Table 1 : Selected transgene for gene therapy of retinal dystrophies.

Preferably, a transgene for gene therapy of retinitis pigmentosa is selected from the group consisting of a gene encoding Rho, PDE6P, RPE65, RLBP1 , MERKT or ChR2. When the retinitis pigmentosa is X-linked retinitis pigmentosa, the transgene preferably encodes RPGR.

Preferably, a transgene for gene therapy of achromatopsia encodes CNGB3 or CNGA3.

Preferably, a transgene for gene therapy of Leber's congenital amaurosis (LCA) encodes RPE65. Preferably, a transgene for gene therapy of Usher syndrome encodes MYO7A.

Preferably, a transgene for gene therapy of choroideremia encodes REP1 . Table 2: Selected transgene for gene therapy of inherited ocular diseases.

In an embodiment, the ocular disease is Leber hereditary optic neuropathy (LHON), and the transgene encodes MT-ND4. In an embodiment, the ocular disease is X-linked retinoschisis and the transgene encodes RS1 (retinoschisin 1 ).

In an embodiment, the ocular disease is a macular dystrophy, in particular Stargardt's disease, Vitelliform macular dystrophy, or North Carolina macular dystrophy. Examples of possible transgenes for therapy of macular dystrophies are listed in T able 3.

Table 3: Selected transgene for gene therapy of macular distrophies.

In some embodiments, the ocular disease is caused by blood vessel proliferation or abnormal growth, or swelling. Examples of such ocular diseases include wet age-related macular degeneration, macular edema caused by retinal vein occlusion, diabetic retinopathy, or myopic choroidal neovascularization, Coats’ disease, Eales’ disease, and central serous retinopathy. In these embodiments, the transgene may encode an anti- angiogenic agent, such as an anti-VEGF agent (e.g. soluble VEGF receptor (sFLT-1 )), Pigment epithelium derived factor (PEDF), endostatin, angiostatin or tissue inhibitor of metalloproteinase-3 (TIMP3).

In a particular embodiment, the ocular disease is age-related macular dystrophy (AMD). Age-related macular dystrophy includes wet AMD (also called neovascular AMD) and dry AMD. As used herein, age-related macular dystrophy is preferably wet or neovascular AMD.

In some embodiments the transgene encode an antibody, such as an anti-VEGF antibody, an anti-TNF antibody, or an anti-IL-6 antibody, or a fragment thereof, such as a VH or VL chain of the antibody.

Vectors

In some embodiments, the vector is a viral vector suitable for gene therapy of an ocular disease, i.e. the vector comprises the essential elements for achieving the expression of the transgene in a target cell, in particular in a retinal cell.

In some embodiments, the viral vector is selected from the group consisting of an adenovirus, an adeno-associated virus (AAV), an alphavirus, a herpesvirus, a lentivirus (LV), a non-integrative lentivirus, a retrovirus and a vaccinia virus. Preferably, the viral vector is a lentivirus, an adeno-associated virus (AAV), or an adenovirus. In some embodiments, the viral vector is a recombinant viral vector, in particular a recombinant AAV vector.

For instance, an AAV (rAAV) vector containing a genomic component of serotype 2 can be used, combined with a capsid of another AAV serotype. The capsid serotype determines the tropism and efficacy of the vector. For example, AAV2/2 and rAAV2/5 vectors transduce both the retinal pigment epithelium (RPE) and photoreceptors, but rAAV2/1 , rAAV2/4 and rAAV4/4 vectors exclusively transduce the RPE. Deliberate serotype selection in the construction of rAAV vectors allows for enhanced specificity and performance.

In some embodiments, the vector is non-viral vector for ocular gene transfer such as a particle, and in particular a nanoparticle (NP). Examples of particles for the delivery of a nucleic acid includes lipoplex NPs, polyplex NPs, pegylated NPs as reviewed by Zulliger et al., J Control Release. 2015 December 10; 219: 471-487. Further examples include a particle comprising cationic lipids; a lipid nano-emulsion; a solid lipid nanoparticle; a peptide based particle; a polymer based particle, in particular comprising natural and/or synthetic polymers.

In some embodiments, a polymer based particle may comprise a protein; a peptide; a polysaccharide, in particular chitosan.

In some embodiments, a polymer based particle may comprise a synthetic polymer, in particular, a polyethylene imine (PEI), a dendrimer, a poly (DL-Lactide) (PLA), a poly(DL- Lactide-co-glycoside) (PLGA), a polymethacrylate and a polyphosphoesters.

In some embodiments, the delivery particle further comprises at its surface one or more ligands suitable for binding to a target receptor exposed at the membrane of a targeted cell. Host cell

In a further aspect, the invention concerns an isolated host cell comprising the nucleic acid or nucleic acid vector, as defined herein.

According to the instant disclosure, the host cell is an ocular cell, in particular a retinal cell, or a progenitor thereof. Retinal cells include in particular rod and/or cone photoreceptors, and/or ganglion cells.

According to some embodiments, the host cell is a progenitor of retinal cells. Such progenitors can be produced from induced pluripotent stem cells (iPS) and differentiated into all retinal cell types, including retinal ganglion cells and precursors of photoreceptors. Reference is made for instance to the method described in the publication Reichman and Goureau, Methods Mol Biol. 2016;1357:339-51.

Pharmaceutical composition

Another aspect of the present invention concerns a pharmaceutical composition comprising (i) a nucleic acid, or a vector or host cell as defined herein, and (ii) a pharmaceutically acceptable vehicle.

The formulation of pharmaceutical compositions according to the instant invention is well known to persons skilled in the art.

As referred herein, a nucleic acid comprising: i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, said regulatory polynucleotide being activated in an individual upon consumption of a diet deficient in at least one essential amino acid; and ii) a transgene for gene therapy of an ocular disease, which is placed under the control of the said regulatory polynucleotide, or a vector or host cell comprising, said nucleic acid represents the at least one active agent.

In some embodiments, the pharmaceutical composition comprises a nucleic acid, or a vector or host cell, as defined in the present disclosure, as the only active agent.

In some embodiments, a suitable pharmaceutically acceptable vehicle according to the invention includes any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

In certain embodiments, suitable pharmaceutically acceptable vehicles may include, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and a mixture thereof. In some embodiments, pharmaceutically acceptable vehicles may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the cells. The preparation and use of pharmaceutically acceptable vehicles is well known in the art.

Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated.

In some embodiments, the pharmaceutical composition may be administered to the eye of an individual in need thereof by any suitable route, e.g. by topical administration, intravenous injection, periocular injection (e.g. in the subconjunctival space), intraocular injection (intravitreal injection, or subretinal injection, suprachoroidal injection). The administration may be combined with electroporation to improve nucleic acid delivery, especially when the nucleic acid is naked or comprised in a non-viral vector.

In certain embodiments, the administration of the pharmaceutical composition by injection may be directly performed in the target tissue of interest, in particular in order to avoid spreading of the nucleic acid or the nucleic acid vector comprised in the said pharmaceutical composition.

In some embodiments, an oral formulation according to the invention includes usual excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

In some embodiments, an effective amount of said compound is administered to said individual in need thereof.

Within the scope of the instant invention, an "effective amount" refers to the amount of said compound that alone stimulates the desired outcome, i.e. alleviates or eradicates the symptoms of the ocular disease.

It is within the common knowledge of a skilled artisan to determine the effective amount of a nucleic acid comprising a transgene for gene therapy of an ocular disease, or a vector comprising said nucleic acid, in order to observe the desired outcome.

Within the scope of the instant invention, the effective amount of the compound to be administered may be determined by a physician or an authorized person 5 skilled in the art and can be suitably adapted within the time course of the treatment.

In certain embodiments, the effective amount to be administered may depend upon a variety of parameters, including the material selected for administration, whether the administration is in single or multiple doses, and the individual's parameters including age, physical conditions, size, weight, gender, and the severity of the disease to be treated. In certain embodiments, an effective amount of the active agent may comprise from about 0.001 mg to about 3000 mg, per dosage unit, preferably from about 0.05 mg to about 100 mg, per dosage unit.

Within the scope of the instant invention, from about 0.001 mg to about 3000 mg includes, from about 0.002 mg, 0.003 rng, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 20 900 mg, 950 mg, 1000 mg, 1 100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg,

1450 mg, 1500 mg, 1550 mg, 1600 mg, 1650 mg, 1700 mg, 1750 mg, 1800 mg, 1850 mg,

1900 mg, 1950 mg, 2000 mg, 2100 mg, 2150 mg, 2200 mg, 2250 mg, 2300 mg, 2350 mg,

2400 mg, 2450 mg, 2500 mg, 2550 mg, 2600 mg, 2650 mg, 2700 mg, 2750 mg, 2800 mg,

2850 mg, 2900 mg and 2950 mg, per dosage unit.

In certain embodiments, the active agent may be at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day.

In some particular embodiments, an effective amount of the active agent may comprise from about 1 x10⁵ to about 1x10¹⁵ copies of the nucleic acid for the controlled expression of a nucleic acid encoding a pro-apoptotic protein, or the nucleic acid vector or the delivery particle, as defined in the present disclosure, per dosage unit.

Within the scope of the instant invention, from about 1 x10⁵ to about 1x10¹⁵ copies includes 2x10⁵, 3x10⁵, 4x10⁵, 5x10⁵, 6x10⁵, 7x10⁵, 8x10⁵, 9x10⁵, 1 x10⁶, 2x10⁶, 3x10⁶, 4x10⁶, 5x10⁶, 6x10⁶, 7x10⁶, 8x10⁶, 9x10⁶, 1x10⁷, 2x10⁷, 3x10⁷, 4x10⁷, 5x10⁷, 6x10⁷, 7x10⁷, 8x10⁷, 9x10⁷, 1 x10⁸, 2x10⁸, 3x10⁸, 4x10⁸, 5x10⁸, 6x10⁸, 7x10⁸, 8x10⁸, 9x10⁸, 1x10⁹, 2x10⁹, 3x10⁹, 4x10⁹, 5x10⁹, 6x10⁹, 7x10⁹, 8x10⁹, 9x10⁹, 1x10¹°, 2x10¹°, 3x10¹°, 4x1 O¹⁰, 5x10¹°, 6x10¹°, 7x10¹°, 8x10¹°, 9x10¹°, 1x10¹¹, 2x10¹¹, 3x10¹¹, 4x10¹¹, 5x10¹¹, 6x10¹¹, 7x10¹¹, 8x10¹¹, 9x10¹¹, 1x10¹², 2x10¹², 3x10¹², 4x10¹², 5x10¹², 6x10¹², 7x10¹², 8x10¹², 9x10¹², 1x10¹³, 2x10¹³, 3x10¹³, 4x10¹³, 5x10¹³6x10¹³, 7x10¹³, 8x10¹³, 9x10¹³, 1x10¹⁴, 2x10¹⁴, 3x10¹⁴, 4x10¹⁴, 5x10¹⁴, 6x10¹⁴, 7x10¹⁴, 8x10¹⁴, 9x10¹⁴ copies, per dosage unit.

Therapeutic applications Another aspect of the invention concerns the nucleic acid comprising a transgene for gene therapy of an ocular disease, which is placed under the control of the regulatory polynucleotide, as defined herein, or the vector or host cell comprising said nucleic acid for use as a medicament, in particular by gene therapy.

In one aspect, the invention also relates to the use of the nucleic acid comprising a transgene for gene therapy of an ocular disease, which is placed under the control of the regulatory polynucleotide, as defined herein, or the vector or host cell comprising said nucleic acid for the preparation or the manufacture of a medicament.

In a still other aspect, the invention concerns the nucleic acid comprising a transgene for gene therapy of an ocular disease, which is placed under the control of the regulatory polynucleotide, as defined herein, or the vector or host cell comprising said nucleic acid for use (as an active agent) for encoding a protein having defective or partial function into at least one ocular target cell.

In a still other aspect, the invention concerns the nucleic acid comprising a transgene for gene therapy of an ocular disease, which is placed under the control of the regulatory polynucleotide, as defined herein, or the vector or host cell comprising said nucleic acid for use (as an active agent) for transcribing a RNA nucleic acid that inhibits or reduces expression of a protein having defective or partial function into at least one ocular target cell.

According to an embodiment, the ocular target cell is a retinal cell. According to some embodiments, the retinal cell is a rod and/or cone photoreceptor, and/or a ganglion cell.

The invention further relates to a method for treating an ocular disease by gene therapy which comprises administering to a subject in need thereof an effective amount of a nucleic acid comprising a transgene for gene therapy of an ocular disease, which is placed under the control of the regulatory polynucleotide, as defined herein, or the vector or host cell comprising said nucleic acid.

In particular, the ocular disease to be treated by gene therapy includes:

- ocular diseases caused by a mutated or defective gene that encodes a protein having defective or partial function in retinal cells

- ocular diseases caused by a gain-of-function mutation in a gene that encodes a protein having function in retinal cells;

- an ocular diseases selected from the group consisting of color blindness, blue cone monochromacy, lysosomal storage disease IV or VII, ocular or oculocutaneous albinism, maculopathies, glaucoma, early onset severe retinal dystrophy, age-related macular degeneration (AMD), retinoschisis (juvenile or X- linked retinoschisis), Leber hereditary optic neuropathy (LHON), retinal dystrophy (such as retinitis pigmentosa (RP), Congenital stationary night blindness, Leber's congenital amaurosis, progressive cone and cone-rod dystrophies, achromatopsia, choroideremia and Usher syndrome), and macular dystrophy (such as Stargardt's disease, Vitelliform macular dystrophy, and North Carolina macular dystrophy); and

- ocular diseases caused by blood vessel proliferation or abnormal growth, or swelling, such as wet age-related macular degeneration, macular edema caused by retinal vein occlusion, diabetic retinopathy, or myopic choroidal neovascularization, Coats’ disease, Eales’ disease, or central serous retinopathy.

According to some embodiments, the nucleic acid comprising a transgene for gene therapy of an ocular disease, which is placed under the control of the regulatory polynucleotide, as defined herein, or the vector or host cell comprising said nucleic acid, is administered only once to the subject.

According to some embodiments, the nucleic acid comprising a transgene for gene therapy of an ocular disease, which is placed under the control of the regulatory polynucleotide, as defined herein, or the vector or host cell comprising said nucleic acid, is administered chronically to the subject, e.g. several months or years apart.

The medical indication or method of treatment further comprises administering a diet deficient in at least one essential amino acid to the individual. Exposing the individual to a deficiency in at least one essential amino acid induces transgene expression in the individual that/who was administered with the nucleic acid comprising the transgene for gene therapy of an ocular disease, which is placed under the control of the regulatory polynucleotide, as defined herein, or the vector or host cell comprising said nucleic acid.

A diet deficient in at least one essential amino acid is chronically administered to the individual, in order to induce repeated transgene expression over time. Depending on the desired frequency of transgene expression in order to reach therapeutic efficacy, the individual may be exposed to a deficiency in at least one essential amino acid for instance every day, every two days, once a week, twice a week, etc..

Accordingly, the system of the invention enables to substitute chronic administration of a diet deficient in an essential amino acid, to induce expression of the transgene end encoded therapeutic protein, for the repeated intraocular injections that are currently implemented for treating patients and that are both traumatic for the patient and at risk for the integrity of the retina.

The invention will be further illustrated in view of the following examples. Example 1 : Proof of concept of regulated expression of a transgene in the eye using a lentiviral vector

C57BL6 mice were injected (intravitreal injection) into the left eye with: (1 ) AARE-TK Luc lentivirus (2 pL of pure vector injected only into the left eye, titer 1.05x10^A9 TU/mL i.e. a dose of 2x10^A6 TU - transduction units) (n=2), or (2) PBS (n=3). The experiment was performed twice.

Three weeks post-injection, mice are fasted and fed 8 h with a control (Ctrl) or Isoleucine (-lie) deficient diet. Luciferase activity is measured by bioluminometry on the anaesthetised live animal. The measurement is made after consumption of a control diet or a -lie diet (each mouse is its own control).

Figure 1 shows that Luciferase expression in the eye injected with the AARE-TK Luc lentivirus (LV-Luc) is 8-fold increased when the mice are fed with the isoleucine deficient diet compared to control diet.

Example 2: Proof of concept of regulated expression of a transgene in the eye using a transgenic mouse that has integrated the Luciferase gene under the control of the AARE-TK promoter

Transgenic mice, in which the AARE-TK-Luc construct has been integrated into the genome, were generated as disclosed previously in Chaveroux et al. (Sci. Signal. 8, rs5 (2015)).

As in the previous experiment, the mice were fasted overnight and fed with a control (Ctrl) or lie deficient (-lie) diet, here for 6h. Luciferase activity was measured by bioluminometry after sacrifice of the animal and removal of the eye. This experiment was repeated 3 times (the repetitions were spread over several years). The picture of Figure 2 shows a representative experiment.

Example 3: Histochemical analyses of Luciferase protein expression in the eye

In order to measure the areas of the eye where the transgene is regulated, we used the transgenic mouse described in example 2.

The transgenic mice were fasted overnight, fed with a control (Ctrl) or lie deficient (- lie) diet for 8 hours and then sacrificed. The eye was extracted from the orbital cavity, fixed with paraformaldehyde, incubated in various sucrose baths and cryo-frozen. Sections are taken to measure the level of Luciferase protein expression by Immuno-Histo-Chemistry (using an anti-luciferase antibody). The secondary antibody is an anti-rabbit-HRP. Peroxidase labelling is revealed with DAB (Di Amino Benzinide). DAB realizes a non- fluorescent labelling that appears as a brown deposit.

The induction of Luciferase expression is very clear identifiable in the area containing the photoreceptors (outer and inner segment) and in the ganglion cell layer (left and right arrows respectively on the panels of Figure 3), in contrast with the other retinal structures (Retinal pigment epithelium (RPE), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL) and nerve fiber layer (NFL), and with the choroid.

Claims

1 . An isolated nucleic acid comprising: i) a regulatory polynucleotide comprising a minimal promoter and at least one AARE (amino acid response element) nucleic acid sequence, said regulatory polynucleotide being activated in an individual upon consumption of a diet deficient in at least one essential amino acid; and ii) a transgene for gene therapy of an ocular disease, which is placed under the control of the said regulatory polynucleotide.

2. The nucleic acid according to claim 1 , wherein the amino acid response element (AARE) nucleic acid sequence is selected in a group consisting of sequences SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

3. The nucleic acid according to claim 1 or 2, wherein the regulatory polynucleotide comprises at least two AARE nucleic acid sequences.

4. The nucleic acid according to any one of claims 1 to 3, wherein the transgene for gene therapy of an ocular disease does not encode a pro-apoptotic protein, or the transgene is for gene therapy of an inherited ocular disease.

5. A vector comprising the nucleic acid according to any one of claims 1 to 4.

6. The vector according to claim 5, wherein said viral vector is a lentivirus, an adeno-associated virus (AAV), or an adenovirus.

7. An isolated host cell comprising the nucleic acid according to any one of claims 1 to 4, or the vector according to any one of claims 5 to 6.

8. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1 to 3, or a vector according to claim 4 or 5, or a host cell according to claim 6, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable carrier is a lipid-based, polymer-based, or peptide-based particle carrier that comprises the nucleic acid according to any one of claims 1 to 4, or the vector according to claim 5 or 6, or the host cell according to claim 7.

10. The nucleic acid according to any one of claims 1 to 4, the vector according to claim 5 or 6, the host cell according to claim 7, or the pharmaceutical composition according to claim 8 or 9, for use for gene therapy of an ocular disease.

1 1 . The nucleic acid, the vector, the host cell, or the pharmaceutical composition for the use according to claim 10, wherein the ocular disease is caused: i) by a mutated or defective gene that encodes a protein having defective or partial function in retinal cells; or ii) by a gain-of-function mutation in a gene that encodes a protein having function in retinal cells.

12. The nucleic acid, vector, the host cell, or pharmaceutical composition for the use according to any one of claims 10-11 , wherein the ocular disease is selected from the group consisting of color blindness, blue cone monochromacy, lysosomal storage disease IV or VII, ocular or oculocutaneous albinism, maculopathies, glaucoma, early onset severe retinal dystrophy, age-related macular degeneration (AMD), retinoschisis (juvenile or X- linked retinoschisis), Leber hereditary optic neuropathy (LHON), retinal dystrophy (such as retinitis pigmentosa (RP), Congenital stationary night blindness, Leber's congenital amaurosis, progressive cone and cone-rod dystrophies, achromatopsia, choroideremia and Usher syndrome), and macular dystrophy (such as Stargardt's disease, Vitelliform macular dystrophy, and North Carolina macular dystrophy).

13. The nucleic acid, vector, the host cell, or pharmaceutical composition for the use according to claim 10, wherein the ocular disease is caused by blood vessel proliferation or abnormal growth, or swelling, and the transgene encodes an anti-angiogenic agent.