Methods and medicaments for treating neurotrophic keratitis

Abstract

According to the present disclosure, there is provided a method of treating neurotrophic keratitis in a patient by administering to the patient an agent that inhibits figetin-like 2 activity (eg, an RNA interfering agent such as an siRNA).

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 423,745, filed November 8, 2022, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Neurotrophic keratitis (NK; also called neuroplegic keratitis) is a degenerative corneal disease caused by damage to the trigeminal nerve, resulting in decreased corneal sensitivity, spontaneous corneal epithelial defects, poor corneal healing, and the development of corneal ulcers, melting, or perforations. In addition to its primary sensory role, the trigeminal nerve also plays a role in maintaining corneal integrity by supplying nutrients to the cornea and regulating tissue metabolism.

[0003] Neurotrophic keratitis (NK) is classified as a rare disease, with an estimated prevalence of less than 50 per 100,000 people. An average of 6% of cases of herpetic keratitis have been reported to progress to this disease, with a peak of 12.8% reported in cases of varicella-zoster virus-induced keratitis. While the causes of NK are diverse, herpes simplex virus (HSV) and varicella-zoster virus (HZV) infection are the most common cause of herpetic keratitis, accounting for up to 27% of NK cases.

[0004] Neurotrophic keratitis (NK) is not adequately treated with current therapies, and therefore there is a need to develop novel therapies to effectively treat NK. Summary of the Invention [Means for solving the problem]

[0005] The disclosed methods and medicaments for use in patients with neurotrophic keratitis (NK) are intended to treat a degenerative disease of the cornea caused by damage to the trigeminal nerve, which is triggered by a variety of precipitating events (described in more detail below) but does not typically progress to NK. Therefore, the use of the disclosed methods and medicaments differs from their use for treating the cause of NK. As described herein, treatment of NK according to the present disclosure is generally initiated at the time of diagnosis of NK and is not sequential with treatment of the cause of NK.

[0006] According to one aspect of the present disclosure, there is provided a method for treating neurotrophic keratitis, the method comprising administering to a subject in need of treatment for neurotrophic keratitis an effective amount of an agent that inhibits or reduces the activity of figetin-like 2 (FL2), the agent being formulated for ocular administration. In some embodiments, the stage of neurotrophic keratitis is stage 1, stage 2, or stage 3. In some embodiments, the agent that acts as an inhibitor of figetin-like 2 is an RNA interfering agent. In some embodiments, the RNA interfering agent is an siRNA. In some embodiments, the RNA interfering agent is an shRNA. In some embodiments, treatment of neurotrophic keratitis includes one or more of: complete corneal healing, a reduction in the area of ​​persistent epithelial defect, no residual fluorescein staining in the corneal lesion area, no persistent fluorescein staining, complete corneal clearing, improved visual acuity, and improved corneal sensitivity.

[0007] In some embodiments, the siRNA comprises a sequence selected from the following: UUACACAGUAUUAAAGCGAUU (SEQ ID NO: 1); UCGCUUUAAUACUGUGUAAUU (SEQ ID NO: 2); CAUCUGAAACCUAGGGUCUUU (SEQ ID NO: 3); AGACCCUAGGUUUCAGAUGUU (SEQ ID NO: 4); GUGACUUAUGCUAGGAGGAUU (SEQ ID NO: 5); UCCUCCUAGCAUAAGUCACUU (SEQ ID NO: 6); GGUCAGAAGCAGAAUGUAUUU (SEQ ID NO: 7); AUACAUUCUGCUUCUGACCUU (SEQ ID NO: 8); CGCCGGCCCACAAGUUGGAdTdT (SEQ ID NO: 9); UCCAACUUGUGGGCCGGCGdTdT (SEQ ID NO: 10); CAGCUCGAGCCCUUUGACAdTdT (SEQ ID NO: 11); UGUCAAAGGGCUCGAGCUGdTdT (SEQ ID NO: 12); CCUCCAACCUCCUCAAGAGdTdT (SEQ ID NO: 13); CUCUUGAGGAGGUUGGAGGdTdT (SEQ ID NO: 14); CGUUGCUGCUCAUCAGCGAdTdT (SEQ ID NO: 15); UCGCUGAUGAGCAGCAACGdTdT (SEQ ID NO: 16); fUfUmAfCmAfCAGUAUUAAAGCGATT (SEQ ID NO: 17); (Phos)UCGCUUUAAUACUGUGUAATT (SEQ ID NO: 18); 5′-UUACACAGUAUUAAAGCGATT-3′ (SEQ ID NO: 34); (Phos)5′-mUmCGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 35); (Phos)5′-mU(s)mC(s)GCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 36); (Phos)5′-fUfCGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 37); (Phos)5′-fU(s)fC(s)GCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 38); (Phos)5′-mU(s)mC(s)GCUUUAAUAmCfUmGfUmGfUmAmATT-3′ (SEQ ID NO: 39); (Phos)5′-U(s)CGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 40); (Phos)5′-mUfCmGfCmUfUmUAAfUmAfCmUGmUmGfUmAmATT (SEQ ID NO: 41); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGmCmGmAmUmU-3' (SEQ ID NO: 42); (Phos)5′-mUmCmGmCmUmUmUmAmAmUmAmCmUmGmUmGmUmAmAmUmU-3′ (SEQ ID NO: 43); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGdCdGdATT-3' (SEQ ID NO: 44); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGdCmGmATT-3' (SEQ ID NO: 45); 5'UUACACAGUAUUAAAGCGA-3' (SEQ ID NO: 46); (Phos)5′-U(s)CGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 47); (Phos)5′-UCGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 48); (Phos)5'-U(s)C(s)GCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 49); 5′-mUmUACACAGUAUUAAAGCGA-3′ (SEQ ID NO: 50); (Phos)5′-U(s)CGCUUUAAUACUGUGUmAmATT-3′ (SEQ ID NO: 51); (Phos)5′-UCGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 52); (Phos)5'-U(s)C(s)GCUUUAAUACUGUGUAAT(s)T-3' (SEQ ID NO: 53); 5′lUlUlAlClACAGUAUUAAAGCGATT-3′ (SEQ ID NO: 54); (Phos)5′-UCGCUUUAAUACUGlUlGlUlAlATT-3′ (SEQ ID NO: 55); 5'fUfUlAfClACAGUAUUAAAGCGA-3' (SEQ ID NO: 56); and (Phos)5'-mU(s)mCmGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 57). In the above sequence, d (nucleotide) = deoxy-(nucleotide), m (nucleotide) = 2'-O-methyl nucleotide, T = thymidine, f (nucleotide) = 2'-fluorodeoxynucleotide, (Phos) = phosphodiester cap; uppercase nucleotide = RNA nucleotide, l (nucleotide) = locked nucleotide, (s) = phosphorothioate.

[0008] In some embodiments, the siRNA has at least one modification selected from a 3' overhang, a 5' overhang, a 5' phosphorylation, a 2' sugar modification, a nucleobase modification, a phosphate backbone modification, and any combination thereof. In some embodiments, the siRNA or shRNA having the above modifications has at least one additional modification selected from a 3' overhang, a 5' overhang, a 5' phosphorylation, a 2' sugar modification, a nucleobase modification, a phosphate backbone modification, and any combination thereof.

[0009] In some embodiments, the figetin-like 2 is human figetin-like 2. In some embodiments, the siRNA or shRNA is encapsulated within a nanoparticle. In some embodiments, the siRNA or shRNA is delivered by nanoparticles, electroporation / nuclear transfection, Accel siRNA, viral vectors, peptides, proteins, or aptamers. In some embodiments, the siRNA or shRNA is delivered by eye drops. In some embodiments, the siRNA or shRNA is delivered by an ophthalmic wafer. In some embodiments, the siRNA or shRNA is delivered by a drug-releasing contact lens.

[0010] In some embodiments, the siRNA comprises a duplex of SEQ ID NO:17 and SEQ ID NO:18.

[0011] In some embodiments, the shRNA comprises SEQ ID NO:23.

[0012] According to another aspect of the present disclosure, there is provided an ophthalmic composition for treating neurotrophic keratitis, the ophthalmic composition comprising an effective amount of an agent that inhibits or reduces the activity of figetin-like 2 (FL2). In some embodiments, the stage of neurotrophic keratitis is stage 1, stage 2, or stage 3. In some embodiments, the agent that acts as an inhibitor of figetin-like 2 is an RNA interfering agent. In some embodiments, the RNA interfering agent is an siRNA. In some embodiments, the RNA interfering agent is an shRNA. In some embodiments, the treatment of neurotrophic keratitis includes one or more of complete corneal healing, a reduction in the area of ​​persistent epithelial defect, no residual fluorescein staining in the corneal lesion area, no persistent fluorescein staining, complete corneal clearing, improved visual acuity, and improved corneal sensitivity.

[0013] In some embodiments, the siRNA comprises a sequence selected from the following: UUACACAGUAUUAAAGCGAUU (SEQ ID NO: 1); UCGCUUUAAUACUGUGUAAUU (SEQ ID NO: 2); CAUCUGAAACCUAGGGUCUUU (SEQ ID NO: 3); AGACCCUAGGUUUCAGAUGUU (SEQ ID NO: 4); GUGACUUAUGCUAGGAGGAUU (SEQ ID NO: 5); UCCUCCUAGCAUAAGUCACUU (SEQ ID NO: 6); GGUCAGAAGCAGAAUGUAUUU (SEQ ID NO: 7); AUACAUUCUGCUUCUGACCUU (SEQ ID NO: 8); CGCCGGCCCACAAGUUGGAdTdT (SEQ ID NO: 9); UCCAACUUGUGGGCCGGCGdTdT (SEQ ID NO: 10); CAGCUCGAGCCCUUUGACAdTdT (SEQ ID NO: 11); UGUCAAAGGGCUCGAGCUGdTdT (SEQ ID NO: 12); CCUCCAACCUCCUCAAGAGdTdT (SEQ ID NO: 13); CUCUUGAGGAGGUUGGAGGdTdT (SEQ ID NO: 14); CGUUGCUGCUCAUCAGCGAdTdT (SEQ ID NO: 15); UCGCUGAUGAGCAGCAACGdTdT (SEQ ID NO: 16); fUfUmAfCmAfCAGUAUUAAAGCGATT (SEQ ID NO: 17); (Phos)UCGCUUUAAUACUGUGUAATT (SEQ ID NO: 18); 5′-UUACACAGUAUUAAAGCGATT-3′ (SEQ ID NO: 34); (Phos)5′-mUmCGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 35); (Phos)5′-mU(s)mC(s)GCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 36); (Phos)5′-fUfCGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 37); (Phos)5′-fU(s)fC(s)GCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 38); (Phos)5′-mU(s)mC(s)GCUUUAAUAmCfUmGfUmGfUmAmATT-3′ (SEQ ID NO: 39); (Phos)5′-U(s)CGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 40); (Phos)5′-mUfCmGfCmUfUmUAAfUmAfCmUGmUmGfUmAmATT (SEQ ID NO: 41); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGmCmGmAmUmU-3' (SEQ ID NO: 42); (Phos)5′-mUmCmGmCmUmUmUmAmAmUmAmCmUmGmUmGmUmAmAmUmU-3′ (SEQ ID NO: 43); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGdCdGdATT-3' (SEQ ID NO: 44); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGdCmGmATT-3' (SEQ ID NO: 45); 5'UUACACAGUAUUAAAGCGA-3' (SEQ ID NO: 46); (Phos)5′-U(s)CGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 47); (Phos)5′-UCGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 48); (Phos)5'-U(s)C(s)GCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 49); 5′-mUmUACACAGUAUUAAAGCGA-3′ (SEQ ID NO: 50); (Phos)5′-U(s)CGCUUUAAUACUGUGUmAmATT-3′ (SEQ ID NO: 51); (Phos)5′-UCGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 52); (Phos)5'-U(s)C(s)GCUUUAAUACUGUGUAAT(s)T-3' (SEQ ID NO: 53); 5′lUlUlAlClACAGUAUUAAAGCGATT-3′ (SEQ ID NO: 54); (Phos)5′-UCGCUUUAAUACUGlUlGlUlAlATT-3′ (SEQ ID NO: 55); 5'fUfUlAfClACAGUAUUAAAGCGA-3' (SEQ ID NO: 56); and (Phos)5'-mU(s)mCmGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 57). In the above sequence, d (nucleotide) = deoxy-(nucleotide), m (nucleotide) = 2'-O-methyl nucleotide, T = thymidine, f (nucleotide) = 2'-fluorodeoxynucleotide, (Phos) = phosphodiester cap; uppercase nucleotide = RNA nucleotide, l (nucleotide) = locked nucleotide, (s) = phosphorothioate.

[0014] In some embodiments, the siRNA has at least one modification selected from a 3' overhang, a 5' overhang, a 5' phosphorylation, a 2' sugar modification, a nucleobase modification, a phosphate backbone modification, and any combination thereof. In some embodiments, the siRNA or shRNA having the above modifications has at least one additional modification selected from a 3' overhang, a 5' overhang, a 5' phosphorylation, a 2' sugar modification, a nucleobase modification, a phosphate backbone modification, and any combination thereof. In some embodiments, the figetin-like 2 is human figetin-like 2. In some embodiments, the siRNA or shRNA is encapsulated within a nanoparticle. In some embodiments, the siRNA or shRNA is delivered by nanoparticles, electroporation / nuclear transfection, Accel siRNA, a viral vector, a peptide, a protein, or an aptamer. In some embodiments, the siRNA or shRNA is delivered by eye drops. In some embodiments, the siRNA or shRNA is delivered by an ophthalmic wafer. In some embodiments, the siRNA or shRNA is delivered by a drug-releasing contact lens. In some embodiments, the siRNA comprises a duplex of SEQ ID NO: 17 and SEQ ID NO: 18. In some embodiments, the shRNA comprises SEQ ID NO:23. DETAILED DESCRIPTION OF THE INVENTION

[0015] The subject matter of the present invention will be more readily understood from a reading of the following detailed description of exemplary embodiments, which form a part of this disclosure. The present invention is not limited to the specific products, methods, conditions, or parameters described and / or illustrated herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments by way of example only, and is not intended to limit the invention as defined by the appended claims.

[0016] Unless otherwise defined, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0017] As used above, and throughout this disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings:

[0018] In this disclosure, unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include the plural, and a reference to a particular numerical value includes at least that particular value. Thus, for example, a reference to "a compound" includes a reference to one or more of such compounds and equivalents thereof known to those of skill in the art. As used herein, the term "plurality" means two or more. When a range of values ​​is recited, another embodiment includes a range from one particular value to another particular value.

[0019] Similarly, when values ​​are stated as approximations, use of the term "about" will be understood to indicate that the particular value forms another embodiment. All ranges are inclusive and combinable. In the context of the present disclosure, the term "about" encompasses deviations from the indicated numerical value within ±20%, preferably within ±10%, and more preferably within ±5%.

[0020] As used herein, the terms "treat," "treatment," or "therapy" (and variations thereof) refer to therapeutic treatment, including prophylactic treatment, aimed at preventing or slowing (reducing) undesirable physiological changes associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease or condition, stabilization of the disease or condition (i.e., when the disease or condition does not worsen), delaying or reducing the progression of the disease or condition, improvement or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition. Those in need of treatment include those already suffering from the disease or condition, as well as those susceptible to the disease or condition and those in whom the disease or condition is to be prevented.

[0021] As used herein, the terms "ingredient," "composition," "formulation," "composition of compound," "compound," "agent," "pharmaceutical active agent," "active agent," "treatment," "therapy," "treatment," or "drug" are used interchangeably herein, depending on the context, and refer to a compound or composition that, when administered to a subject (human or animal), elicits a desired pharmacological and / or physiological effect through local and / or systemic action. A personalized composition or method refers to a product or its use in a dosing regimen that is tailored or individualized to meet the specific needs identified or anticipated in the subject.

[0022] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to an animal, e.g., a human, to which treatment with a composition or formulation according to the subject matter of the present disclosure is provided. As used herein, the term "subject" refers to a human and a non-human animal. The terms "non-human animal" and "non-human mammal" are used interchangeably herein and include all vertebrates, e.g., mammals such as non-human primates (e.g., higher primates), sheep, dogs, rodents (e.g., mice, rats), guinea pigs, goats, pigs, cats, rabbits, cows, and horses, and non-mammals such as reptiles, amphibians, chickens, and turkeys. The compositions described herein can be used to treat any suitable mammal, including primates (e.g., monkeys, humans), horses, cows, cats, dogs, rabbits, and rodents (e.g., mice, rats). In one embodiment, the mammal being treated is a human. The human can be of any age. In one embodiment, the human is an adult. In another embodiment, the human is a child. The human can be male, female, pregnant, middle-aged, adolescent, or elderly. According to any of the methods of the presently disclosed subject matter, in one embodiment, the subject is a human. In another embodiment, the subject is a non-human primate. In another embodiment, the subject is a rodent, in one embodiment a mouse, and in another embodiment a rat. In another embodiment, the subject is a dog, cat, cow, horse, rabbit, or pig. In another embodiment, the subject is a mammal.

[0023] As used herein, the conditions and disorders in a subject for which a particular drug, compound, composition, formulation (or any combination thereof) is "indicated (prescribed)" are not limited to those conditions and disorders for which the drug, compound, composition, or formulation has been expressly approved by a regulatory agency, but also include other conditions and disorders known or reasonably believed by a physician or other health or nutritional professional to be suitable for treatment with the drug, compound, composition, formulation, or any combination thereof.

[0024] Other abbreviations used herein include: Fi2, figetin-like 2; FL2, figetin-like 2; FL2-NPsi, rRat analog of SiFi2, silencing of FL2 by siRNA; HSV, herpes simplex virus; HZV, varicella-zoster virus; HZO, varicella-zoster ophthalmicus; MT, microtubules; NK, neurotrophic keratitis; SiFi2, small interfering ribonucleic acid targeting figetin-like 2; siRNA, small interfering ribonucleic acid.

[0025] The present disclosure relates to treating neurotrophic keratitis (NK) by administering to a subject (patient) an RNA interfering agent that targets figetin-like 2 (FL2).

[0026] Neurotrophic keratitis

[0027] Neurotrophic keratitis (NK; also known as neuroplegic keratitis) is a rare degenerative corneal disease caused by damage to the trigeminal nerve and characterized by reduced or absent corneal sensitivity accompanied by defects in the corneal epithelium, which can lead to corneal ulceration, infection, melting, and perforation, secondary to poor corneal healing and ultimately to severe visual impairment or blindness.

[0028] NK develops when persistent impairment of corneal sensory innervation leads to dysfunction of posterior ganglion nerve fibers. As shown in Table 1, there are multiple etiologies that share a common pathway of altered corneal sensory innervation. The disclosed methods target all causes of NK, including but not limited to those listed in Table 1. The most common causes include herpes simplex virus (HSV) and herpes zoster virus (HZV) infection, which accounts for up to 27% of NK cases (Bonini et al. 2003, Sacchetti et al. 2014, Versura et al. 2018). Chemical burns, corneal surgery, and other corneal trauma are other common ophthalmic causes. Genetic factors and systemic conditions may also lead to the development of NK.

[0029] [Table 1]

[0030] The cornea is innervated by the ophthalmic branch of the trigeminal nerve and autonomic nerves. Corneal nerves are essential for maintaining a healthy corneal epithelium by activating protective reflexes after injury and providing trophic factors (e.g., calcitonin gene-related peptide, acetylcholine, serotonin, substance P, and neuropeptide Y) that promote the proliferation, migration, adhesion, and differentiation of corneal epithelial cells. Furthermore, corneal epithelial cells play an important role in maintaining corneal homeostasis by secreting various neurotrophic growth factors, such as ciliary neurotrophic factor, glial cell line-derived neurotrophic factor, and nerve growth factor. Epithelial cells interact with corneal nerves in a mutually supportive relationship, promoting their development and survival. Therefore, impaired sensory innervation disrupts crosstalk between corneal nerves and epithelial cells, leading to NK, resulting in epithelial destruction, poor wound healing, and persistent corneal defects.

[0031] The diagnosis of NK is based on a medical history, ocular examination, and tests to assess for corneal sensitivity loss and nerve damage (Sacchetti et al. 2014, Feroze et al. 2022). The clinical picture of NK is primarily unilateral and characterized by progressive ocular surface changes. The severity of corneal damage in NK is classified into three stages using the Mackie system: relatively mild Stage 1 (corneal epithelial changes), moderate Stage 2 (current or persistent corneal epithelial defect), and more severe Stage 3 (corneal ulcer, perforation, or melting) (Sacchetti et al. 2014).

[0032] [Table 2]

[0033] Patients also commonly experience redness, dry eyes, decreased vision, and blurred vision due to chronic epithelial defects (Sacchetti et al. 2014, Versura et al. 2018, Feroze et al. 2022). The prognosis for NK depends on the severity of the disease and the stage of the disease at the time of diagnosis. Given the natural history of NK, it is essential to inhibit the progression of corneal damage and promote epithelial healing. The present disclosure provides methods and agents for treating NK, including, but not limited to, inhibiting the progression of corneal damage and promoting epithelial healing.

[0034] The methods and medicaments for treating NK according to the present disclosure may be combined with one or more other therapies for NK. In one embodiment, the other therapy is the use of senegelmine (Oxervate™), a recombinant human nerve growth factor (Oxervate 2019). In one embodiment, the other therapy is the use of topical antibiotics to prevent infection. In one embodiment, the other therapy is the use of therapeutic contact lenses to promote corneal healing. In one embodiment, the other therapy is the use of surgical procedures to treat corneal ulcers that are resistant to drug therapy.

[0035] The disclosed methods and medicaments for use in NK patients are intended to treat degenerative diseases of the cornea caused by damage to the trigeminal nerve, which can be caused by a variety of precipitating events (such as, but not limited to, those listed in Table 1), but which do not typically progress to NK. Thus, the use of the disclosed methods and medicaments differs from treating the causes of NK, such as those listed in Table 1. As described herein, treatment of NK according to the present disclosure is generally initiated at the time of diagnosis of NK and is not sequential with treatment of the causes listed in Table 1.

[0036] In one embodiment, treatment of NK according to the present disclosure is initiated at the time of diagnosis of NK. In some embodiments, treatment of NK according to the present disclosure is initiated when the corneal defect does not resolve or heal. In some embodiments, treatment of NK according to the present disclosure is initiated when the corneal defect does not heal, in some embodiments, 1 month, 2 months, 3 months, 4 months, or more than 4 months from the time of diagnosis of NK. In some embodiments, treatment of NK according to the present disclosure is initiated when the corneal defect does not heal 1 month, 2 months, 3 months, 4 months, or more than 4 months from the time of onset. In some embodiments, the onset of NK can precede the diagnosis of NK.

[0037] Clinical evaluation of RNAi agents in NK

[0038] In one embodiment, an 8-week Phase I / II, multicenter, randomized, double-blind, vehicle-controlled, parallel-group study was conducted to evaluate the activity and efficacy of two dose frequencies of 20 μg of liposomally formulated siRNA against figetin-like 2 for the treatment of NK (compared to eye drops and vehicle control groups) in patients with stage 2 and stage 3 neurotrophic keratitis.

[0039] In some embodiments, for siRNA 9 (e.g., duplexes of SEQ ID NO: 17 and SEQ ID NO: 18 in a liposomal formulation), the siRNA is 20 μg. One 35 μl drop corresponds to 9.37 μg of siRNA duplex. In some embodiments, the solvent control is an ophthalmic solution of the same composition as the test product, but without the siRNA. In some embodiments, the test product and solvent are formulated as single-use formulations (frozen solutions packaged in glass vials) and administered using the sterile polyethylene dropper provided in the kit. In one embodiment, the siRNA is stored frozen. In some embodiments (at home), the test drug is stored in a refrigerator at 2-8°C for up to 7 days. In some embodiments, one drop (35 μl) of the test drug is administered to the affected eye six times daily during the 8-week randomized, double-blind, controlled treatment period and the 8-week uncontrolled treatment period of eligible subjects (see Study Design below).

[0040] In some embodiments, the diagnosis of NK is based on a review of the medical history, an eye examination, and tests to assess for decreased corneal sensitivity and nerve damage (Sacchetti, M. and A. Lambiase, Diagnosis and management of neurotrophic keratitis. Clinical ophthalmology (Auckland, NZ), 2014. 8: pp. 571-579, Feroze KB, Patel BC,; Neurotrophic keratitis, StatPearls 2022 May 24). In one embodiment, the clinical presentation of NK is characterized by predominantly unilateral, progressive ocular surface changes. In some embodiments, the severity of corneal damage in NK is classified into three stages using the Mackie system, as shown in Table 1 above: relatively mild Stage 1 (corneal epithelial changes), moderate Stage 2 (current or persistent corneal epithelial defects), and more severe Stage 3 (corneal (stromal) ulceration, perforation, or melting) (Sacchetti et al. 2014). In some embodiments, patients typically present with redness, dry eyes, decreased vision, and blurred vision due to chronic epithelial defects (Sacchetti et al. 2014, Versura, P., et al., Neurotrophic keratitis: current challenges and future prospects. Eye and brain, 2018. 10: pp. 37-45. Feroze et al. 2022). In some embodiments, the prognosis for NK depends on the severity of the disease and the stage of the disease at the time of diagnosis. Considering the natural history of NK, it is essential to inhibit the progression of corneal damage and promote epithelial healing.

[0041] In some embodiments, Phase I and II studies begin with an 8-week randomized, controlled, double-blind treatment period. In some embodiments, after the 8-week controlled treatment period, patients are assessed as completely cured (CH) or not completely cured (NCH) and then enter a 48-week or 56-week follow-up period. In some embodiments, the follow-up period is 48 weeks for patients who: 1. Patients who were originally randomized to the siRNA group, regardless of whether they achieved complete or incomplete healing at 8 weeks after administration. 2. Patients who were initially randomized to the vehicle control group and achieved complete recovery after 8 weeks of treatment.

[0042] In some embodiments, the follow-up period will be 56 weeks for patients who: 1. Patients who were initially randomized to the vehicle control group and who have not completely recovered after 8 weeks will be administered siRNA during an 8-week uncontrolled treatment period (weeks 8 to 16). Patients will be randomized to one of two regimens. After the uncontrolled treatment period, these patients will be followed for 48 weeks, resulting in a total follow-up period of 56 weeks (i.e., 8 weeks of siRNA treatment + 48 weeks of follow-up).

[0043] In one embodiment, 20 μg of siRNA is administered once every 48 hours for 8 weeks (regimen 1) or 20 μg of siRNA is administered once daily for 8 weeks (regimen 2).

[0044] Primary Objectives and Endpoints: In some embodiments, the objective of the Phase I study is to evaluate the safety and pharmacokinetic profile of the siRNA eye drops administered at the proposed clinical dose in the target patient population (one 20 μg drop administered every other day or daily for Stage 2 and Stage 3 NK patients). In some embodiments, the incidence of ophthalmic adverse events (AEs) is assessed by routine ophthalmic examinations performed on all subjects throughout the treatment and follow-up periods. In some embodiments, such ophthalmic examinations include visual acuity assessment, corneal photography, manifest refraction, confocal microscopy (for a subset of subjects), corneal sensitivity testing, slit-lamp examination, and dilated fundus examination. In some embodiments, ophthalmic examinations are performed weekly during the treatment period, monthly (12 weeks + / - 3 days) during the follow-up period, and then every three months for the remainder of the follow-up period. Systemic adverse events (AEs) occurring in patients during treatment or the 48-week follow-up period are recorded.

[0045] In some embodiments, primary endpoints of the Phase I and / or Phase II trials include one or more of the following: The incidence of ophthalmic adverse events in the treatment and vehicle control groups during the treatment or follow-up period. Ophthalmic adverse events included corneal epithelial defects, corneal neovascularization, corneal thinning, ocular inflammation, eye pain, anterior chamber congestion, keratitis, decreased visual acuity, corneal deposits, ophthalmia, eye pain, increased lacrimation, ocular redness, and increased intraocular pressure. - The incidence of systemic adverse events in the treatment and control groups during the treatment or follow-up period. ·Determining the pharmacokinetic profile of siRNA and collecting data on the potential systemic exposure of siRNA in stage 2 and stage 3 NK patients treated with siRNA via topical application to the eye.

[0046] In some embodiments, secondary endpoints of the Phase I and / or Phase II trials include one or more of the following: · Clinical examinations (hematological and serum chemistry parameters) will be performed at the beginning and end of the study. Heart rate and blood pressure will be measured at baseline, 4 weeks, and 8 weeks, and electrocardiograms will be measured at baseline, 4 weeks, and 8 weeks.

[0047] In some embodiments, the primary objective of the Phase II study is to evaluate the efficacy of siRNA eye drops (20 μg administered every other day or once daily) compared to a control in inducing complete healing of Stage 2 (PED) and Stage 3 (corneal ulcer) NK by centrally assessing clinical features of corneal fluorescein staining. In some embodiments, secondary objectives of the Phase II study are to evaluate one or more of the following: investigator assessment of complete healing, duration of complete healing, improvement in visual acuity, improvement in corneal sensitivity, and proportion of patients achieving complete corneal clearing, defined as complete disappearance of staining on the modified Oxford Scale.

[0048] In some embodiments, the main secondary objective is assessment of complete corneal healing (lesion size less than 0.5 mm) by clinical assessment of corneal fluorescein staining at a central reading center.

[0049] In some embodiments, other secondary objectives include one or more of the following: -Evaluation of visual acuity improvement using the ETDRS chart. Assessment of improvement in corneal sensitivity as measured by Cochet-Bonnet aesthesiometer.

[0050] In some embodiments, the primary efficacy endpoint is the proportion of patients achieving complete healing (lesion size of 0 mm and no residual staining) in stage 2 (PED) and stage 3 (corneal ulcer) NK at 8 weeks based on assessment of corneal fluorescein staining images at a central reading center. In some embodiments, corneal fluorescein staining is assessed with a slit lamp using a yellow barrier filter and cobalt blue illumination using the modified Oxford corneal scale.

[0051] In one embodiment, secondary efficacy endpoints include one or more of the following: The proportion of patients with complete healing of PED or corneal ulcer as assessed by corneal fluorescein staining at 4, 6, 8, 12, 20, 32, 44, and 56 weeks as defined by the investigator. The proportion of patients with complete corneal clearing (grade 0 on the modified Oxford Scale) at 4, 6, 8, 12, 20, 32, 44, and 56 weeks. -Mean change in best corrected distance visual acuity (BCDVA) from baseline to 8 weeks after treatment. The proportion of patients achieving a 15-letter or greater gain in BCDVA at 4, 6, and 8 weeks. The proportion of patients who achieved improvement in corneal sensitivity as measured by Cochet-Bonnet aesthesiometer at 4, 6, and 8 weeks (binary variable of target attainment: "corneal sensitivity at 4, 6, and 8 weeks" - "baseline corneal sensitivity" > 0 [Yes / No]). The proportion of patients with stage 2 or 3 NK whose lesions worsened (increase in lesion size of ≥1 mm, decrease in BCDVA by ≥5 letters on ETDRS, progression of lesion depth (corneal melting or perforation), or development of infection) from baseline to 4, 6, and 8 weeks after treatment. Time to onset of exacerbation from baseline to 8 weeks after treatment.

[0052] In some embodiments, one or more exploratory efficacy variables are evaluated, including time to complete corneal clearing and time to onset of healing (defined as a 20% or greater reduction in the maximum diameter of the lesion), change in unanesthetized Schirmer score, change in tear film osmolality, and change in National Eye Institute Visual Function Questionnaire-25 (NEI-VFQ) and / or EuroQol 5D (EQ-5D) score.

[0053] In some embodiments, the target number of participants for the study is calculated based on an estimated 60% complete healing rate of PED or corneal ulcers in patients receiving the siRNA eye drops at 4 weeks compared to 30% in the vehicle control group. In some embodiments, a Phase II study requires 141 evaluable subjects to have 80% power to detect such a difference. In some embodiments, at least 156 subjects are randomized into the Phase II study, assuming a dropout rate of 10-20%. In some embodiments, this number of participants is determined to be sufficient to evaluate safety for progression to the Phase II study. In some embodiments, a total of at least 174 subjects (including the 18 patients in the Phase I study) are planned to be randomized.

[0054] In some embodiments, Phase II studies include a baseline randomization scheme for all subjects randomized to vehicle control and active second-line treatment, defined as an active regimen administered during the 56-week follow-up period if the vehicle control patient has not fully healed after 8 weeks or if PED or corneal ulcer recurrence occurs.

[0055] In some embodiments, the selection criteria include one or more of the following: Patients aged 18 years or older. Patients with stage 2 PED or stage 3 (corneal ulcer) NK. Patients with stage 2 or 3 NK in only one eye are accepted. Patients with PED or corneal ulcers that have persisted for more than 2 weeks and are resistant to one or more conventional non-surgical treatments for NK (e.g., preservative-free artificial tears, gels, ointments; preservative-containing eye drops and discontinuation of corneal desensitizing medications; therapeutic contact lenses). Patients with evidence of decreased corneal sensitivity (≤4 cm as measured by a Cochet-Bonnet aesthesiometer) in at least one corneal quadrant within the area of ​​PED or corneal ulcer and outside the defect area. Patients with a best-corrected distance visual acuity (BCDVA) score of 75 or less in Early Treatment Diabetic Retinopathy Study (ETDRS) letters (LogMAR ≥ +0.2, Snellen ≤ 20 / 32, or decimal ≤ 0.625 equivalent). Patients without objective clinical evidence of improvement of PED or corneal ulcer within 2 weeks prior to study enrollment.

[0056] In some embodiments, the exclusion criteria include any one or more of the following: The affected eye has an active ocular infection (bacterial, viral, fungal, or protozoal) or active ocular inflammation that is not related to NK. - If you have any other ocular disease that requires topical ophthalmic treatment of the affected eye during the study treatment period. Topical ophthalmic treatments other than the investigational drug provided by the study sponsor or permitted by the study protocol must not be administered to the affected eye during the study period. -Patients with severe visual impairment in the affected eye who are judged by the investigator to have no chance of improving visual acuity in the affected eye with the administration of the investigational drug. -Patients whose Schirmer test result under unanesthetized conditions is 3mm / 5 minutes or less in the affected eye. Patients with severe blepharitis and / or severe meibomian gland disease in the affected eye. Patients who have had ophthalmic surgery (such as laser surgery or refractive surgery) on the affected eye within 3 months prior to study enrollment. Patients with a history of surgery to treat NK in the affected eye (e.g., complete conjunctival blepharoplasty or conjunctival flap, excluding amniotic membrane transplantation). Patients who have previously been treated with Botox (botulinum toxin) injections are accepted only if the last injection was administered at least 90 days prior to enrollment in this study. -Patients who use refractive contact lenses during treatment of the affected eye. - If the need for punctal occlusion is anticipated during the treatment period. Patients who had punctal occlusion or eye plugs inserted before the study were eligible for enrollment if punctal occlusion was maintained during the study period. Patients with evidence of corneal ulcer, corneal melt, or perforation involving the posterior third of the cornea in the affected eye. Patients with or a history of ocular or systemic disorders or diseases that may interfere with the administration or evaluation of the investigational product, may interfere with the interpretation of study results, or that the investigator determines are incompatible with the scheduling or conduct of study visits (e.g., progressive or degenerative corneal or retinal disease, uveitis, optic neuritis, uncontrolled diabetes, autoimmune disease, systemic infection, neoplastic disease). Anticipated need to change the dose of systemic medications known to inhibit trigeminal nerve function (e.g., neuroleptics, antipsychotics, antihistamines) unless initiated at least 30 days before study enrollment and stable throughout the study treatment period. Patients with known hypersensitivity to RNA-based therapeutics.

[0057] In some embodiments, "complete healing" includes Dompe's original definition: a maximum diameter of corneal fluorescein staining (measured at the baseline visit) in the area of ​​persistent epithelial defect (PED) or corneal ulcer of less than 0.5 mm at the time of evaluation. In some embodiments, "complete absence of staining" refers to any one or more or all of the following: At the time of evaluation, no fluorescein staining remains in the corneal lesions. · No persistent staining in other areas of the cornea in photographs taken at different times during the study period (i.e., no change in shape and / or position at different times).

[0058] In one embodiment, corneal images that require review at a central review center without disclosure to the patient were taken at the visit corresponding to each image. In some embodiments, all images are reviewed for patients who are determined to have completely healed (Dompe definition) or have a lesion size of 0 mm at any time point, and the following is determined for each patient: Is there residual staining (i.e., is there staining outside the lesion area)? (Yes / No) If residual staining is present, is it persistent? (Persistent means that the staining persists in a specific area of ​​the cornea and has not faded or changed in shape / location between images) (Yes / No)

[0059] [Table 3]

[0060] In some embodiments, all post-hoc efficacy analyses are performed using the ITT population. Analysis of post-hoc efficacy endpoints for Phase II studies used data from the 8-week controlled treatment period. In some embodiments, for the chi-squared analyses of achievement of complete cure at 4 and 8 weeks, missing data are imputed using the last observation carried forward (LOCF) method. In some embodiments, these analyses impute missing data as failure (worst-case scenario). In this case, data are considered missing at a particular visit, regardless of the reason for the missing data. In some embodiments, all other post-hoc analyses are performed based on observed cases, and no further imputation of missing data is performed unless explicitly stated otherwise.

[0061] Test product: 20 μg of SiFi2 was formulated in a liposomal formulation. One 35 μl drop corresponds to 9.37 μg of siRNA dimer.

[0062] Solvent control: An ophthalmic solution of the same composition as the test product, except that it does not contain siRNA.

[0063] Preparation of test product and vehicle control:

[0064] The test product and vehicle were formulated as single-use formulations (frozen solutions packaged in glass vials) and administered using sterile polyethylene droppers (a dropper was provided for each vial in the kit box). The study medications were supplied in identical boxes. Each treatment kit contained seven boxes, each containing six frozen single-use vials of randomly assigned medication for each day of treatment. Thus, each treatment kit contained a total of 42 vials. At home, the study medication was stored in a refrigerator at 2–8°C for a maximum of 7 days. When patients began using the study medication, they removed one day's supply box (containing six single-use vials) from the refrigerator for that day's use.

[0065] Eligible subjects will receive one drop (35 μl) of study medication six times daily in the affected eye during an 8-week randomized, double-blind, controlled treatment period and an 8-week uncontrolled treatment period (see Study Design below). The estimated time to complete enrollment is 3 months.

[0066] FL2 inhibitors

[0067] In one embodiment, treating NK comprises administering to a patient in need thereof an agent that decreases the expression or activity of figetin-like 2 (FL2) in the cornea.

[0068] Non-limiting examples of inhibitors that inhibit the expression or activity of FL2 include aptamers, nucleic acids, oligonucleotides, and small molecules (2000 daltons or less). In one embodiment, agents that inhibit the expression or activity of FL2 include, but are not limited to, nucleic acids, such as RNA interference agents. Non-limiting examples of RNA interference agents include shRNA and siRNA.

[0069] siRNA

[0070] In one embodiment, the RNA interference agent is a siRNA (small interfering RNA). In one embodiment, the siRNA used in the methods or compositions described herein comprises a portion complementary to an mRNA sequence encoding a figetin-like 2 protein. In one embodiment, the figetin-like 2 protein is human figetin-like 2 protein. In one embodiment, the mRNA is encoded by the DNA sequence NCBI Reference Sequence: NM_1013690.4 (SEQ ID NO: 19), and the siRNA is effective to inhibit the expression of the figetin-like 2 protein. In one embodiment, the figetin-like 2 protein comprises consecutive amino acid residues having the sequence set forth in SEQ ID NO: 20.

[0071] In one embodiment, the siRNA comprises a double-stranded portion (duplex). In one embodiment, the siRNA is 20-25 nucleotides in length. In one embodiment, the siRNA comprises a 19-21 core RNA duplex with one or two nucleotide 3' overhangs on one or both strands, independently of each other.

[0072] siRNA oligonucleotides may be modified to enhance their activity or inhibit their degradation according to teachings in the art, particularly, for example, Chakraborty et al., 2017, Mol Ther Nucleic Acids 8:132-143, which is incorporated herein by reference. In one embodiment, the siRNA may or may not be 5' phosphorylated and may be modified with any of the modifications known in the art to improve efficacy and / or resistance to nuclease degradation. In one embodiment, the siRNA may be administered such that it is transfected into one or more cells. In one embodiment, the siRNA is 5' phosphorylated.

[0073] In one embodiment, the 5'-terminal residue of the siRNA strand is phosphorylated. In one embodiment, the 5'-terminal residue of the antisense strand of the siRNA is phosphorylated. In one embodiment, the siRNA of the present disclosure comprises double-stranded RNA, one strand of which is 80, 85, 90, 95, or 100% complementary to a portion of an RNA transcript of a gene encoding a figetin-like 2 protein. In one embodiment, the RNA transcript of a gene encoding a figetin-like 2 protein is mRNA. In one embodiment, the figetin-like 2 protein is human figetin-like 2 protein. In one embodiment, the siRNA of the present disclosure comprises double-stranded RNA, one strand of which comprises a portion having a sequence identical to a portion of 18 to 25 contiguous nucleotides of an RNA transcript of a gene encoding a figetin-like 2 protein. In one embodiment, the figetin-like 2 protein is human figetin-like 2 protein. In yet another embodiment, the siRNA of the present disclosure comprises double-stranded RNA, in which both strands of RNA are linked by a non-nucleotide linker. Alternatively, the siRNA of the present disclosure comprises a double-stranded RNA in which both strands of the RNA are connected by a nucleotide linker, such as a loop or stem-loop structure.

[0074] In one embodiment, the single-stranded component of the siRNA of the present disclosure is 14 to 50 nucleotides in length. In another embodiment, the single-stranded component of the siRNA of the present disclosure is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides in length. In yet another embodiment, the single-stranded component of the siRNA of the present disclosure is 21 nucleotides in length. In yet another embodiment, the single-stranded component of the siRNA of the present disclosure is 22 nucleotides in length. In yet another embodiment, the single-stranded component of the siRNA of the present disclosure is 23 nucleotides in length. In one embodiment, the siRNA of the present disclosure is 28 to 56 nucleotides in length. In another embodiment, the siRNA of the present disclosure is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 nucleotides in length.

[0075] In one embodiment, the siRNA of the present disclosure comprises at least one modification selected from a 3' overhang, a 5' overhang, a 5' phosphorylation, a 2' sugar modification, a nucleobase modification, a phosphate backbone modification, and any combination thereof. Such one or more modifications may be made in addition to any of the modifications in any of the sequences disclosed herein.

[0076] In another embodiment, the siRNA of this disclosure comprises a 3' overhang. In another embodiment, the siRNA of this disclosure comprises a 5' overhang. In another embodiment, the siRNA of this disclosure comprises at least one 2' sugar modification. Examples of 2' sugar modifications include, but are not limited to, 2'-azido-2'-deoxycytidine ribonucleic acid, 2'-azido-2'-deoxyuridine ribonucleic acid, 2'-azido-2'-deoxyadenosine ribonucleic acid, 2'-azido-2'-deoxyguanosine ribonucleic acid, 2'-fluoro-2'-deoxyadenosine ribonucleic acid, 2'-fluoro-2'-deoxycytidine ribonucleic acid, 2'-fluoro-2'-deoxyuridine ribonucleic acid, 2'-fluorothymidine ribonucleic acid, 2'-O-methyladenosine ribonucleic acid, 2'-O-methylcytidine ribonucleic acid, 2'-O-methylguanosine ribonucleic acid, 2'-O-methyluridine ribonucleic acid, etc. In another embodiment, the siRNA of the present disclosure comprises at least one nucleobase modification. Nucleobase modifications include, but are not limited to, 2'-fluorodeoxycytidine ribonucleic acid, 2'-fluorodeoxyuracil ribonucleic acid, 2'-O-methyladenosine ribonucleic acid, etc. Other nucleotide modifications are described in Chiu et al., 2003, RNA 9(9):1034-1048, and Peacock et al., 2011, J Org Chem 76(18):7295-7300, which are incorporated herein by reference.

[0077] In another embodiment, the siRNA of the present disclosure comprises at least one phosphate backbone modification. In another embodiment, the siRNA of the present disclosure comprises at least one 5' phosphorylation. As used herein, "at least one" means one or more.

[0078] Such one or more modifications may be made in addition to any such modifications in any of the sequences disclosed herein.

[0079] NCBI reference sequence: NM_1013690.4 (nucleic acid encoding human figetin-like 2) 1 agtgagctat ggggacacta ctgcactgta gcctgggcaa cagagcaaga ccttgtctca 61 aaaatgtata tatattttgg gctttttttc ctaaaacggg aactacaaca gcatatttgc 121 gagctgatga gagtgacca gcagagaggg aaatggatca gctctgttga agatgcactg 181 gacaccagaa cacgcccagc ccctcaacca gtggccagag cagcacctgg acgtctcctc 241 caccaccccg tcgccggccc acaagttgga gttgccccct gggggtcgcc aacgctgcca 301 ctacgcttgg gcacacgacg acatctcagc cctcactgcc tccaacctcc taaagcgcta 361 tgcagagaag tactctgggg tcttggattc tccctacgag cgtccggccc tgggcgggta 421 cagcgacgcc tccttcctca acggcgccaa aggggatccc gagccctggc cagggccgga 481 gccaccctac ccctggcct cactccacga aggcctccca ggaaccaaat cgggcggtgg 541 cggcggttcc ggggccctgg ggggctcccc agttttagcc gggaacctcc ctgaacccct 601 ctacgccggc aatgcgtgcg ggggcccatc ggcggcgccc gagtacgcgg ccggctacgg 661 cggggggtac ctggcgccgg gttactgcgc gcagacgggc gccgcgctgc ccccgccgcc 721 cccggccgcg ctcctgcagc ccccaccgcc tccggggtac gggccctcag cgccgctgta 781 caactatccc gcagggggct acgcagcgca gcccggctat ggcgcgctcc cgccgccccc 841 aggcccaccc ccggccccct acctgacccc gggcctgccc gcgcccacgc ccctgcccgc 901 gccggcaccg cccaccgcct atggcttccc cacggccgcg ccgggtgccg aatccgggct 961 gtcgctgaag cgcaaggccg ccgacgaggg gcccgagggc cgctaccgca agtacgcgta 1021 cgagcccgcc aaggcccccg tggctgacgg agcctcctac cccgccgcgg acaacggcga 1081 atgtcggggc aacgggttcc gggccaagcc gccaggagcc gcggaggagg cgtcgggcaa 1141 gtacggtggc ggcgtccccc tcaaggtcct gggctccccc gtctacggcc cgcaactgga 1201 gccctttgaa aagttcccgg agcgggcccc ggctcctcgt ggggggttcg ccgtgccgtc 1261 gggggagact cccaaaggcg tggaccctgg ggccctggag ctggtgacga gcaagatggt 1321 ggactgcggg cccccggtgc agtgggcgga tgtggcgggc cagggcgcgc tcaaggcggc 1381 gctggaggag gagctggtgt ggcccctgct caggccgccc gcctacccgg gcagcctgcg 1441 cccgccgcgg accgtcctgc tctttgggcc gcggggcgcg ggcaaagcgc tgctgggccg 1501 ctgcctcgcc acgcagctgg gcgccacgct gttgcgcctg cgcggcgcga ccctggctgc 1561 gcccggcgcc gccgagggcg cgcgcctcct ccaggccgcc ttcgcggccg cgcgctgccg 1621 cccaccctcc gtactcctca tcagcgagct agaggcgctg ctccccgccc gggacgacgg 1681 cgcggcggca gggggcgcgc tgcaggtgcc gctcctggcc tgcctggacg ggggctgcgg 1741 cgcgggggct gacggcgtgc tggttgtggg caccacctcg cggcccgcgg ctctggacga 1801 ggcgacccgc cggcgcttct ctctccgctt ctacgtggcg ctgcccgaca gcccggcccg 1861 cgggcagatc ctgcagcggg cgctggccca gcagggctgc gcgctcagtg agcgggaact 1921 ggcggcgctg gtgcagggca cgcagggctt ctctgggggc gagctggggc agctgtgcca 1981 gcaggcggcg gccggggcgg gcctcccggg gctgcagcgc cccctctcct acaaggacct 2041 ggaggcggcg ctggccaagg tgggccctag ggcctctgcc aaggaactgg actcgttcgt 2101 ggagtgggac aaaatgtacg gctccggaca ctgacggcgc gcgggggagg ccgcgggagc 2161 cgcagtccct ccgtccccgc cgcctccgcg tgggagggat gtcactgact aaacccggct 2221 ggcaggggct ggagtggtga atgtgggatc ggggacagga ggggtctgcc ggtggatatt 2281 ttttttttcg tgggaaggaa aatgcttctg ccaggcagat gccatatgcg ccgtgtactc 2341 aggtttttcc tatttattgt ggactggaag ctcgccatct ccgcccggca gaccgggcag 2401 atccggcatg ggctggcacc cggggcctta agaactcctg ctctcttgcc acaacgcttt 2461 tgtctcctcg ctatctgaat ggcaccctcc ttctccctca ctctctccat cccattctct 2521 gcattctctt ggttttctct cccttttgct ttgtcgctga cacccctgcc caccccatgc 2581 tggccctgtt tctctcctgc ccctccctcc ccagctctcc atccctcacc ctctgtgctt 2641 ctgtctccat ccctggctct ccagcgtccc tggccttttg gtccctgagc tttaatgcct 2701 ttccctgcct tctgttctta tttggactgc agtggccctt tgcaggagct ctggaggccc 2761 aggggctgag gaggagggtt acccctctac ccatctgaaa cctagggtct agggggatca 2821 aggaaaaaaa gtccccaaag aaggggaatt ttttgtttgt ttttgagggg agatcccaga 2881 aatgtagctt gtttcatatt ttagtcttct tatttttgta aaatgtgtag aatttgctgt 2941 3001 gctgctctcc tgtgtgcccc tcacacctgc cccctccccc ccactccatc caggggacca 3061 aattctccca gacactcaaa aaatgagact tacggggaaag gggagaggaa gacccagagg 3121 cctcagtgaa accccagcta ttcctggtca gaagcagaat gtattcctaa gggcttctc 3181 cccagggccg aggcctaggc atgaatgtgg ggagtgggct gtggggtttg agaaaggga 3241 ggccttattc ctctcctgct gctccccacc ccctgcccca cccaacccct ccgctgagtg 3301 ttttctgtga agggctatcc agagttagga tgcccttgcc caattccttc ctgagaccca 3361 gaaggtaggg tgggagggcc caaatgggaa ggtgacctaa gcagaaagtc tccagaaagg 3421 tcatgtcccc tggccctgcc ttggcagagg tccccagtga cttatgctag gaggattcca 3481 tctgggtaga cagtctggcc acaaaatcag ctactggacc tcagccatct ctgctggagg 3541 ctctgaggag gagtgagcat ccctcacttg tgggggctct gtgaggaaat gtgccttccc 3601 cattcccccg gagtcctagg tctggagctc cagggctggg agagggtgag ggagatgggc 3661 aggggtgttt tctctgacct tgggggctta gtctcagtcc tgcctgaact ttccactagg 3721 cttggaaccc ttccaagaac catatttctc tccttcccac caattttccc ttgatgaggc 3781 tttagcagtt tgctcccacc acccccagcc catttcacaa ctctgatctt agtccaaagc 3841 aggggacacg cccccccacc accacttttt ctctctccca tctcagcctc ctgtgcagtt 3901 ccttgcctgc ccgtgcattt cctagagtct actgcctccc ccctggctgg gagggtgtct 3961 gggggggatc tttcaggggc cctggcaccc agggcctgtg ctggcctagg agtgctgacc 4021 agaaggctgc tctgttcccc cccacccccg ttgctttctg gccccctctt tggagccagc 4081 cacccacagg gctttggtgc ctcagaagca gtgggctgcc gggtcacagc cgcaggctgc 4141 aaaagaccct cggagggagc atggagtgag gggttctctc tcaggtgtgt atgtattggg 4201 gggtgggggt gggtggaggg tgtcagggaa gttggggtgg gatcccagcc ttcccttcaa 4261 gaggcaggga gctctgggag gtggagtccc caccgctttc tctactaggc tcctcctgtt 4321 ccccaggctt ggggagcttt gcacaaggag actgccccca gcctagtggc acctacctca 4381 tgggctctgg ggcaggtagg ggaagggcca gtccagctct ggtaatgctg gggggaggca 4441 taccaaagaa tccaggggca gggagtgggg agggtgactt ccgagctggc ctctcccctt 4501 cctctaccca gactggggct gggatcctct cctcccgctg taaccatttc tacctcattt 4561 tgctgcgtgt tgtacatgga cgtatttatc tcctgtctga cgatgctctg cagttgtggt 4621 ctgtctacct cagaagagac tgtattttaa aagaaagtat tacacagtat taaagcgatg 4681 acatgtggtt tgcaaaaaaa aaaaaaaaaa a (SEQ ID NO: 19), and MHWTPEHAQPLNQWPEQHLDVSSTTPSPAHKLELPPGGRQRCHYAWAHD DISALTASNLLKRYAEKYSGVLDSPYERPALGGYSDASFLNGAKGDPEP WPGPEPPYPLASLHEGLPGTKSGGGGGSGALGGSPVLAGNLPEPLYAGN ACGGPSAAPEYAAGYGGGYLAPGYCAQTGAALPPPPPAALLQPPPPPGY GPSAPLYNYPAGGYAAQPGYGALPPPPGPPPAPYLTPGLPAPTPLPAPA PPTAYGFPTAAPGAESGLSLKRKAADEGPEGRYRKYAYEPAKAPVADGA SYPAADNGECRGNGFRAKPPGAAEEASGKYGGGVPLKVLGSPVYGPQLE PFEKFPERAPAPRGGFAVPSGETPKGVDPGALELVTSKMVDCGPPVQWA DVAGQGALKAALEEELVWPLLRPPAYPGSLRPPRTVLLFGPRGAGKALL GRCLATQLGATLLRLRGATLAAPGAAEGARLLQAAFAAARCRPPSVLLI SELEALLPARDDGAAAGGALQVPLLACLDGGCGAGADGVLVVGTTSRPA ALDEATRRRFSLRFYVALPDSPARGQILQRALAQQGCALSERELAALVQ GTQGFSGGELGQLCQQAAAGAGLPGLQRPLSYKDLEAALAKVGPRASAK Encodes ELDSFVEWDKMYGSGH (SEQ ID NO: 20) (human figetin-like 2).

[0080] In one embodiment, the FL2-inhibiting siRNA comprises a sense or antisense sequence selected from the table below.

[0081] [Table 4-1]

[0082] [Table 4-2]

[0083] In the above sequence, d (nucleotide) = deoxy-(nucleotide), m (nucleotide) = 2'-O-methyl nucleotide, T = thymidine, f (nucleotide) = 2'-fluorodeoxynucleotide, (Phos) = phosphodiester cap; capitalized nucleotide = RNA nucleotide, l (nucleotide) = locked nucleotide, (s) = phosphorothioate. Thus, for example, dT represents deoxythymidine, dC represents deoxycytidine, fC represents 2'-fluorodeoxycytidine ribonucleic acid, fU represents 2'-fluorodeoxyuracil ribonucleic acid, mA represents 2'-O-methyladenosine ribonucleic acid, mU represents 2'-O-methyluracil ribonucleic acid, mC represents 2'-O-methylcytosine ribonucleic acid, and mG represents 2'-O-methylguanosine ribonucleic acid.

[0084] In some embodiments, the siRNA has a 5'-phosphodiester cap, as abbreviated as "(Phos)" in the sequences above. In some embodiments, the siRNA does not have a 5'-phosphodiester cap. siRNA sequences that do not have a 5'-phosphodiester cap are fully encompassed herein.

[0085] Phosphorothioate internucleotide linkages are represented in the sequence by "(s)".

[0086] In one embodiment, a locked nucleotide comprises a ribose with a 2'-O, 4'-C methylene bridge, e.g., 2'-O, 4'-C methylene adenosine (lA) ribonucleoside, 2'-O, 4'-C methylene guanosine (lG) ribonucleoside, 2'-O, 4'-C methylene cytidine (lC) ribonucleoside, 2'-O, 4'-C methylene uridine (lU) ribonucleoside, and 2'-O, 4'-C methylene thimine (lT) ribonucleoside. In another embodiment, a locked nucleic acid comprises a methyl group linked to the methylene group. Other types of locked nucleic acids are also encompassed herein.

[0087] In one embodiment, the FL2 siRNA is double-stranded and comprises any complementary sense and antisense sequences selected from the table above.

[0088] Non-limiting examples of such double-stranded sequences include SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and SEQ ID NO: 17 and SEQ ID NO: 18. In one embodiment, the siRNA is a single-stranded sequence selected from SEQ ID NOs: 1 to 18 above.

[0089] In one embodiment, there is provided a double-stranded nucleic acid consisting of complementary nucleic acid molecules selected from SEQ ID NOs: 34 to 57, or from SEQ ID NOs: 1 to 18, or 34 to 57. In one embodiment, the double-stranded nucleic acid comprises a sense strand and an antisense strand. In one embodiment, the double-stranded nucleic acid consists of a sense strand and an antisense strand.

[0090] In one embodiment, a double-stranded nucleic acid is provided, which consists of a sense strand selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 and an antisense strand selected from SEQ ID NOs: 2, 4, 6, 8, 10, 1, 2, 14, 16, and 18.

[0091] In one embodiment, a double-stranded nucleic acid is provided, which consists of a sense strand selected from SEQ ID NOs: 1, 17, 34, 42, 44, 45, 46, 50, and 54 and an antisense strand selected from SEQ ID NOs: 2, 18, 35, 36, 37, 38, 39, 40, 41, 43, 47, 48, 49, 51, 52, 53, 55, and 57.

[0092] In one embodiment, a double-stranded nucleic acid consisting of a sense strand selected from SEQ ID NOs: 1, 17, 34, 42, 44, 45, 46, 50, 54 and 56 and an antisense strand selected from SEQ ID NOs: 2, 4, 6 and 8 is provided.

[0093] In one embodiment, a double-stranded nucleic acid is provided, which consists of a sense strand selected from SEQ ID NOs: 1, 3, 5, and 7 and an antisense strand selected from SEQ ID NOs: 18, 35, 36, 37, 38, 39, 40, 41, 43, 47, 48, 49, 51, 52, 53, 55, and 57.

[0094] In one embodiment, a double-stranded nucleic acid is provided, which consists of a sense strand selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 34, 42, 44, 45, 46, 50, and 54, and an antisense strand selected from SEQ ID NOs: 2, 4, 6, 8, 10, 1, 2, 14, 16, 18, 35, 36, 37, 38, 39, 40, 41, 43, 47, 48, 49, 51, 52, 53, 55, and 57.

[0095] In one embodiment, a double-stranded nucleic acid is provided comprising a sense strand selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 34, 42, 44, 45, 46, 50, and 54, and an antisense strand selected from SEQ ID NOs: 2, 4, 6, 8, 10, 1, 2, 14, 16, 18, 35, 36, 37, 38, 39, 40, 41, 43, 47, 48, 49, 51, 52, 53, 55, and 57.

[0096] In one embodiment, there is provided a double-stranded nucleic acid consisting of SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:34 and SEQ ID NO:35, SEQ ID NO:34 and SEQ ID NO:36, SEQ ID NO:34 and SEQ ID NO:37, SEQ ID NO:34 and SEQ ID NO:38, SEQ ID NO:34 and SEQ ID NO:39, SEQ ID NO:17 and SEQ ID NO:40, SEQ ID NO:34 and SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:43, SEQ ID NO:46 and SEQ ID NO:47, SEQ ID NO:46 and SEQ ID NO:48, SEQ ID NO:46 and SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, SEQ ID NO:46 and SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, or SEQ ID NO:56 and SEQ ID NO:57.

[0097] In one embodiment, a double-stranded nucleic acid is provided that includes at least one nucleic acid molecule selected from SEQ ID NOs: 1 to 18 or 34 to 57.

[0098] In one embodiment, there is provided a double-stranded nucleic acid comprising two nucleic acid molecules selected from SEQ ID NOs: 1 to 18 or 34 to 57. In one embodiment, the double-stranded nucleic acid comprises a sense strand and an antisense strand.

[0099] In one embodiment, each strand of the double-stranded nucleic acid has 52 or fewer nucleotides.

[0100] In one embodiment, a double-stranded nucleic acid is provided, comprising a sense strand comprising a nucleic acid molecule selected from SEQ ID NOs: 1, 17, 34, 42, 44, 45, 46, 50, 54, and 56, and an antisense strand comprising a nucleic acid molecule selected from SEQ ID NOs: 2, 18, 35, 36, 37, 38, 39, 40, 41, 43, 47, 48, 49, 51, 52, 53, 55, and 57.

[0101] In one embodiment, a double-stranded nucleic acid is provided, comprising a sense strand comprising a nucleic acid molecule selected from SEQ ID NOs: 1, 17, 34, 42, 44, 45, 46, 50, 54, and 56, and an antisense strand comprising a nucleic acid molecule selected from SEQ ID NOs: 4, 6, 8, and 10.

[0102] In one embodiment, a double-stranded nucleic acid is provided, comprising a sense strand comprising a nucleic acid molecule selected from SEQ ID NOs: 1, 3, 5, 7, and 9, and an antisense strand comprising a nucleic acid molecule selected from SEQ ID NOs: 18, 35, 36, 37, 38, 39, 40, 41, 43, 47, 48, 49, 51, 52, 53, 55, and 57.

[0103] In one embodiment, the double-stranded nucleic acid comprises a nucleic acid molecule comprising SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:34 and SEQ ID NO:35, SEQ ID NO:34 and SEQ ID NO:36, SEQ ID NO:34 and SEQ ID NO:37, SEQ ID NO:34 and SEQ ID NO:38, SEQ ID NO:34 and SEQ ID NO:39, SEQ ID NO:17 and SEQ ID NO:40, SEQ ID NO:34 and SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:43, SEQ ID NO:46 and SEQ ID NO:47, SEQ ID NO:46 and SEQ ID NO:48, SEQ ID NO:46 and SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, SEQ ID NO:46 and SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55, or SEQ ID NO:56 and SEQ ID NO:57.

[0104] In one embodiment, each strand of the double-stranded nucleic acid has 52 or fewer nucleotides.

[0105] In one embodiment, any one of the above nucleic acids has at least one nucleotide that is modified or further modified. In one embodiment, the modified nucleotide is selected from 2'-O-methyl-adenosine, 2'-O-methyl-uridine, 2'-O-methyl-cytosine, 2'-O-methyl-guanosine, 2'-O-methyl-thymidine, 2'-fluoro-adenosine, 2'-fluoro-cytidine, 2'-fluoro-guanosine, 2'-fluoro-uracil, 2'-fluoro-thymidine, deoxycytosine, deoxyguanosine, deoxyadenosine, deoxythymidine, deoxyuridine, locked adenosine, locked uridine, locked guanosine, locked cytidine, phosphorothioate, and phosphodiester cap. In one embodiment, at least one additional or modified nucleotide is added to the end of the nucleic acid.

[0106] As discussed above, in one embodiment, a locked nucleotide comprises a ribose with a 2'-O, 4'-C methylene bridge, e.g., 2'-O, 4'-C methylene adenosine (lA) ribonucleoside, 2'-O, 4'-C methylene guanosine (lG) ribonucleoside, 2'-O, 4'-C methylene cytidine (lC) ribonucleoside, 2'-O, 4'-C methylene uridine (lU) ribonucleoside, and 2'-O, 4'-C methylene thimine (lT) ribonucleoside. In another embodiment, the locked nucleic acid comprises a methyl group linked to the methylene group. Other types of locked nucleic acids are also encompassed herein.

[0107] In other examples, the siRNA targeting FL2 may be selected from the table below.

[0108] [Table 5]

[0109] In one embodiment, a double-stranded nucleic acid is provided which consists of a sense strand selected from SEQ ID NOs: 1, 17, 34, 42, 44, 45, 46, 50, and 54 and an antisense strand selected from any one of SEQ ID NOs: 58 to 72.

[0110] In one embodiment, a double-stranded nucleic acid is provided which consists of a sense strand selected from SEQ ID NOs: 1, 3, 5, and 7 and an antisense strand selected from any one of SEQ ID NOs: 58 to 72.

[0111] In one embodiment, a double-stranded nucleic acid is provided which consists of a sense strand selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 34, 42, 44, 45, 46, 50, and 54, and an antisense strand selected from any one of SEQ ID NOs: 58 to 72.

[0112] In one embodiment, a double-stranded nucleic acid is provided which consists of a sense strand selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 34, 42, 44, 45, 46, 50, and 54, and an antisense strand selected from any one of SEQ ID NOs: 58 to 72.

[0113] In one embodiment, there is provided a double-stranded nucleic acid consisting of SEQ ID NO:34 and SEQ ID NO:58, SEQ ID NO:34 and SEQ ID NO:59, SEQ ID NO:34 and SEQ ID NO:60, SEQ ID NO:17 and SEQ ID NO:61, SEQ ID NO:34 and SEQ ID NO:62, SEQ ID NO:42 and SEQ ID NO:63, SEQ ID NO:44 and SEQ ID NO:63, SEQ ID NO:45 and SEQ ID NO:63, SEQ ID NO:46 and SEQ ID NO:64, SEQ ID NO:46 and SEQ ID NO:65, SEQ ID NO:46 and SEQ ID NO:66, SEQ ID NO:50 and SEQ ID NO:67, SEQ ID NO:46 and SEQ ID NO:69, SEQ ID NO:54 and SEQ ID NO:70, SEQ ID NO:17 and SEQ ID NO:72, or SEQ ID NO:56 and SEQ ID NO:71.

[0114] In one embodiment, a double-stranded nucleic acid is provided that includes at least one nucleic acid molecule selected from SEQ ID NOs: 58 to 72.

[0115] In one embodiment, there is provided a double-stranded nucleic acid comprising two nucleic acid molecules selected from SEQ ID NOs: 1 to 18 or 34 to 72. In one embodiment, the double-stranded nucleic acid comprises a sense strand and an antisense strand.

[0116] In one embodiment, each strand of the double-stranded nucleic acid has 52 or fewer nucleotides.

[0117] In one embodiment, a double-stranded nucleic acid is provided, comprising a sense strand comprising a nucleic acid molecule selected from SEQ ID NOs: 1, 17, 34, 42, 44, 45, 46, 50, 54, and 56, and an antisense strand comprising a nucleic acid molecule selected from any one of SEQ ID NOs: 58 to 72.

[0118] In one embodiment, a double-stranded nucleic acid is provided, comprising a sense strand comprising a nucleic acid molecule selected from SEQ ID NOs: 1, 3, 5, 7, and 9, and an antisense strand comprising a nucleic acid molecule selected from any one of SEQ ID NOs: 58 to 72.

[0119] In one embodiment, the double-stranded nucleic acid comprises a nucleic acid molecule comprising SEQ ID NO:34 and SEQ ID NO:58, SEQ ID NO:34 and SEQ ID NO:59, SEQ ID NO:34 and SEQ ID NO:60, SEQ ID NO:17 and SEQ ID NO:61, SEQ ID NO:34 and SEQ ID NO:62, SEQ ID NO:42 and SEQ ID NO:63, SEQ ID NO:44 and SEQ ID NO:63, SEQ ID NO:45 and SEQ ID NO:63, SEQ ID NO:46 and SEQ ID NO:64, SEQ ID NO:46 and SEQ ID NO:65, SEQ ID NO:46 and SEQ ID NO:66, SEQ ID NO:50 and SEQ ID NO:67, SEQ ID NO:46 and SEQ ID NO:69, SEQ ID NO:54 and SEQ ID NO:70, SEQ ID NO:17 and SEQ ID NO:72, or SEQ ID NO:56 and SEQ ID NO:71.

[0120] Any of the FL2-targeting siRNA compositions and uses described elsewhere herein can utilize any of the single-stranded nucleic acid sequences of SEQ ID NOs: 58-72 above, or double-stranded nucleic acids comprising or consisting of any of SEQ ID NOs: 58-72.

[0121] In one embodiment, RNAi inhibition of figetin-like 2 protein is achieved by short hairpin RNA ("shRNA"). The shRNA is introduced into suitable cells by vector transduction. In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector includes a promoter. In one embodiment, the promoter is a U6 promoter or an H1 promoter. In one embodiment, the shRNA encoded by the vector is a first nucleotide sequence ranging from 19 to 29 nucleotides that is complementary to a target gene / mRNA, where the mRNA encodes figetin-like 2 protein. In one embodiment, the figetin-like 2 protein is human figetin-like 2 protein. In one embodiment, the shRNA encoded by the vector includes a short spacer (non-hybridizing loop) of 4 to 15 nucleotides and a 19 to 29 nucleotide sequence that is the reverse complement of the first nucleotide sequence. In one embodiment, the siRNA resulting from intracellular processing of the shRNA has a 1- or 2-nucleotide overhang. In one embodiment, the siRNA overhangs generated by intracellular processing of the shRN have two 3' overhangs. In one embodiment, the overhangs are UU.

[0122] shRNA

[0123] In one non-limiting example, an shRNA useful for purposes herein comprises CACGCTGGGCCCTTTGAAGTTCGAAACTTGTCAAAGGGCCGAAAACGGGCCCCCCTTT (SEQ ID NO: 23). In one embodiment, the shRNA sequence consists of CACGCTGGGGCCCTTTGAAGTTCGAAGAACTTGTCAAAGGGCCCCGCTTT (SEQ ID NO: 23).

[0124] preparation

[0125] Any of the nucleic acid sequences described herein can be prepared by any method known in the art and purified by HPLC or any other method to provide an inhibitor suitable for in vitro, ex vivo, or in vivo use as described herein. In some embodiments, the purity of the inhibitor is 85% or greater. In some embodiments, the purity of the inhibitor is 90% or greater. In some embodiments, the purity of the inhibitor is 95% or greater. In some embodiments, the purity of the inhibitor is 98% or greater. In some embodiments, the purity of the inhibitor is 99% or greater. In some embodiments, where the inhibitor is double-stranded or comprises a double strand, the purity of the double strand is 85% or greater. In some embodiments, where the inhibitor is double-stranded or comprises a double strand, the purity of the double strand is 90% or greater. In some embodiments, where the inhibitor is double-stranded or comprises a double strand, the purity of the double strand is 95% or greater. In some embodiments, where the inhibitor is double-stranded or comprises a double strand, the purity of the double strand is 98% or greater. In some embodiments, the inhibitor is double-stranded or comprises double strands, the purity of the double strands is 99% or greater. In some embodiments, the inhibitor is prepared according to current good manufacturing practice. In some embodiments, the inhibitor is prepared for human use. In one embodiment, the inhibitor is prepared for in vitro or ex vivo use for subsequent administration to a human. In one embodiment, the inhibitor is prepared for human administration.

[0126] Pharmaceutical compositions and methods for ocular administration

[0127] In one embodiment, provided is a composition comprising any of the above-mentioned nucleic acid molecules or double-stranded nucleic acids and pharmaceutically acceptable carrier, solvent, excipient or diluent.In one embodiment, the siRNA described herein is formulated in preservative-free sterile liquid for ocular administration.

[0128] As described herein, in one embodiment, one or more agents that inhibit FL2 are administered to the eye for the treatment of NK.

[0129] In one embodiment, the FL2 inhibitor, such as the siRNA described herein, is formulated into an ophthalmic solution for administration to the eye. In one embodiment, the FL2 inhibitor is encapsulated in nanoparticles or liposomes in the formulation.

[0130] In one embodiment, the agent that inhibits the activity or expression of FL2 is provided in an ocular delivery form. Non-limiting examples of such ocular delivery forms include eye drops, drug-releasing contact lenses, and implants. As described herein, the agent formulation may include nanoparticles, liposomes, or other carriers. The present disclosure is not limited by a particular delivery method for treating NK.

[0131] eye drops

[0132] In one embodiment, the agent is provided as an eye drop. Guidance for such formulations is well known in the art. For example, Li et al., 2014, Current Molecular Medicine 14(9):1215-1225, describes how cationic complexing agents such as polyethyleneimine (PEI) increase corneal delivery of siRNA. Baran-Rachwalska et al., 2020, J Controlled Release 326:192-202, describes how a silicon-based hybrid nanoparticle formulation according to U.S. Patent No. 9,132,083 B2 provides siRNA in porous silicon nanoparticles and lipids that penetrate the cornea. Schhiroli et al., 2019, Mol Ther Nucleic Acids 17:891-906, describes cell-penetrating peptide derivatives, such as trifluoromethylquinoline-palmitoyl modification of CGGG[ARKKAAKA]4, to aid in siRNA penetration into the corneal layer. Additionally, Nie et al., 2009, Bioscience Hypotheses 2(4):223-225, suggest various localized means for siRNA delivery that do not involve injection. The above examples and the documents incorporated herein by reference are merely illustrative.

[0133] Drug-releasing contact lenses

[0134] Contact lens drug delivery is generally described in "Barnett in Review of Optometry, August 15, 2021," which describes other current and future means of ocular delivery, such as immersion, molecular imprinting, and colloidal nanoparticle-containing lenses. Also, "Choi et al., 2018, Materials (Basel) 11(7):1125" describes therapeutic contact lenses using polymer solvents for ocular drug delivery.

[0135] wafer

[0136] In one embodiment, a wafer containing an FL2 inhibitor, such as siRNA, is implanted at a site, such as under the eyelid. In one embodiment, the composition of the FL2-siRNA-wafer is 2.5% collagen, 7.5% chondroitin sulfate, 82.5% polyvinylpyrrolidone (PVP), and 7.5% PEG400. The siRNA is then incorporated into the wafer. The siRNA is then measured and optimized for, for example, size (by scanning electron microscope, light scattering microscope, or atomic force microscope), pH, charge, delivery rate, polyplex delivery volume, minimum loading volume, and leakage to obtain an appropriate formulation for achieving good delivery and siRNA knockdown efficacy. The test formulation is applied to the implantation site. The FL2-siRNA-wafer (or a control containing nonsense siRNA) is cut to the appropriate size and applied to the implantation site as a gel-like matrix using forceps.

[0137] One non-limiting example is the sustained-release, biodegradable, bioengineered product NanoM Wafer™, which is a tissue-adhesive product that can be placed under the upper or lower eyelid to release the agents described herein; see Barman et al., 2017, Invest Ophthal. Vis Sci 58(8):4103.

[0138] In one non-limiting example, the nanowafers described in Yuan et al., 2015, ACS Nano, 9(2):1749-1758 are used.

[0139] In another embodiment, the siRNA is delivered by an implantable delivery device such as that described in Zhang et al., 2019, Sci Adv 5:eaaw5296, which is incorporated herein by reference.

[0140] Other means for ocular delivery of the agents disclosed herein are described in Gote et al., 2019, J Pharmacol Exp. Ther. 370(3):602-624. Additional non-limiting examples include the Surodex® Epi-scleral implant and the DSP-Visulex cul-de-sac implant.

[0141] nanoparticles

[0142] In one embodiment, nanoparticles are used to deliver drugs to the eye.The preparation of siRNA nanoparticles is known in the art, as described in Kim et al., 2019, Adv Mater (49) e1903637, epub; Ickenstein et al., 2019, Expert Opin Drug Deliv 16(11): 1205-1226 (September 17 epub).Various methods can be used to prepare nanoparticles for effectively delivering payload siRNA or shRNA into the eye.

[0143] In one embodiment, nanoparticles containing tetramethyl orthosilicate (TMOS) are used. The TMOS nanoparticles contain FL2 siRNA. For example, 500 μl of tetramethyl orthosilicate (TMOS) is hydrolyzed in the presence of 100 μl of 1 mM HCl by sonication on ice for about 15 minutes until a single phase is formed. Then, 100 μl of the hydrolyzed TMOS is added to 900 μl of a solution of 20 μM siRNA (mouse FL2 (Sigma-Aldrich, SASI_Mm02_00354635) or negative control) containing 10 mM phosphate at pH 7.4. A gel is formed within 10 minutes. The gel is frozen at -80°C for 15 minutes and lyophilized.

[0144] Figetin, figetin-like 1, or FL2 inhibitors can be used in compositions containing additives. Examples of suitable additives include sodium alginate as a gelling agent to prepare a suitable base, cellulose derivatives such as guar or xanthan gum, inorganic gelling agents (called thixotropic gel-forming agents) such as aluminum hydroxide or bentonite, polyacrylic acid derivatives such as Carbopol®, polyvinylpyrrolidone, microcrystalline cellulose, and carboxymethylcellulose. Amphiphilic low- and high-molecular-weight compounds and phospholipids are also suitable. Gels can exist as either aqueous hydrogels or hydrophobic organogels, for example, based on mixtures of low- and high-molecular-weight paraffin hydrocarbons and petrolatum. Hydrophilic organogels can be prepared, for example, based on high-molecular-weight polyethylene glycol. These gelatinous forms can be washed. Hydrophobic organic gels are also suitable. Hydrophobic additives such as petrolatum, wax, oleyl alcohol, propylene glycol monostearate, and / or propylene glycol monopalmitostearate, especially isopropyl myristate, may be included. The composition may be in any suitable form. Emulsifiers that may be used in compositions containing an inhibitor of figetin-like 2 include anionic, cationic, or neutral surfactants, such as alkali metal soaps, metal soaps, amine soaps, sulfur compounds, sulfonated compounds, inverse soaps, higher fatty alcohols, partial fatty acid esters of sorbitan and polyoxyethylene sorbitan, such as lanet type, wool wax, lanolin, or other synthetic products for preparing oil / water and / or water / oil emulsions.

[0145] Additionally, compositions containing agents that inhibit figetin-like 2 can contain petrolatum, natural or synthetic waxes, fatty acids, fatty acid alcohols, fatty acid esters, such as monoglycerides, diglycerides, or triglycerides, paraffin oil or vegetable oils, hydrogenated castor oil or coconut oil, porcine fat, synthetic fats (e.g., based on caprylic acid, capric acid, lauric acid, stearic acid, etc., e.g., Softisan®), or triglyceride mixtures such as Miglyol®, which can be used as lipids in the form of fatty and / or oily and / or waxy components for preparing ointments, creams, or emulsions of compositions containing inhibitors of figetin-like 2 used in the methods described herein.

[0146] Osmotically activated acid and alkali solutions, such as hydrochloric acid, citric acid, sodium hydroxide solution, potassium hydroxide solution, and sodium bicarbonate, can also be used as components of the composition, as well as buffer systems such as citric acid, phosphoric acid, Tris buffer, or triethanolamine to adjust the pH. Preservatives such as methyl benzoate, propyl benzoate (paraben), or sorbic acid can also be added to enhance stability.

[0147] Pastes, powders, and solutions are further forms of compositions containing agents that inhibit figetin, figetin-like 1, or figetin-like 2. As a consistency-imparting base, pastes often contain hydrophobic and hydrophilic auxiliary substances, but preferably contain lipophilic auxiliary substances with a very high solids content. To improve dispersibility, flowability, and slip properties and prevent clumping, powders or topically applicable powders can contain, for example, wheat or rice starch, flame-dispersed silicon dioxide, or siliceous earth, which also function as diluents.

[0148] In one embodiment, the composition comprises additional active ingredients, such as one or more antibiotics, antiseptics, vitamins, anesthetics, antihistamines, anti-inflammatory agents, moisturizers, penetration enhancers, and / or anti-irritants.

[0149] Dosing regimen

[0150] The agents described herein that inhibit the expression or activity of FL2 are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, "dosage unit form" refers to a physically discrete unit of agent appropriate for the patient being treated. However, it should be understood that the total daily dosage of the compounds and compositions of the present disclosure will be determined within the scope of sound medical judgment of the attending physician. The specific therapeutically effective dose level for a particular patient will depend on factors well known in the medical field, such as the type and severity of the disease being treated; the activity of the specific compound used; the specific composition used; the patient's age, weight, health, sex, and diet; the timing and route of administration; the duration of treatment; and drugs used in combination or co-administration with the specific compound used.

[0151] In one embodiment, the formulation comprises an siRNA, such as the duplex of SEQ ID NO: 17 and SEQ ID NO: 18. In one embodiment, the siRNA is contained in a liposomal formulation. In some embodiments, the formulation for ocular administration comprises the siRNA in a sterile solution, with one 35 μl drop corresponding to 9.37 μg of the siRNA duplex.

[0152] In one embodiment of the methods and compositions described herein, the subject is a mammal, hi one embodiment, the subject is a human.

[0153] In one embodiment, nanoparticles comprising an RNA interfering agent are used for administration in in vitro, ex vivo, or in vivo applications of the methods described herein.

[0154] The following examples are presented to more fully illustrate preferred embodiments of the invention, but should not be construed as limiting the broad scope of the invention.

[0155] Example

[0156] Example 1: Ophthalmic eye drops for NK treatment

[0157] Ophthalmic drops containing nanoparticles incorporating siRNA containing sense and antisense SEQ ID NOs: 17 and 18 were formulated.

[0158] [Table 6]

[0159] siRNA was formulated as a preservative-free, sterile liquid for topical ophthalmic administration. This formulation was administered every other day for 8 weeks to patients with NK. Improvement of NK (neurotrophic keratitis), including corneal healing and increased corneal sensitivity, was observed.

[0160] Example 2: Ophthalmic drops for NK treatment

[0161] An ophthalmic preparation containing sense and antisense siRNAs containing SEQ ID NOs: 17 and 18 and polyethyleneimine was formulated. One drop of this preparation was administered to the eyes of a patient with NK every other day. As a result, improvement of NK was observed.

[0162] Example 3: siRNA-releasing contact lens formulation for NK therapy

[0163] An ophthalmic preparation was formulated incorporating sense and antisense sequences SEQ ID NOs: 17 and 18 into soft contact lenses. These contact lenses were worn in the eyes of NK patients and replaced daily. As a result, improvement in NK was observed.

[0164] Example 4: Wafer ophthalmic formulation for NK treatment

[0165] An ophthalmic formulation containing sense and antisense SEQ ID NOs: 17 and 18 in a wafer formulation was formulated. This wafer ophthalmic formulation was placed under the lower eyelid of a patient with NK. As a result, improvement in NK was observed.

[0166] Example 5: Evaluation of the efficacy of FL2-targeting siRNA in stage 2 and 3 NK

[0167] The efficacy of ophthalmic formulations containing siRNAs of SEQ ID NO: 17 and SEQ ID NO: 18 was assessed for each patient based on a review of medical history, ocular examination, and tests to assess corneal sensitivity loss and nerve damage. The clinical picture of NK is primarily unilateral and characterized by progressive ocular surface changes. Patients with NK disease in stages 2 or 3, as described above, were selected. Two dosing regimens were evaluated: 20 μg of siRNA administered once every 48 hours for 8 weeks (Regimen 1) or 20 μg of siRNA administered once daily for 8 weeks (Regimen 2). After an 8-week control treatment period, patients were assessed as completely healed (CH) or not completely healed (NCH) and then entered a 48-week or 56-week follow-up period.

[0168] Efficacy was assessed by clinical assessment of corneal fluorescein staining. Secondary objectives included the investigator's assessment of complete healing, duration of complete healing, improvement in visual acuity, improvement in corneal sensitivity, and the proportion of patients who achieved complete corneal clearing, defined as complete disappearance of staining on the modified Oxford corneal scale. The main secondary objective was assessment of complete corneal healing (lesion size less than 0.5 mm) by centrally assessing clinical corneal fluorescein staining. Other secondary objectives included assessment of visual acuity improvement by the ETDRS chart and improvement in corneal sensitivity by the Cochet-Bonnet corneal sensitivity test.

[0169] The primary efficacy outcome measure was the proportion of patients achieving complete healing (lesion size 0 mm and no residual staining) in stage 2 (PED) and stage 3 (corneal ulcer) NK at 8 weeks, based on assessment of corneal fluorescein staining images at a central reading center. Corneal fluorescein staining was assessed using a slit lamp with a yellow barrier filter and cobalt blue illumination using the modified Oxford corneal scale.

[0170] Efficacy: "Complete healing" includes the original Dompe definition: a maximum diameter of corneal fluorescein staining (measured at the baseline visit) in the area of ​​persistent epithelial defect (PED) or corneal ulcer of less than 0.5 mm at the time of evaluation. "Complete absence of staining" refers to the absence of residual fluorescein staining in the corneal lesion at the time of evaluation or the absence of persistent staining in other areas of the cornea in photographs taken at different time points during the study (i.e., no change in shape and / or location at different time points).

[0171] Secondary efficacy endpoints were as follows: The proportion of patients with complete healing of PED or corneal ulcer as assessed by corneal fluorescein staining at 4, 6, 8, 12, 20, 32, 44, and 56 weeks as defined by the investigator. The proportion of patients with complete corneal clearing (grade 0 on the modified Oxford Scale) at 4, 6, 8, 12, 20, 32, 44, and 56 weeks. -Mean change in best corrected distance visual acuity (BCDVA) from baseline to 8 weeks after treatment. The proportion of patients achieving a 15-letter or greater gain in BCDVA at 4, 6, and 8 weeks. The proportion of patients who achieved improvement in corneal sensitivity as measured by Cochet-Bonnet aesthesiometer at 4, 6, and 8 weeks (binary variable of target attainment: "corneal sensitivity at 4, 6, and 8 weeks" - "baseline corneal sensitivity" > 0 [Yes / No]). The proportion of patients with stage 2 or 3 NK whose lesions worsened (increase in lesion size of ≥1 mm, decrease in BCDVA by ≥5 letters on ETDRS, progression of lesion depth (corneal melting or perforation), or development of infection) from baseline to 4, 6, and 8 weeks after treatment. Time to onset of exacerbation from baseline to 8 weeks after treatment.

[0172] Exploratory efficacy variables will also be evaluated, including time to complete corneal clearing and time to onset of healing (defined as a 20% or greater reduction in the maximum diameter of the lesion), change in unanesthetized Schirmer score, change in tear film osmolality, and change in National Eye Institute Visual Function Questionnaire-25 (NEI-VFQ) and / or EuroQol 5D (EQ-5D) scores.

[0173] The main selection criteria were as follows: Patients aged 18 years or older. Patients with stage 2 PED or stage 3 (corneal ulcer) NK. Patients with stage 2 or 3 NK in only one eye are accepted. Patients with PED or corneal ulcers that have persisted for more than 2 weeks and are resistant to one or more conventional non-surgical treatments for NK (e.g., preservative-free artificial tears, gels, ointments; preservative-containing eye drops and discontinuation of corneal desensitizing medications; therapeutic contact lenses). Patients with evidence of decreased corneal sensitivity (≤4 cm as measured by a Cochet-Bonnet aesthesiometer) in at least one corneal quadrant within the area of ​​PED or corneal ulcer and outside the defect area. Patients with a best-corrected distance visual acuity (BCDVA) score of 75 or less in Early Treatment Diabetic Retinopathy Study (ETDRS) letters (LogMAR ≥ +0.2, Snellen ≤ 20 / 32, or decimal ≤ 0.625 equivalent). Patients without objective clinical evidence of improvement of PED or corneal ulcer within 2 weeks prior to study enrollment.

[0174] The main exclusion criteria were as follows: The affected eye has an active ocular infection (bacterial, viral, fungal, or protozoal) or active ocular inflammation that is not related to NK. - If you have any other ocular disease that requires topical ophthalmic treatment of the affected eye during the study treatment period. Topical ophthalmic treatments other than the investigational drug provided by the study sponsor or permitted by the study protocol must not be administered to the affected eye during the study period. -Patients with severe visual impairment in the affected eye who are judged by the investigator to have no chance of improving visual acuity in the affected eye with the administration of the investigational drug. -Patients whose Schirmer test result under unanesthetized conditions is 3mm / 5 minutes or less in the affected eye. Patients with severe blepharitis and / or severe meibomian gland disease in the affected eye. Patients who have had ophthalmic surgery (such as laser surgery or refractive surgery) on the affected eye within 3 months prior to study enrollment. Patients with a history of surgery to treat NK in the affected eye (e.g., complete conjunctival blepharoplasty or conjunctival flap, excluding amniotic membrane transplantation). Patients who have previously been treated with Botox (botulinum toxin) injections are accepted only if the last injection was administered at least 90 days prior to enrollment in this study. -Patients who use refractive contact lenses during treatment of the affected eye. - If the need for punctal occlusion is anticipated during the treatment period. Patients who had punctal occlusion or eye plugs inserted before the study were eligible for enrollment if punctal occlusion was maintained during the study period. Patients with evidence of corneal ulcer, corneal melt, or perforation involving the posterior third of the cornea in the affected eye. Patients with or a history of ocular or systemic disorders or diseases that may interfere with the administration or evaluation of the investigational product, may interfere with the interpretation of study results, or that the investigator determines are incompatible with the scheduling or conduct of study visits (e.g., progressive or degenerative corneal or retinal disease, uveitis, optic neuritis, uncontrolled diabetes, autoimmune disease, systemic infection, neoplastic disease). Anticipated need to change the dose of systemic medications known to inhibit trigeminal nerve function (e.g., neuroleptics, antipsychotics, antihistamines) unless initiated at least 30 days before study enrollment and stable throughout the study treatment period. Patients with known hypersensitivity to RNA-based therapeutics.

[0175] Eligible subjects will receive one drop (35 μl) of study medication six times daily in the affected eye during an 8-week randomized, double-blind, controlled treatment period and an 8-week uncontrolled treatment period.

[0176] Subjects enrolled in the study according to the inclusion and exclusion criteria described above and treated with the siRNA eye drops experienced improvement in one or more of the efficacy endpoints, secondary objectives, primary efficacy endpoints, secondary efficacy endpoints, and exploratory efficacy variables, i.e., NK cure.

Claims

1. 1. A method for treating neurotrophic keratitis, comprising: A method comprising administering to a subject in need of treatment for neurotrophic keratitis an effective amount of an agent that inhibits or reduces the activity of figetin-like 2 (FL2), the agent being formulated for ocular administration.

2. 10. The method of claim 1, The method, wherein the stage of the neurotrophic keratitis is stage 1, stage 2, or stage 3.

3. 10. The method of claim 1, The method, wherein the agent that inhibits or reduces the activity of figetin-like 2 is an RNA interference agent.

4. 4. The method of claim 3, The method, wherein the RNA interfering agent is an siRNA.

5. 4. The method of claim 3, The method, wherein the RNA interfering agent is an shRNA.

6. 10. The method of claim 1, The method, wherein the treatment of neurotrophic keratitis includes one or more of complete corneal healing, a reduction in the area of ​​persistent epithelial defect, no residual fluorescein staining in the affected corneal area, no persistent fluorescein staining, complete corneal clearing, improved visual acuity, and improved corneal sensitivity.

7. 5. The method of claim 4, The method, wherein the siRNA comprises a sequence selected from the following: UUACACAGUAUUAAAGCGAUU (SEQ ID NO: 1); UCGCUUUAAUACUGUGUAAUU (SEQ ID NO: 2); CAUCUGAAACCUAGGGUCUUU (SEQ ID NO: 3); AGACCCUAGGUUUCAGAUGUU (SEQ ID NO: 4); GUGACUUAUGCUAGGAGGAUU (SEQ ID NO: 5); UCCUCCUAGCAUAAGUCACUU (SEQ ID NO: 6); GGUCAGAAGCAGAAUGUAUUU (SEQ ID NO: 7); AUACAUUCUGCUUCUGACCUU (SEQ ID NO: 8); CGCCGGCCCACAAGUUGGAdTdT (SEQ ID NO: 9); UCCAACUUGUGGGCCGGCGdTdT (SEQ ID NO: 10); CAGCUCGAGCCCUUUGACAdTdT (SEQ ID NO: 11); UGUCAAAGGGCUCGAGCUGdTdT (SEQ ID NO: 12); CCUCCAACCUCCUCAAGAGdTdT (SEQ ID NO: 13); CUCUUGAGGAGGUUGGAGGdTdT (SEQ ID NO: 14); CGUUGCUGCUCAUCAGCGAdTdT (SEQ ID NO: 15); UCGCUGAUGAGCAGCAACGdTdT (SEQ ID NO: 16); fUfUmAfCmAfCAGUAUUAAAGCGATT (SEQ ID NO: 17); (Phos)UCGCUUUAAUACUGUGUAATT (SEQ ID NO: 18); 5'-UUACACAGUAUUAAAGCGATT-3' (SEQ ID NO: 34); (Phos) 5'-mUmCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 35); (Phos)5'-mU(s)mC(s)GCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 36); (Phos) 5'-fUfCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 37); (Phos)5'-fU(s)fC(s)GCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 38); (Phos)5'-mU(s)mC(s)GCUUUAAUAmCfUmGfUmGfUmAmATT-3' (SEQ ID NO: 39); (Phos)5'-U(s)CGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 40); (Phos)5'-mUfCmGfCmUfUmUAAfUmAfCmUGmUmGfUmAmATT (SEQ ID NO: 41); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGmCmGmAmUmU-3' (SEQ ID NO: 42); (Phos)5'-mUmCmGmCmUmUmUmAmAmUmAmCmUmGmUmGmUmAmAmUmU-3' (SEQ ID NO: 43); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGdCdGdATT-3' (SEQ ID NO: 44); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGdCmGmATT-3' (SEQ ID NO: 45); 5'UUACACAGUAUUAAAGCGA-3' (SEQ ID NO: 46); (Phos)5'-U(s)CGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 47); (Phos) 5'-UCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 48); (Phos)5'-U(s)C(s)GCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 49); 5'-mUmUACACAGUAUUAAAGCGA-3' (SEQ ID NO: 50); (Phos)5'-U(s)CGCUUUAAUACUGUGUmAmATT-3' (SEQ ID NO: 51); (Phos) 5'-UCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 52); (Phos)5'-U(s)C(s)GCUUUAAUACUGUGUAAT(s)T-3' (SEQ ID NO: 53); 5′ lUlUlAlClACAGUAUUAAAGCGATT-3′ (SEQ ID NO: 54); (Phos) 5'-UCGCUUUAAUACUGlUlGlUlAlATT-3' (SEQ ID NO: 55); 5'fUfUlAfClACAGUAUUAAAGCGA-3' (SEQ ID NO: 56); (Phos)5'-mU(s)mCmGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 57); 5′-fUfCGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 58); 5′-fU(s)fC(s)GCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 59); 5′-mU(s)mC(s)GCUUUAAUAmCfUmGfUmGfUmAmATT-3′ (SEQ ID NO: 60); 5′-U(s)CGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 61); 5'-mUfCmGfCmUfUmUAAfUmAfCmUGmUmGfUmAmATT (SEQ ID NO: 62); 5'-mUmCmGmCmUmUmUmAmAmUmAmCmUmGmUmGmUmAmAmUmU-3' (SEQ ID NO: 63); 5′-U(s)CGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 64); 5'-UCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 65); 5'-U(s)C(s)GCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 66); 5′-U(s)CGCUUUAAUACUGUGUmAmATT-3′ (SEQ ID NO: 67); 5'-UCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 68); 5'-U(s)C(s)GCUUUAAUACUGUGUAAT(s)T-3' (SEQ ID NO: 69); 5'-UCGCUUUAAUACUGlUlGlUlAlATT-3' (SEQ ID NO: 70); 5'-mU(s)mCmGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 71); and 5'-UCGCUUUAAUACUGUGUAATT (SEQ ID NO: 72). In the above sequence, d (nucleotide) = deoxy-(nucleotide), m (nucleotide) = 2'-O-methyl nucleotide, T = thymidine, f (nucleotide) = 2'-fluorodeoxynucleotide, (Phos) = phosphodiester cap; uppercase nucleotide = RNA nucleotide, l (nucleotide) = locked nucleotide, (s) = phosphorothioate.

8. 5. The method of claim 4, The method, wherein the siRNA has at least one modification selected from a 3' overhang, a 5' overhang, a 5' phosphorylation, a 2' sugar modification, a nucleobase modification, a phosphate backbone modification, and any combination thereof.

9. 8. The method of claim 7, The method, wherein the siRNA has at least one additional modification selected from a 3' overhang, a 5' overhang, a 5' phosphorylation, a 2' sugar modification, a nucleobase modification, a phosphate backbone modification, and any combination thereof.

10. 10. The method of claim 1, The method, wherein said figetin-like 2 is human figetin-like 2.

11. 6. The method of claim 4 or 5, The method, wherein the siRNA or the shRNA is encapsulated in a nanoparticle.

12. 6. The method of claim 4 or 5, The method, wherein the siRNA or shRNA is delivered by nanoparticles, electroporation / nuclear transfection, Accel siRNA, viral vectors, peptides, proteins, or aptamers.

13. 6. The method of claim 4 or 5, The method, wherein the siRNA or the shRNA is delivered by eye drops.

14. 6. The method of claim 4 or 5, The method, wherein the siRNA or the shRNA is delivered by an ophthalmic wafer.

15. 6. The method of claim 4 or 5, The method, wherein the siRNA or the shRNA is delivered by a drug-releasing contact lens.

16. 5. The method of claim 4, The method, wherein the siRNA comprises a duplex of SEQ ID NO:17 and SEQ ID NO:

18.

17. 6. The method of claim 5, The method, wherein the shRNA comprises SEQ ID NO:

23.

18. 7. The method of claim 6, The method, wherein treatment of said neurotrophic keratitis is initiated upon diagnosis of said neurotrophic keratitis.

19. 7. The method of claim 6, The method, wherein the treatment of the neurotrophic keratitis is initiated about 1 month, about 2 months, or about 3 months after the onset of the neurotrophic keratitis.

20. 7. The method of claim 6, The method, wherein the treatment of the neurotrophic keratitis is initiated about 1 month, about 2 months, or about 3 months after the diagnosis of the neurotrophic keratitis.

21. 1. An ophthalmic composition for treating neurotrophic keratitis, comprising: An ophthalmic composition comprising an effective amount of an agent that inhibits or reduces the activity of figetin-like 2 (FL2).

22. 22. The ophthalmic composition of claim 21, The ophthalmic composition, wherein the neurotrophic keratitis is at stage 1, stage 2, or stage 3.

23. 22. The ophthalmic composition of claim 21, The ophthalmic composition, wherein the agent that inhibits or reduces the activity of figetin-like 2 is an RNA interference agent.

24. 24. The ophthalmic composition of claim 23, An ophthalmic composition, wherein the RNA interference agent is siRNA.

25. 24. The ophthalmic composition of claim 23, An ophthalmic composition, wherein the RNA interference agent is an shRNA.

26. 22. The ophthalmic composition of claim 21, The ophthalmic composition, wherein the treatment of neurotrophic keratitis includes one or more of complete corneal healing, a reduction in the area of ​​persistent epithelial defect, no residual fluorescein staining in the corneal diseased area, no persistent fluorescein staining, complete corneal clearing, improved visual acuity, and improved corneal sensitivity.

27. 25. The ophthalmic composition of claim 24, An ophthalmic composition, wherein the siRNA comprises a sequence selected from the following: UUACACAGUAUUAAAGCGAUU (SEQ ID NO: 1); UCGCUUUAAUACUGUGUAAUU (SEQ ID NO: 2); CAUCUGAAACCUAGGGUCUUU (SEQ ID NO: 3); AGACCCUAGGUUUCAGAUGUU (SEQ ID NO: 4); GUGACUUAUGCUAGGAGGAUU (SEQ ID NO: 5); UCCUCCUAGCAUAAGUCACUU (SEQ ID NO: 6); GGUCAGAAGCAGAAUGUAUUU (SEQ ID NO: 7); AUACAUUCUGCUUCUGACCUU (SEQ ID NO: 8); CGCCGGCCCACAAGUUGGAdTdT (SEQ ID NO: 9); UCCAACUUGUGGGCCGGCGdTdT (SEQ ID NO: 10); CAGCUCGAGCCCUUUGACAdTdT (SEQ ID NO: 11); UGUCAAAGGGCUCGAGCUGdTdT (SEQ ID NO: 12); CCUCCAACCUCCUCAAGAGdTdT (SEQ ID NO: 13); CUCUUGAGGAGGUUGGAGGdTdT (SEQ ID NO: 14); CGUUGCUGCUCAUCAGCGAdTdT (SEQ ID NO: 15); UCGCUGAUGAGCAGCAACGdTdT (SEQ ID NO: 16); fUfUmAfCmAfCAGUAUUAAAGCGATT (SEQ ID NO: 17); (Phos)UCGCUUUAAUACUGUGUAATT (SEQ ID NO: 18); 5'-UUACACAGUAUUAAAGCGATT-3' (SEQ ID NO: 34); (Phos) 5'-mUmCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 35); (Phos)5'-mU(s)mC(s)GCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 36); (Phos) 5'-fUfCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 37); (Phos)5'-fU(s)fC(s)GCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 38); (Phos)5'-mU(s)mC(s)GCUUUAAUAmCfUmGfUmGfUmAmATT-3' (SEQ ID NO: 39); (Phos)5'-U(s)CGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 40); (Phos)5'-mUfCmGfCmUfUmUAAfUmAfCmUGmUmGfUmAmATT (SEQ ID NO: 41); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGmCmGmAmUmU-3' (SEQ ID NO: 42); (Phos)5'-mUmCmGmCmUmUmUmAmAmUmAmCmUmGmUmGmUmAmAmUmU-3' (SEQ ID NO: 43); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGdCdGdATT-3' (SEQ ID NO: 44); 5'mUmUmAmCmAmCmAmGmUmAmUmUmAmAmAmGdCmGmATT-3' (SEQ ID NO: 45); 5'UUACACAGUAUUAAAGCGA-3' (SEQ ID NO: 46); (Phos)5'-U(s)CGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 47); (Phos) 5'-UCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 48); (Phos)5'-U(s)C(s)GCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 49); 5'-mUmUACACAGUAUUAAAGCGA-3' (SEQ ID NO: 50); (Phos)5'-U(s)CGCUUUAAUACUGUGUmAmATT-3' (SEQ ID NO: 51); (Phos) 5'-UCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 52); (Phos)5'-U(s)C(s)GCUUUAAUACUGUGUAAT(s)T-3' (SEQ ID NO: 53); 5′ lUlUlAlClACAGUAUUAAAGCGATT-3′ (SEQ ID NO: 54); (Phos) 5'-UCGCUUUAAUACUGlUlGlUlAlATT-3' (SEQ ID NO: 55); 5'fUfUlAfClACAGUAUUAAAGCGA-3' (SEQ ID NO: 56); (Phos)5'-mU(s)mCmGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 57); 5′-fUfCGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 58); 5′-fU(s)fC(s)GCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 59); 5′-mU(s)mC(s)GCUUUAAUAmCfUmGfUmGfUmAmATT-3′ (SEQ ID NO: 60); 5′-U(s)CGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 61); 5'-mUfCmGfCmUfUmUAAfUmAfCmUGmUmGfUmAmATT (SEQ ID NO: 62); 5'-mUmCmGmCmUmUmUmAmAmUmAmCmUmGmUmGmUmAmAmUmU-3' (SEQ ID NO: 63); 5′-U(s)CGCUUUAAUACUGUGUAATT-3′ (SEQ ID NO: 64); 5'-UCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 65); 5'-U(s)C(s)GCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 66); 5′-U(s)CGCUUUAAUACUGUGUmAmATT-3′ (SEQ ID NO: 67); 5'-UCGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 68); 5'-U(s)C(s)GCUUUAAUACUGUGUAAT(s)T-3' (SEQ ID NO: 69); 5'-UCGCUUUAAUACUGlUlGlUlAlATT-3' (SEQ ID NO: 70); 5'-mU(s)mCmGCUUUAAUACUGUGUAATT-3' (SEQ ID NO: 71); and 5'-UCGCUUUAAUACUGUGUAATT (SEQ ID NO: 72). In the above sequence, d (nucleotide) = deoxy-(nucleotide), m (nucleotide) = 2'-O-methyl nucleotide, T = thymidine, f (nucleotide) = 2'-fluorodeoxynucleotide, (Phos) = phosphodiester cap; uppercase nucleotide = RNA nucleotide, l (nucleotide) = locked nucleotide, (s) = phosphorothioate.

28. 25. The ophthalmic composition of claim 24, The ophthalmic composition, wherein the siRNA has at least one modification selected from a 3' overhang, a 5' overhang, a 5' phosphorylation, a 2' sugar modification, a nucleobase modification, a phosphate backbone modification, and any combination thereof.

29. 25. The ophthalmic composition of claim 24, The ophthalmic composition, wherein the siRNA has at least one additional modification selected from a 3' overhang, a 5' overhang, a 5' phosphorylation, a 2' sugar modification, a nucleobase modification, a phosphate backbone modification, and any combination thereof.

30. 22. The ophthalmic composition of claim 21, An ophthalmic composition, wherein the figetin-like 2 is human figetin-like 2.

31. 26. The ophthalmic composition of claim 24 or 25, An ophthalmic composition, wherein the siRNA or the shRNA is encapsulated in nanoparticles.

32. 26. The ophthalmic composition of claim 24 or 25, The ophthalmic composition, wherein the siRNA or shRNA is delivered by nanoparticles, electroporation / nuclear transfection, Accel siRNA, viral vectors, peptides, proteins, or aptamers.

33. 26. The ophthalmic composition of claim 24 or 25, An ophthalmic composition, wherein the siRNA or the shRNA is delivered by eye drops.

34. 26. The ophthalmic composition of claim 24 or 25, An ophthalmic composition, wherein the siRNA or the shRNA is delivered by an ophthalmic wafer.

35. 26. The ophthalmic composition of claim 24 or 25, An ophthalmic composition, wherein the siRNA or the shRNA is delivered by a drug-releasing contact lens.

36. 26. The ophthalmic composition of claim 25, An ophthalmic composition, wherein the shRNA comprises SEQ ID NO:23.

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