Bdnf for the treatment of rhodopsin-mediated retinitis pigmentosa

Abstract

The present invention relates to brain-derived neurotrophic factor (BDNF) for use in treating rhodopsin-mediated retinal pigment degeneration in a subject, said BDNF being administered as a protein.

Description

Technical Field

[0001] This invention relates to the treatment of rhodopsin-mediated retinitis pigmentosa. Background Technology

[0002] Retinitis pigmentosa encompasses a heterogeneous group of inherited retinal dystrophys characterized by the degeneration of rod and cone photoreceptors, affecting approximately 1 in 4,000 people worldwide.

[0003] The disease is characterized by the initial progressive degeneration and death of rod photoreceptors, leading to night vision loss (night blindness) and progressive narrowing of the visual field. In a later stage, rod cell depletion induces secondary degeneration of cone cells, possibly through the bystander effect, ultimately resulting in complete blindness.

[0004] Retinitis pigmentosa exhibits genetic heterogeneity, displaying autosomal recessive, autosomal dominant, and X-linked inheritance patterns. To date, pathogenic mutations leading to retinitis pigmentosa have been identified in more than 80 genes (Verbakel et al., Progress in Retinal and Eye Research 2018, 66: 157-186).

[0005] Rhodopsin (RHO) is a G protein-coupled receptor located in the extracellular segment of rod cells in the retina, and is responsible for visual light transduction in these cells.

[0006] Mutations in the rhodopsin-encoding gene are among the earliest identified molecular defects in retinitis pigmentosa, accounting for approximately 30% of autosomal dominant RP (adRP) cases in the United States and 8% to 10% of all RP cases (Hartong et al., Lancet 2006, 368, 1795-1809; Athanasiou et al., ProgRetin Eye Res 2018, 62:1-23). ​​To date, more than 150 mutations have been associated with the adRP phenotype, with the P23H mutation being the most common (Yan et al., Molecular Therapy: Nucleic Acids 2023, Vol. 33, pp. 750-761).

[0007] Rhodopsin mutations associated with adRP are classified into six classes based on their biochemical and cellular defects, with most belonging to class I or II. Class I mutations are located only at the C-terminus of the protein, enabling it to fold correctly and form a functional photoreceptor, but preventing proper transport to the outer segment. Class II mutations occur in the disc, transmembrane, and cytoplasmic domains of the protein, causing misfolding that prevents it from reconstituted with 11-cis-retinal to form functional rhodopsin, and instead traps it in the endoplasmic reticulum (Athanasiou et al., Prog Retin Eye Res 2018, 62:1-23; Xiao et al., Eye 2019 33:592-599). The P23H mutation belongs to class II.

[0008] Although uncommon, some rhodopsin mutations associated with autosomal recessive RP (arRP) have been identified.

[0009] Currently, only one approved treatment is available for retinitis pigmentosa, and this treatment is available for a very small number of patients. voretigene neparvovec (Luxturna®) is a gene therapy based on a recombinant adeno-associated virus vector that delivers a copy of the gene for the functional human retinal pigment epithelium-specific 65 kDa protein (RPE65) to the retinal cells of patients with RPE65 mutation-associated retinitis pigmentosa. However, this patient population accounts for only 0.3% to 1% of all RP cases. Currently, there is no standard treatment for other patients, and treatment relies solely on supportive care, such as vitamin supplementation, sun protection, and visual aids.

[0010] Therefore, there is a strong sense of need to develop new treatments for retinitis pigmentosa, especially for the most common form of this retinal dystrophy, such as rhodopsin-mediated retinitis pigmentosa.

[0011] Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor that supports the differentiation, maturation and survival of neurons in the nervous system and exhibits neuroprotective effects under adverse conditions (such as glutamatergic stimulation, cerebral ischemia, hypoglycemia and neurotoxicity) (Bathina et al., Arch Med Sci. 2015; 11(6): 1164-1178).

[0012] Given its pro-survival and neuroprotective activities, intravitreal administration of BDNF has been tested in models of retinal degeneration. However, in studies on the effects of BDNF on different hereditary retinal degeneration (RP) models, modest partial rescue was observed in only one of seven models (LaVail MM et al., Proc Natl Acad Sci USA 1992, 89:11249-11253; LaVail MM et al., Invest Ophthalmol Vis Sci 1998, 39:592-602). These data were confirmed by subsequent studies (Ogilvie JM et al., Experimental Neurology 2000, 161, 676-685). Similarly, in a cone-rod dystrophy model, BDNF failed to prevent photoreceptor degeneration (Chong et al., Invest Ophthalmol Vis Sci. 1999, 40(6):1298-305.).

[0013] In the above study, it was also hypothesized that different pathological mechanisms associated with different genetic alterations in retinitis pigmentosa could lead to specific susceptibility to the activity of survival-promoting protective factors (LaVail MM et al., Invest Ophthalmol Vis Sci 1998, 39:592-602). Summary of the Invention

[0014] As will be described in the experimental section, the inventors have now demonstrated that intravitreal administration of BDNF protects photoreceptors from degeneration in rhodopsin-mediated retinitis pigmentosa and salvages retinal function and morphology in a dose-dependent manner, inducing a significant recovery of visual ability.

[0015] Furthermore, the inventors have demonstrated that BDNF administration improves intracellular rhodopsin clearance and transport in photoreceptor cells with rhodopsin mutations associated with rhodopsin-mediated retinitis pigmentosa, as shown in the following in vitro examples.

[0016] Therefore, the first object of the present invention is BDNF for treating rhodopsin-mediated retinitis pigmentosa in subjects, preferably, said BDNF is administered as a protein.

[0017] Another object of the present invention is an ophthalmic composition comprising BDNF and at least one ophthalmologically acceptable excipient for treating rhodopsin-mediated retinitis pigmentosa in subjects, wherein the BDNF is preferably administered as a protein. Attached Figure Description

[0018] Figure 1The ERG a-wave amplitude (Fig. a) and b-wave amplitude (Fig. b) responses recorded at P30 in untreated WT mice (WT), WT mice treated with a loading agent (WT loading agent), RHO-P23H mice treated with a loading agent (RHO-P23H loading agent), and RHO-P23H mice treated with a single injection of rhBDNF (RHO-P23H BDNF) according to Example 1 are shown. Error bars represent SEM; statistical significance is indicated by asterisks (unpaired Student t-test, loading agent vs WT (hexagonal asterisk), and rhBDNF vs loading agent (pentagonal asterisk)). ).

[0019] Figure 2 The ERG a-wave amplitude (Fig. a) and b-wave amplitude (Fig. b) responses recorded at P60 in untreated WT mice (WT), WT mice treated with a loading agent (WT loading agent), RHO-P23H mice treated with a loading agent (RHO-P23H loading agent), and RHO-P23H mice treated with a single injection of rhBDNF (RHO-P23H BDNF) according to Example 1 are shown. Error bars represent SEM; statistical significance is indicated by asterisks (unpaired Student t-test, loading agent vs WT (hexagonal asterisk), and rhBDNF vs loading agent (pentagonal asterisk)). ).

[0020] Figure 3 The figures show the ONL thickness (in µm) (Fig. a), ONL density (in cells / area) (Fig. b), and cone cell / area (number of cone cells in ONL / 0.006 mm²) in the retinas of WT (WT carrier), RHO-P23H (RHO-P23H BDNF), and RHO-P23H (RHO-P23H carrier) retinas according to Example 1. 2 Figure c), OS thickness in µm (Figure d). Statistical significance is indicated by asterisks (unpaired Student t-test, carrier vs WT (hexagonal asterisk), and rhBDNF vs carrier (pentagonal asterisk)). ).

[0021] Figure 4 The figure shows the number of TUNEL-positive cells in the ONL region of mice treated with WT (WT carrier), RHO-P23H (RHO-P23H BDNF), and RHO-P23H (RHO-P23H carrier) according to Example 1, per 0.006 mm². 2The bar chart shows the statistical significance (unpaired Student t-test, carrier vs. WT (hexagonal star), and rhBDNF vs. carrier (pentagonal star)). ).

[0022] Figure 5 Figures (a) and (b) show the ERG a-wave amplitude (Figure a) and b-wave amplitude (Figure b) recorded at P30 after PN18 injection according to Example 2 for the following mice: WT mice treated with the loading agent (WT loading agent), RHO-P23H mice treated with the loading agent (RHO-P23H loading agent), RHO-P23H mice treated with 5 µg / µl BDNF (RHO-P23H BDNF 5 µg / µl), RHO-P23H mice treated with 3.5 µg / µl BDNF (RHO-P23H BDNF 3.5 µg / µl), RHO-P23H mice treated with 2 µg / µl BDNF (RHO-P23H BDNF 2 µg / µl), and RHO-P23H mice treated with 1 µg / µl BDNF (RHO-P23H BDNF 1 µg / µl). Error bars represent SEM. Statistical significance is indicated by an asterisk (unpaired Student t-test, carrier vs WT). (Astro); rhBDNF 5 µg / µl vs. carrier ( (Astro); rhBDNF 3.5 µg / µl vs. carrier ( (Astro); rhBDNF 2 µg / µl vs. carrier ( (Astro); rhBDNF 1 µg / µl vs. carrier ( (asterisk) These p-values ​​also apply to other asterisks.

[0023] Figure 6Figures (a) and (b) show the ERG a-wave amplitude (Figure a) and b-wave amplitude (Figure b) recorded at P60 after PN18 injection according to Example 2 for the following mice: WT mice treated with the loading agent (WT loading agent), RHO-P23H mice treated with the loading agent (RHO-P23H loading agent), RHO-P23H mice treated with 5 µg / µl BDNF (RHO-P23H BDNF 5 µg / µl), RHO-P23H mice treated with 3.5 µg / µl BDNF (RHO-P23H BDNF 3.5 µg / µl), RHO-P23H mice treated with 2 µg / µl BDNF (RHO-P23H BDNF 2 µg / µl), and RHO-P23H mice treated with 1 µg / µl BDNF (RHO-P23H BDNF 1 µg / µl). Error bars represent SEM. Statistical significance is indicated by an asterisk (unpaired Student t-test, carrier vs WT). (Astro); rhBDNF 5 µg / µl vs. carrier ( (Astro); rhBDNF 3.5 µg / µl vs. carrier ( (Astro); rhBDNF 2 µg / µl vs. carrier ( (Astro); rhBDNF 1 µg / µl vs. carrier ( (asterisk) These p-values ​​also apply to other asterisks.

[0024] Figure 7 Figures (a) and (b) show the ERG a-wave amplitude (Figure a) and b-wave amplitude (Figure b) recorded at P90 after PN18 injection according to Example 2 for the following mice: WT mice treated with the loading agent (WT loading agent), RHO-P23H mice treated with the loading agent (RHO-P23H loading agent), RHO-P23H mice treated with 5 µg / µl BDNF (RHO-P23H BDNF 5 µg / µl), RHO-P23H mice treated with 3.5 µg / µl BDNF (RHO-P23H BDNF 3.5 µg / µl), RHO-P23H mice treated with 2 µg / µl BDNF (RHO-P23H BDNF 2 µg / µl), and RHO-P23H mice treated with 1 µg / µl BDNF (RHO-P23H BDNF 1 µg / µl). Error bars represent SEM. Statistical significance is indicated by an asterisk (unpaired Student t-test, carrier vs WT). (Astro); rhBDNF 5 µg / µl vs. carrier ( (Astro); rhBDNF 3.5 µg / µl vs. carrier ( (Astro); rhBDNF 2 µg / µl vs. carrier ( (Astro); rhBDNF 1 µg / µl vs. carrier ( (asterisk) These p-values ​​also apply to other asterisks.

[0025] Figure 8 Figures (a) and (b) show the ERG a-wave amplitude (Figure a) and b-wave amplitude (Figure b) recorded at P120 after PN18 injection according to Example 2: WT mice treated with the loading agent (WT loading agent), RHO-P23H mice treated with the loading agent (RHO-P23H loading agent), RHO-P23H mice treated with 5 µg / µl BDNF (RHO-P23H BDNF 5 µg / µl), RHO-P23H mice treated with 3.5 µg / µl BDNF (RHO-P23H BDNF 3.5 µg / µl), RHO-P23H mice treated with 2 µg / µl BDNF (RHO-P23H BDNF 2 µg / µl), and RHO-P23H mice treated with 1 µg / µl BDNF (RHO-P23H BDNF 1 µg / µl). Error bars represent SEM. Statistical significance is indicated by an asterisk (unpaired Student t-test, carrier vs WT). (Astro); rhBDNF 5 µg / µl vs. carrier ( (Astro); rhBDNF 3.5 µg / µl vs. carrier ( (Astro); rhBDNF 2 µg / µl vs. carrier ( (Astro); rhBDNF 1 µg / µl vs. carrier ( (asterisk) These p-values ​​also apply to other asterisks.

[0026] Figure 9 The figures show the ONL thickness (in µm) (Fig. a), ONL density (in cells / area) (Fig. b), and cone cell / area (number of cone cells in ONL / 0.006 mm²) in the retinas of WT (WT carrier), RHO-P23H (RHO-P23H BDNF), and RHO-P23H (RHO-P23H carrier) retinas according to Example 2, according to Example 2. 2Figure c) and OS thickness in µm (Figure d). Statistical significance is indicated by asterisks (unpaired Student t-test, carrier vs WT (black asterisk), and rhBDNF vs carrier (light gray asterisk)). ).

[0027] Figure 10 The figure shows the number of TUNEL-positive cells in the ONL of the retina of mice treated with load-loaded WT (WT load), RHO-P23H treated with rhBDNF (RHO-P23H BDNF), and RHO-P23H treated with load-loaded RHO-P23H (RHO-P23H load) in 0.006 mm². 2 The bar chart shows the statistical significance indicated by asterisks (unpaired Student t-test, carrier vs. WT (black asterisk), and rhBDNF vs. carrier (light gray asterisk)). ).

[0028] Figure 11 The bar chart shows the optical density analysis of RHO protein expression in the soluble (Fig. A) and insoluble (Fig. B) fractions of 661W RHO-P23H cells treated with the carrier or rhBDNF solution. The histogram reports the mean ± SEM optical density values ​​of the protein bands, with each band normalized relative to the relative optical density value of actin. The experiment was repeated N=2 times. (Student T test).

[0029] Figure 12 Bar charts are shown representing the number of lysosomes containing rhodopsin in LAMP1-positive 661W RHO-P23H cells treated with the load (P23H VEH), load and BAF (P23H, VEH + BAF), rhBDNF (P23HBDNF), and rhBDNF and BAF (P23H, BDNF + BAF). Results are expressed as fold change ± SEM, with experiments replicated twice. Mean values ​​are calculated based on at least 10 images, each containing an average of 2 to 3 cells, collected from at least two independent experiments.

[0030] Figure 13Figure A) shows representative Western blot analysis of RHO and actin proteins at 0, 2, 4, and 6 hours after CHX treatment of 661W RHO-P23H cells treated with a carrier (VEH) and rhBDNF solution (BDNF). Figure B) shows normalized quantification of RHO protein relative to the actin loading control at 0, 2, 4, and 6 hours after CHX treatment of 661W RHO-P23H cells treated with a carrier (VEH) and rhBDNF solution (BDNF). The figure shows the mean ± SEM of three independent experiments. Figure C) shows the quantification of RHO protein at 0 and 24 hours after treatment of 661W RHO-P23H cells and 661W WT cells (used as controls) treated with BORT and a carrier (BORT + VEH) or BORT and rhBDNF solution (BORT + BDNF). The bar chart represents the mean ± SEM of two independent experiments. Detailed Implementation

[0031] The first objective of this invention is brain-derived neurotrophic factor (BDNF) for the treatment of rhodopsin-mediated retinitis pigmentosa in subjects.

[0032] Preferably, the BDNF is administered as a protein.

[0033] In the context of degenerative and progressive diseases such as retinitis pigmentosa, the term “treatment” as used herein refers to improving the disease or one or more symptoms associated with it, or to stopping or delaying the progression of the disease.

[0034] Preferably, the object is a human object.

[0035] Preferably, the rhodopsin-mediated retinitis pigmentosa is rhodopsin-mediated autosomal dominant retinitis pigmentosa.

[0036] More preferably, the rhodopsin-mediated autosomal dominant retinitis pigmentosa is rhodopsin-mediated autosomal dominant retinitis pigmentosa associated with type I rhodopsin mutations.

[0037] Preferably, the type I rhodopsin mutation is selected from one of the following mutations:

[0038] L328P, T342M, Q344R, Q344P, Q344ter, V345L, V345M, A346P, P347A, P347R, P347Q, P347L, P347S, P347T, ter349Q, ter349.

[0039] More preferably, the rhodopsin-mediated autosomal dominant retinitis pigmentosa is rhodopsin-mediated autosomal dominant retinitis pigmentosa associated with type II rhodopsin mutations.

[0040] Preferably, the type II rhodopsin mutation is selected from one of the following mutations:

[0041] N15S, T17M, V20G, P23A, P23H, P23L, Q28H, G51R, G51V, P53R, T58R, T58M, V87D, G89D, G106R, G106W, C110F, C110R, C110S, C110Y, E113K, L125R, W161R, A164E, A164V, C167R, C167W, P171 Q, P171L, P171S, Y178N, Y178D, Y178C, E181K, G182S, G182V, C185R, C187G, C187Y, G188R, G188E, D190N, D190G, D190Y, H211R, H211P, C222R, P267R, P267L, S270R, K296N, K296E, and K296M.

[0042] More preferably, the rhodopsin-mediated retinitis pigmentosa is rhodopsin-mediated autosomal dominant retinitis pigmentosa associated with the P23H rhodopsin mutation.

[0043] Preferably, the rhodopsin-mediated retinitis pigmentosa is in its early or middle stages.

[0044] Preferably, the BDNF is a human BDNF, more preferably, it is a recombinant human BDNF (rhBDNF).

[0045] Preferably, the BDNF used as described above is administered to the subject via an ocular drug delivery route capable of delivering the protein to the subject's retina, such as subconjunctival, suprachoroidal, subretinal, transscleral, and intravitreal administration.

[0046] Preferably, the BDNF used as described above is applied to the object via intravitreal injection.

[0047] Preferably, the BDNF is administered to the subject via intravitreal injection, with each dose per eye ranging from 20 μg to 500 μg, more preferably from 50 μg to 400 μg, more preferably from 100 μg to 300 μg, and even more preferably from 150 μg, 175 μg, or 250 μg.

[0048] Also preferably, the BDNF is administered to the subject via intravitreal injection, with each dose per eye ranging from 10 μg to 500 μg, preferably from 15 μg to 250 μg, more preferably from 18 μg to 50 μg, and even more preferably from 20 μg.

[0049] Preferably, the BDNF is administered in a volume of 20 μl to 100 μl, more preferably 50 μl.

[0050] The administration regimen may vary depending on the patient and is selected by a person skilled in the art based on the characteristics of the patient to be treated, the severity of the disease, and the tests performed during treatment to monitor the response and disease progression.

[0051] Preferably, the administration of the drug to the subject via intravitreal injection is repeated every two years, more preferably every six months, and even more preferably every three or four months.

[0052] According to the preferred administration regimen, at the start of treatment, two or three initial intravitreal injections are given every three or four months, and subsequent injections are given at intervals of six months to two years, more preferably at intervals of six months to one year.

[0053] Another object of the present invention is an ophthalmic composition suitable for intravitreal application, comprising the BDNF and at least one ophthalmologically acceptable excipient.

[0054] Preferably, the BDNF is administered as a protein.

[0055] Preferably, the composition is administered to the object via intravitreal injection.

[0056] As used in this article, “ophthalmic acceptable excipients” refers to excipients that are suitable for application to the subject’s eye.

[0057] Preferably, the ophthalmic acceptable excipients are selected from solvents, thickeners, mucosal adhesives, buffers, antioxidants, and preservatives.

[0058] Another object of the present invention relates to the above-described ophthalmic composition for treating rhodopsin-mediated retinitis pigmentosa in subjects.

[0059] Preferably, the rhodopsin-mediated retinitis pigmentosa is rhodopsin-mediated autosomal dominant retinitis pigmentosa, more preferably rhodopsin-mediated autosomal dominant retinitis pigmentosa associated with type I or type II rhodopsin mutations, and even more preferably rhodopsin-mediated autosomal dominant retinitis pigmentosa associated with type II rhodopsin mutations.

[0060] The type I or type II rhodopsin mutations are preferably selected from the mutations reported above.

[0061] More preferably, the rhodopsin-mediated retinitis pigmentosa is rhodopsin-mediated autosomal dominant retinitis pigmentosa associated with the P23H rhodopsin mutation.

[0062] Preferably, the ophthalmic composition is an ophthalmic liquid composition.

[0063] Preferably, the ophthalmic composition is administered in a volume of 20 μl to 100 μl, more preferably 50 μl.

[0064] The ophthalmic liquid composition may be in the form of a solution or a suspension, preferably in the form of a solution.

[0065] Preferably, the ophthalmic liquid composition comprises a physiological saline solution as the main carrier.

[0066] Preferably, the pH of the ophthalmic liquid composition is from 4.5 to 8.0, more preferably about 7.

[0067] Preferably, the concentration of BDNF in the ophthalmic liquid composition is from 0.4 μg / μl to 10 μg / μl, more preferably from 1 μg / μl to 8 μg / μl, more preferably from 2 μg / μl to 6 μg / μl, and even more preferably selected from 3, 3.5 or 5 μg / μl.

[0068] Also preferably, the concentration of BDNF in the ophthalmic liquid composition is from 0.2 μg / μl to 10 μg / μl, more preferably from 0.3 μg / μl to 5 μg / μl, more preferably from 0.35 μg / μl to 1 μg / μl, and even more preferably from 0.4 μg / μl.

[0069] The ophthalmic liquid compositions according to the invention can be appropriately formulated using suitable methods known in the art or by methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing, Easton Pa.

[0070] In another aspect, the present invention relates to a method for treating rhodopsin-mediated retinitis pigmentosa as described above in a subject in need of such treatment, comprising administering a therapeutically effective amount of BDNF as described above to the subject.

[0071] Preferably, the BDNF is administered as a protein.

[0072] Preferably, the BDNF is administered via intravitreal injection.

[0073] Preferably, the method includes the following steps: diagnosing the presence of rhodopsin-mediated retinitis pigmentosa in the subject, and administering an effective amount of BDNF as described above to the subject, preferably via intravitreal injection. Preferably, the rhodopsin-mediated retinitis pigmentosa is rhodopsin-mediated autosomal dominant retinitis pigmentosa.

[0074] More preferably, the rhodopsin-mediated autosomal dominant retinitis pigmentosa is rhodopsin-mediated autosomal dominant retinitis pigmentosa associated with type I or type II rhodopsin mutations.

[0075] Preferably, the type I rhodopsin mutation is selected from one of the following mutations:

[0076] L328P, T342M, Q344R, Q344P, Q344ter, V345L, V345M, A346P, P347A, P347R, P347Q, P347L, P347S, P347T, ter349Q, ter349E.

[0077] Preferably, the method includes administering BDNF formulated in the ophthalmic composition described above.

[0078] More preferably, the rhodopsin-mediated autosomal dominant retinitis pigmentosa is associated with a type I or type II rhodopsin mutation, preferably type II rhodopsin-mediated autosomal dominant retinitis pigmentosa.

[0079] The type I or type II rhodopsin mutations are preferably selected from the mutations reported above.

[0080] More preferably, the rhodopsin-mediated retinitis pigmentosa is rhodopsin-mediated autosomal dominant retinitis pigmentosa associated with the P23H rhodopsin mutation.

[0081] Preferably, in conjunction with the above preferred embodiments, the rhodopsin-mediated retinitis pigmentosa is in the early or middle stage.

[0082] The present invention will be further described in the following embodiments, which do not limit the scope of the invention as defined by the claims.

[0083] Experimental Section

[0084] Example 1

[0085] The pharmacological efficacy of a single intravitreal injection of rhBDNF (3.5 µg / µl) in rescuing retinal degeneration in the eyes of heterozygous RHO-P23H knock-in mice (RHO-P23H mice) was tested.

[0086] The animals tested were grouped as follows:

[0087] - A group of 10 RHO-P23H mice received intravitreal injections of 1 µl of a solution containing 3.5 µg / µl rhBDNF (RHO-P23H BDNF) in both eyes on day 18 after birth ("PN" or "P").

[0088] - As a control for RHO-P23H, 10 RHO-P23H mice received intravitreal injections of 1 µl of the carrier (RHO-P23H carrier) in both eyes on day PN18.

[0089] As a wild-type (WT) control, WT animals were analyzed following these steps:

[0090] - A group of 6 WT mice received intravitreal injections of 1 µl of the carrier (WT carrier) in both eyes on day P18.

[0091] - A group of 6 untreated (uninjected) WT mice were used as control animals to calibrate the instrument and evaluate ERG (WT) under physiological conditions.

[0092] For intravitreal administration, mice were anesthetized by intraperitoneal injection of ketamine / xylazine, and their body temperature was monitored using a rectal thermometer and maintained at a constant 37.5°C. Then, according to the experimental group, mice were intravitreally injected into both eyes with rhBDNF solution (1 µl, 3.5 µg / µl BDNF) or a carrier (50 mM sodium dihydrogen phosphate monohydrate, 100 mM sodium chloride, pH 7.2) using an unsilicified Hamilton 7000 series borosilicate glass syringe.

[0093] The above rhBDNF solution was prepared according to the following: 3.5 µg / µl BDNF, 50 mM sodium dihydrogen phosphate monohydrate, 100 mM sodium chloride, pH 7.2.

[0094] To evaluate photoreceptor function, the animals were subjected to standard electroretinograms (ERG) analysis for scotopic and photopic vision at P30 and P60 days, respectively.

[0095] Electrophysiological analysis was performed using the following procedure: animals were dark-acclimatized for 16 hours, then anesthetized by intraperitoneal injection of ketamine / xylazine, pupils were dilated with 1% tropicamide (Visufarma, Rome, Italy), and body temperature was monitored using a rectal thermometer and maintained constant at 37.5°C.

[0096] Electrophysiological signals from both eyes were recorded using two gold electrodes placed on the cornea. On the other hand, a reference electrode was placed under the skin in the forehead region, while a ground electrode was placed near the tail. Signals were amplified at a 1K gain and filtered from 0.3 to 1kHz. ERG recordings were performed to monitor the functional state of retinal photoreceptors / bipolar cells, monitored by increments of 12 intensity levels (from 10⁻⁴ to 20.0 cd.sm). -2 The retinal response under flashes (with a time frequency of 0.7 Hz) was measured. The ERG was performed using 50 cd / m². -2 Constant background lighting and 10 times with the same intensity (20 cd.sm) -2 The flashing light is used during the examination. The examination lasts an average of 15 minutes.

[0097] ERG results recorded at P30 are reported at Figure 1 A (a wave) and Figure 1 B (b wave), of which a wave: 0.0001 cd·s / m2, 6.1 mV; 0.1 cd·s / m2, 52.8 mV; 1 cd·s / m2, 213.4 mV; 10 cd·s / m2, 341.3 mV; 20cd·s / m2, 401.1 mV; clear vision, 26.4 mV; b wave: 0.0001 cd·s / m2, 2.3 mV; 0.1 cd·s / m2, 315.8mV; 1 cd·s / m2, 506.3 mV; 10 cd·s / m2, 732 mV; 20 cd·s / m2, 876.8 mV; photopic vision, 188.1 mV.

[0098] In particular, although a-wave amplitude analysis reflecting the response of cone and rod cells to light stimulation showed no significant difference in scotopic (rod cells), interocular (rod-cone cells), and photopic (cone cells) responses between RHO-P23H mice treated with rhBDNF and those treated with the loading agent, Figure 1 A), but b-wave amplitude analysis, which reflects photoreceptor-mediated postsynaptic bipolar cell depolarization, showed that RHO-P23H mice treated with rhBDNF exhibited a statistically significant improvement in photopic response (A). Figure 1 B).

[0099] The ERG results recorded at P60 are shown below. Figure 2 A (a wave) and Figure 2 B (b wave), where a wave: 0.0001 cd·s / m 2 6.1 mV; 0.1 cd·s / m 2 52.8 mV; 1 cd·s / m 2213.4 mV; 10 cd·s / m 2 341.3 mV; 20 cd·s / m 2 401.1 mV; visible light, 26.4 mV; b-wave: 0.0001 cd·s / m 2 2.3 mV; 0.1 cd·s / m 2 315.8 mV; 1 cd·s / m 2 506.3 mV; 10 cd·s / m 2 732 mV; 20 cd·s / m 2 , 876.8 mV; visible vision, 188.1 mV.

[0100] Notably, at P60 (42 days after injection), compared with the control group, the ERG spectrum of RHO-P23H mice treated with rhBDNF showed that progressive retinal degeneration had stopped.

[0101] Specifically, analysis of the a-wave amplitude showed that, compared with RHO-P23H mice treated with the carrier (RHO-P23H carrier), RHO-P23H mice treated with rhBDNF (RHO-P23H BDNF) exhibited statistically significant improvements in both interocular and photopic responses. Figure 2 A). Similar results were obtained when analyzing the b-wave amplitude: compared with RHO-P23H animals treated with the carrier, RHO-P23H mice treated with rhBDNF showed statistically significant improvements in scotopic, interocular, and photopic responses to light stimuli. Figure 2 B).

[0102] Therefore, the data obtained indicate that a single intravitreal administration of rhBDNF (3.5 µg / µl) can alleviate photoreceptor degeneration and restore visual function in RHO-P23H knock-in mice 42 days after injection. The improved ERG response is mainly attributed to better preservation of photoreceptor function and reduction in degeneration.

[0103] At P60, the morphological and phenotypic effects of rhBDNF administration to RHO-P23H knock-in mice were also investigated.

[0104] Specifically, the morphological and phenotypic effects of rhBDNF administration were assessed by evaluating the following frozen sections obtained from the above animal groups (excluding the untreated WT animal group): the length (i.e., thickness) of the outer segment (OS) of rod cells, the length (i.e., thickness) and density of the outer nuclear layer (ONL), and the structure and number of cone cells.

[0105] In addition, to quantify the protective effect of rhBDNF treatment against retinal degeneration, the level of apoptosis in frozen retinal sections was evaluated by TdT-mediated dUTP nick end labelling (TUNEL) assay, which identifies cell nuclei containing fragmented DNA (i.e., apoptotic nuclei) in each intact retinal section.

[0106] The aforementioned retinal cryosections were obtained as follows: Retinal tissue and retinal tissue were isolated from a single eye of each of the treated animals and fixed overnight at 4°C in PBS containing 4% paraformaldehyde. The tissues were then cryopreserved, first with 15% phosphate-buffered saline and then with 30% sucrose, and embedded in OCT. 20 µm cryosections were collected on glass slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA).

[0107] The number and structure of cone cells in the frozen retinal sections were evaluated by counting cone-inhibitory protein immunofluorescence-positive cells in a standard region at a comparable distance from the optic nerve.

[0108] The cone-inhibitor protein immunofluorescence protocol was performed as follows: The frozen retinal sections were permeabilized with 1% NP40 at room temperature for 15 minutes. The retina was washed three times with PBS / 0.1% Tween-20 for 30 minutes each time, and blocked for 1 hour at room temperature in blocking buffer (PBS / 0.1% Tween-20 containing 10% FBS). The (thawed) retina was then incubated overnight at 4°C with the primary antibody in the blocking buffer on a shaker. The next day, the retina was washed three times with PBS / 0.1% Tween-20 for 30 minutes each time, and placed in secondary antibody in PBS / 0.1% Tween-20 containing 5% FBS at RT for 1 hour. The retina was rinsed three times in PBS for 30 minutes each time, counterstained with 4,6-diamidinyl-2-phenylindole (DAPI, Vector Laboratories H-1200), and then laid flat between two coverslips for confocal imaging. The following primary antibodies were used: mouse anti-rhodopsin (1:100, Abcam ab5417) and rabbit anti-cone-inhibitor protein (1:1000, EMD Millipore AB15282). The secondary antibody was a goat anti-rabbit / mouse antibody conjugated to Alexa 488 (1:1000, Invitrogen A-11008, A-11001). Coverslips were mounted on the slides with PBS containing 70% glycerol, or dehydrated before mounting (Eukitt Mounting Medium; EMS, Fort Washington, PA). The slides were imaged using an LSM710 Zeiss confocal microscope system. All samples were imaged, and the images were processed in the same manner. ImageJ software was used to process the images, and the average number of photoreceptor cells per 200 µm of retina was calculated. Cone cell count / area was evaluated by manual counting using a Leica DM-6000 microscope with objectives of Leica ∞ / 0.17 / D, HCX PL FLUOTAR, 40X / 0.75, and an area of ​​0.31 mm². 2 .

[0109] The same retinal slice used for cone cell counting was imaged using a Leica TCS SPE 40× confocal microscope at DAPI wavelengths, acquiring Z-stacked images. The images were used to calculate the number of ONL cell nuclei (ONL cell density) and ONL thickness. The number of ONL cell nuclei was evaluated by counting DAPI-stained nuclei in regions equidistant from the optic nerve. Selection of each region and nucleus counting were performed manually using the ITEM analysis image processing program, which stores and subsequently processes the results. ONL was manually measured in both the superior and inferior retina, close to the optic nerve head, using the ITEM analysis image processing program.

[0110] Evaluation of the extracellular rod segment (OS) was performed by measuring the length (i.e., thickness) of the OS of anti-rhodopsin positive cells in a standard region at a comparable distance from the optic nerve using a Leica DM-6000 microscope.

[0111] The results obtained are Figure 3 As shown in A to 3D.

[0112] Specifically, compared with RHO-P23H (RHO-P23H carrier) eyes treated with rhBDNF, RHO-P23H (RHO-P23H BDNF) retinas showed a slight increase in rod cell OS length (OS thickness) on both the ventral and dorsal sides. Figure 3 D), while on both sides of the RHO-P23H retina, no outer nuclear layer (ONL) thickness (i.e., ONL length) was observed after treatment with rhBDNF. Figure 3 A) and density (i.e., the density of cell nuclei within the ONL, Figure 3 The changes in B) suggest that rhBDNF may not be sufficient to protect RHO-P23H rod cells from the rapid phase of retinal degeneration.

[0113] Unexpectedly, as confirmed by quantitative cone cell / area measurements, a significant recovery in the number of cone cells in the retinas of RHO-P23H mice treated with rhBDNF was observed compared to that in RHO-P23H control mice treated with the loading agent. Figure 3 C); Notably, the cone cells surviving in the rhBDNF-treated RHO-P23H retina were longer and healthier than those in the load-treated RHO-P23H retina, and were similar to those in the load-treated WT.

[0114] Consistent with the ERG results, these data indicate that, compared to RHO-P23H control mice treated with the loading agent, the number of rod cell nuclei and OS length in the retinas of rhBDNF-treated RHO-P23H mice were slightly increased, while, most importantly, the number of cone cells was significantly restored. Specifically, the surviving cone cells in the rhBDNF-treated RHO-P23H retinas exhibited a similar number and morphology to those in the WT retinas. Notably, this significant rescue of cone cells is consistent with the retinal function rescue observed by ERG analysis in Example 1.

[0115] For the assessment of the degree and distribution of apoptotic cell death in the above-mentioned frozen retinal sections, the TUNEL assay was performed using the In Situ Cell Death Detection Kit (POD 11684817910) according to the manufacturer's instructions. To evaluate possible nonspecific results, fixed and permeabilized retinal sections were incubated with a reaction mixture without TUNEL reactants as a negative control. Sections were observed using a Leica DM-6000 microscope, and confocal images were subsequently acquired using an LSM710 Zeiss confocal microscope system.

[0116] The results of the TUNEL measurement were in Figure 4 As shown in the image.

[0117] In particular, compared with RHO-P23H mice treated with the loading agent, rhBDNF treatment induced a significant reduction in TUNEL-positive cells in RHO-P23H mice treated with rhBDNF; unexpectedly, the number of TUNEL-positive cells in the retinas of RHO-P23H mice treated with rhBDNF was basically similar to that in the WT control group treated with the loading agent.

[0118] Therefore, the TUNEL assay results indicate that rhBDNF administration reduces the number of apoptotic cells in the retina of RHO-P23H mice, thus supporting the efficacy of rhBDNF administration.

[0119] In summary, the results obtained support the following conclusion: therapeutic application of rhBDNF produces morphological and functional rescue of retinal degeneration, characterized by delaying rod cell degeneration, blocking secondary cone cell death, and functionally rescuing photoreceptor function.

[0120] Example 2

[0121] The dose-response of intravitreal (IVT) administration of rhBDNF to block retinal degeneration in heterozygous RHO-P23H knock-in mice (RHO-P23H mice) was evaluated, and the duration of the beneficial effects of rhBDNF treatment was assessed.

[0122] The animals were divided into the following experimental groups:

[0123] - Group 1 (G1, WT carrier): 6 WT animals received an intravitreal injection of 1 µl of carrier in the left eye at PN18 (the right eye was used as a control eye without injection).

[0124] - Group 2 (G2, RHO-P23H carrier): 6 RHO-P23H mice received an intravitreal injection of 1 µl of the carrier in their left eye at PN18 (the right eye was used as a control eye without injection).

[0125] - Group 3 (G3, RHO-P23H BDNF 5 µg / µl): Six RHO-P23H mice received intravitreal injections of 1 µl of a solution containing 5 µg / µl rhBDNF in both eyes at PN18.

[0126] - Group 4 (G4, RHO-P23H BDNF 3.5 µg / µl): Six RHO-P23H mice received intravitreal injections of 1 µl of a solution containing 3.5 µg / µl rhBDNF in both eyes at PN18.

[0127] - Group 5 (G5, RHO-P23H BDNF 2 µg / µl): Six RHO-P23H mice received intravitreal injections of 1 µl of a solution containing 2 µg / µl rhBDNF in both eyes at PN18.

[0128] - Group 6 (G6, RHO-P23H BDNF 1 µg / µl): Six RHO-P23H mice received intravitreal injections of 1 µl of a solution containing 1 µg / µl rhBDNF in both eyes at PN18.

[0129] The above-mentioned carrier consists of 50 mM sodium dihydrogen phosphate monohydrate and 100 mM sodium chloride (pH 7.2).

[0130] The above rhBDNF solution was prepared according to the following: BDNF at the concentration specified for each of the above animal groups, 50 mM sodium dihydrogen phosphate monohydrate, 100 mM sodium chloride, pH 7.2.

[0131] For intravitreal administration, animals were anesthetized as described in Example 1.

[0132] The photoreceptor function of each animal group was evaluated by standard electroretinography (ERG) as described in Example 1 at the following time points after administration: PN30, PN60, PN90, and PN120.

[0133] ERG results in Figures 5 to 8 The results show that scotopic vision (rod cells, a-wave: 0.0001 cd·s / m2, 6.1 mV; 0.1 cd·s / m2, 52.8 mV; b-wave: 0.0001 cd·s / m2, 2.3 mV; 0.1 cd·s / m2, 315.8 mV), interocular vision (rod-cone cells, a-wave: 1 cd·s / m2, 213.4 mV; 10 cd·s / m2, 341.3 mV; 20 cd·s / m2, 401.1 mV; b-wave: 1 cd·s / m2, 506.3 mV; 10 cd·s / m2, 732 mV; 20 cd·s / m2, 876.8 mV) and photopic vision (cone cells, a-wave: 26.4 mV; b-wave: 188.1 mV) were analyzed. mV) response.

[0134] At PN30 (i.e., 12 days after injection), ERG analysis showed that, compared with control mice (G1 and G2), both rod and cone cell responses were improved in RHO-P23H mice treated with rhBDNF in G3 and G4. Figure 5 (A to 5B).

[0135] In particular, analysis of the b-wave amplitude, which reflects photoreceptor-mediated depolarization of postsynaptic bipolar cells, showed that both G3 and G4 treated with rhBDNF-treated RHO-P23H exhibited an improved trend in scotopic and interocular responses.

[0136] Notably, the b-wave amplitude of RHO-P23H treated with rhBDNF in G3 showed a statistically significant increase in both scotopic and indirect visual responses. Importantly, from 0 to 1.3 cd s / m 2 The light intensity-induced responses originating from the inter-visual range (i.e., both rod photoreceptors and cone photoreceptors) were similar between groups G3 and G1.

[0137] As in Example 1, the beneficial effects of 5 µg / µl rhBDNF administration were more pronounced at P60 (i.e., 42 days after injection). Figure 6As shown in A to 6B. Compared with the control, the ERG spectra of both G3 and G4 RHO-P23H mice treated with rhBDNF showed improved photoreceptor function and delayed progression of retinal degeneration. Analysis of α-wave amplitude showed that, compared with RHO-P23H animals treated with the carrier, both G3 and G4 RHO-P23H mice treated with rhBDNF showed statistically significant improvements in both interocular and photopic responses.

[0138] Notably, compared with the G4 group, the RHO-P23H mice in the G3 group treated with rhBDNF showed a statistically significant improvement in interocular response.

[0139] This result was even more evident when analyzing b-wave amplitude. In fact, analysis of b-wave amplitude showed that, compared with control animals, both G3 and G4 RHO-P23H mice treated with rhBDNF exhibited statistically significant improvements in all scotopic, interocular, and photopic responses to light stimuli.

[0140] Importantly, 5 µg / µl rhBDNF induced a statistically significant improvement in b-wave amplitude in G3 animals, with interocular and photopic responses similar to those in WT mice. In particular, the interocular range-derived responses induced by light intensities from 0 to 1.3 cd s / m² were comparable between the G3 and G1 groups, indicating that both rod and cone cell functions were fully salvaged.

[0141] The improvement in photoreceptor function was still significant at PN90 (i.e., 72 days after injection), such as Figure 7 As shown in A to 7B. Analysis of the a-wave amplitude of RHO-P23H mice treated with rhBDNF in G3 and G4 showed that the interocular response showed a slight trend of improvement, while the photopic response showed a statistically significant improvement.

[0142] This result was even more evident when analyzing b-wave amplitude. In fact, analysis of b-wave amplitude showed that, compared with control animals, both G3 and G4 RHO-P23H mice treated with rhBDNF exhibited statistically significant improvements in all scotopic, interocular, and photopic responses to light stimuli.

[0143] Similarly, 5 µg / µl rhBDNF induced a statistically highly significant improvement in b-wave amplitude in G3 animals, with interocular and photopic responses remaining similar to the ERG responses recorded in WT mice. Likewise, above 1.3 cd s / m 2 The light intensity-induced interocular response was comparable between the G3 and G1 groups, indicating the high beneficial effects of high-dose rhBDNF administration, such as those detected in the rescue of both rod and cone cell function.

[0144] like Figure 8 As shown in A to 8B, compared with the control, the ERG spectra of G3 (but not G4) RHO-P23H mice treated with rhBDNF showed a trend toward functional preservation up to PN120 (i.e., 102 days after injection). 1 to 1.3 cd s / m 2 The light intensity-induced interocular response showed a positive increasing trend in G3 RHO-P23H mice treated with rhBDNF compared to the control. This result indicates that 5 µg / µl rhBDNF maintained its beneficial effects on both rod and cone cell function approximately 4 months after injection. In contrast, administration of 3.5 µg / µl rhBDNF no longer showed a beneficial effect approximately 4 months after injection. However, in the range of -1 to 0 cd s / m 2 Even under light intensity, the improvement may still be noticeable.

[0145] Regarding RHO-P23H mice treated with rhBDNF in G5, ERG analysis at PN30 and PN60, when comparing the a and b waves with control mice (G1 and G2), showed that the improvements in rod and cone cell responses in rhBDNF-treated RHO-P23H mice were similar to those observed in RHO-P23H mice treated with rhBDNF in G4. Consistent with the beneficial effects of rhBDNF administration, RHO-P23H mice treated with rhBDNF in G5 showed statistically significant improvements in interocular responses for both a and b waves. However, light intensities above -1 cd s / m² induced a slower response in G5 than those recorded in G4, indicating that low-dose rhBDNF may reduce the beneficial effects of rhBDNF on both rod and cone cell function. Figure 5 and Figure 6 ).

[0146] At PN90 and PN120, the beneficial effects of rhBDNF-treated G5 RHO-P23H mice were no longer evident. Analysis of the a-wave and b-wave amplitudes in both PN90 and PN120 day-old G5 RHO-P23H mice treated with rhBDNF showed that ERG recordings were similar to those recorded in untreated G2 RHO-P23H mice across all scotopic, interocular, and photopic responses to light stimuli. However, similar to G4, residual improvement was still observed at light intensities ranging from -1 to 0 cd s / m², indicating slight preservation of rod cell function approximately 4 months after injection. Figure 7 and Figure 8 ).

[0147] No beneficial effect was identified in G6 RHO-P23H mice treated with rhBDNF at any of the time points analyzed (see [link to relevant documentation]). Figures 5 to 8 ).

[0148] These data indicate that in RHO-P23H knock-in mice, dose-dependent rescue of photoreceptor function is stronger and more durable following a single intravitreal administration of a high dose of rhBDNF (5 µg / µl).

[0149] In particular, intravitreal administration of the high dose of rhBDNF showed the best long-term preservation of photoreceptor function; in contrast, intravitreal administration of the low dose (1 µg / µl) of rhBDNF did not show any improvement in photoreceptor function.

[0150] At P120, the morphological and phenotypic effects of rhBDNF administration on RHO-P23H knock-in mice were studied in the G3 group (RHO-P23H mice treated with 5 µg / µl rhBDNF) using the same analysis as described in Example 1, and compared with the G1 (WT carrier) and G2 (RHO-P23H carrier) groups.

[0151] The results of morphological analysis are in Figure 9 Reports from A to 9D.

[0152] In particular, compared with the control, the RHO-P23H retina treated with rhBDNF (“RHO-P23H BDNF”) showed a statistically significant improvement in the thickness of the outer nuclear layer (ONL) on both sides of the optic nerve (i.e., the dorsal and ventral sides). Figure 9 A). This improvement is accompanied by a statistically significant improvement in the outer nuclear layer density ( Figure 9 B), and in both the ventral and dorsal sides, compared with the carrier-treated RHO-P23H eye, an increase in the length of the extracellular segment (OS) of the photoreceptor (B). Figure 9 (D), thus demonstrating that administration of the rhBDNF at a concentration of 5 µg / µl can protect RHOP23H. + / - Rod cells are protected from retinal degeneration.

[0153] Clearly, compared with RHO-P23H control mice treated with the loading agent, statistically significant salvage was also observed in the cone cell count defect in the retina of RHO-P23H mice treated with rhBDNF. Figure 9 (C) In particular, both the number and structure of surviving cone cells were unexpectedly comparable to those of the WT retina treated with the loading agent. This remarkable rescue of cone cells is consistent with the rescue observed in retinal function as shown in the ERG analysis of the G3 group.

[0154] In summary, these data support the complete rescue of cone cell death in RHO-P23H mice by administration of rhBDNF at a concentration of 5 µg / µl.

[0155] Consistent with the morphological results above, treatment with 5 µg / µl of rhBDNF significantly reduced the number of TUNEL-positive photoreceptor cells compared to RHO-P23H mice treated with the loading agent. Figure 10 This indicates a reduction in photoreceptor cell death.

[0156] In fact, rhBDNF treatment induced a statistically significant reduction in TUNEL-positive cells in rhBDNF-treated RHO-P23H mice (compared to load-treated RHO-P23H mice), which was actually highly similar to the load-treated control group. These data strongly confirm the effectiveness of rhBDNF treatment in rescuing retinal apoptosis in RHO-P23H mice.

[0157] Based on the above results, treatment with rhBDNF at a concentration of 5 µg / µl resulted in improved photoreceptor function, accompanied by salvage of retinal cell death and morphology, up to 4 months of age (i.e., 102 days after injection). Notably, the efficacy of this treatment was demonstrated by statistically significant improvements in ONL thickness and density, and complete recovery of cone cell function and survival, which were essentially identical in number and morphology to those in the WT control retina treated with the carrier.

[0158] Example 3

[0159] The inventors investigated in vitro whether rhBDNF could improve the clearance / transport of intracellular rhodopsin in the 661W photoreceptor cell line overexpressing RHO-P23H.

[0160] Materials and Methods:

[0161] - Cell Culture and Processing

[0162] 661W cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 1% (v / v) fetal bovine serum (FBS) and 5% penicillin-streptomycin.

[0163] The cell lines were kept in a humidified incubator at 37°C and 5% CO2, according to the supplier’s guidelines.

[0164] To analyze potential rhodopsin degradation, cells were plated for 24 hours and then treated with the following: 200 nM bafloxacin A1 (Baf) (Sigma-Aldrich, B1793) for 3 hours, 100 µg / ml cycloheximide (CHX) (Sigma-Aldrich, C4859), 100 mM bortezomib (BTZ) (Sigma-Aldrich, 5043140001), rhBDNF solution (5 ng / ml rhBDNF, 50 mM sodium phosphate pH 7.0, 100 mM NaCl) or a carrier (50 mM sodium phosphate pH 7.0, 100 mM NaCl) for 1 hour or 6 hours, as disclosed below.

[0165] - Plasmids and Transfection

[0166] Cells were transfected with 2.5 µg pCS2-RHO-P23H for 24 hours when they reached 70% to 80% confluence. For plasmid transfection, Lipofectamine 2000 (Invitrogen, 12566014) was used according to the manufacturer's protocol.

[0167] Western blot analysis

[0168] Cells were collected after transfection or treatment to extract total protein. Cell samples were lysed using RIPA buffer (150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0) containing an inhibitor mixture (ThermoFischer Scientific, 78420). Total protein concentration was determined by Bradford assay and quantified using a NanoDrop ND-8000 spectrophotometer (NanoDrop Technologies).

[0169] Proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (EMDMillipore, IPVH00010). The membrane was then blocked in 0.1% Tween-Tris buffered saline containing 5% bovine serum albumin (Sigma-Aldrich, 9048-46-8) at room temperature for 1 hour, and subsequently incubated with primary antibody overnight at 4°C.

[0170] For Western blot analysis, the following antibodies were used: mouse anti-Lamp1 (1:500, Sigma-Aldrich, L1418), rabbit anti-β-actin (1:1000, Cell Signaling Technology, 4967), and mouse anti-RHO (1:500, Santa Cruz, sc-57432).

[0171] After washing with 1% TBS, the membrane was incubated for 1 hour at room temperature with the following secondary antibodies: HPR-conjugated goat anti-rabbit IgG antibody and HPR-conjugated goat anti-mouse IgG antibody (1:10,000, EMD Millipore, 12-348; 12-349), and anti-goat IgG (full molecular weight) (1:10,000, Sigma Aldrich, A5420). Western blot analysis was performed using a Chemidoc imaging system (Bio-Rad) with enhanced chemiluminescence solution (Clarity Western ECL substrate, Bio-Rad, 1705061). Quantification of the Western blot was performed using ImageJ software.

[0172] - Soluble and insoluble rhodopsin extraction

[0173] Transfect 661W cells with 2.5 µg pCS2-RHO-P23H for 24 hours when they are at 70% to 80% confluence, as described above.

[0174] After 24 hours, 661W RHO-P23H cells were treated with rhBDNF or a carrier solution for 6 hours. Cells were collected after transfection or treatment to extract soluble and insoluble protein fractions. Lysed cells were obtained by incubating on ice for 30 minutes with 250 µl of ice-cold lysis buffer (5 mM EDTA, 1% Triton X-100, PBS pH 7.5) and a protease inhibitor mixture (Thermo Fischer Scientific, 78420). The samples were then centrifuged at 13,000 × g for 15 minutes at 4 °C to separate soluble and insoluble fractions. The supernatant containing the soluble fraction was collected and stored at -80 °C until use. The precipitate containing the insoluble fraction was resuspended in 50 µl of 1% SDS PBS for 10 minutes, then an additional 200 µl of lysis buffer was added and gently mixed. Finally, the precipitate is dissolved on ice by acoustic treatment with short ultrasonic pulses and stored at -80°C until use.

[0175] - Immunofluorescence

[0176] Cells were fixed with 4% formaldehyde (Sigma-Aldrich) at room temperature for 15 minutes, followed by washing with 1% PBS. After fixation, cells were permeabilized with blocking buffer (0.5% BSA, 0.005% saponin, 0.02% NaN3) at room temperature for 1 hour. The following primary antibodies were used: rabbit anti-LAMP1 (1:400, abcam, ab24170) and mouse anti-RHO (1:100 Santa Cruz, sc-57432 abcam, ab5417).

[0177] After washing with 1% PBS, the slides were incubated for 1 hour at room temperature with the following secondary antibodies: Alexa 594 goat anti-rabbit / mouse (1:1,000, Invitrogen A-11037 rabbit, A-11032 mouse) or Alexa 488 goat anti-rabbit / mouse (1:1,000, Invitrogen A-11008 rabbit, A-11001 mouse) and DAPI (1:500, Vector Laboratories H-1200); then, the slides were washed with 1% PBS, mounted with PBS / glycerol, and imaged using an Olympus FV3000 confocal microscope. All images were processed in the same manner.

[0178] result:

[0179] Surprisingly, as Figure 11 A (soluble fraction) and Figure 11 According to B (insoluble fraction), the administration of rhBDNF to 661W RHO-P23H cells (P23H VEH) significantly reduced RHO-P23H in both insoluble and soluble cell fractions compared to 661W RHO-P23H cells treated with the carrier (P23H VEH).

[0180] In particular, Figure 11 A and 11B specifically report the following forms of P23H-rhodopsin: mature glycosylated dimer (120 kDa), immature glycosylated dimer (70 kDa), and mature glycosylated monomer (50 kDa).

[0181] It is worth noting that, Figure 11 A and Figure 11 Both B showed a reduction in immature rhodopsin glycosylated dimers (corresponding to 70 kDa), indicating a decrease in RHO-P23H aggregates in BDNF-treated cells (wild-type rhodopsin was not considered as a control because it does not accumulate in an insoluble form and is already expressed in 661W cells).

[0182] These data indicate that rhBDNF administration directly leads to significant changes in rhodopsin degradation.

[0183] To further investigate the role of BDNF in the degradation of rhodopsin in RHO-P23H photoreceptors, the degradation of lysosomal cargo in 661W RHO-P23H cells after BDNF administration was assessed by treating cells with bafloxacin A1 (Baf).

[0184] Baf is a well-known vacuolar H( + Inhibitors of ATPase promote alkalization of the lysosomal cavity and inhibit the degradation of lysosomal cargo, leading to the accumulation of undigested material in the fused autophagosome cavity and preventing the fusion of new autophagosomes with lysosomes.

[0185] The result is Figure 12 Report from the Central Committee.

[0186] Quantification was performed on cell fluorescence images captured at 63× magnification using an LSM700 Zeiss confocal microscope system, which were then converted to grayscale and normalized relative to background staining using ImageJ. The quantification of RHO-P23H positive lysosomal structures in each cell was measured using the JaCoP plugin in ImageJ software (Bolte & Cordelieres, 2006).

[0187] like Figure 12 As shown, compared with cells previously treated with BAF followed by a carrier treatment (“P23H VEH + BAF”), treatment with rhBDNF on 661W RHO-P23H cells previously treated with BAF (“P23H BDNF + BAF”) showed increased accumulation of RHO-P23H in the LAMP1-positive autolysosome cavity.

[0188] LAMP1 is commonly used as a lysosomal marker, and LAMP1-positive organelles are often referred to as lysosomal compartments (colocalization was not performed in this experimental test because RHO-P23H accumulates in the lysosomal lumen, rather than on the membrane of the lysosome stained with lamp1 immunofluorescence).

[0189] Notably, administration of BAF (661WRHO-P23H cells treated with BAF / BDNF) 3 hours after BDNF treatment exacerbated the increase in RHO-P23H-containing autolysosomal structures within the lumen (not shown in immunofluorescence images). This is consistent with the blockade of cargo degradation in the increased number of autolysosomes, further supporting the idea that BDNF treatment increases RHO-P23H degradation via the autophagy pathway.

[0190] The effects of BDNF administration on 661W RHO-P23H cells were evaluated in the presence of cyclohexylimide (CHX), a known protein synthesis inhibitor.

[0191] In 661W RHO-P23H cells treated with a carrier, BDNF, and CHX, Western blot analysis results of P23H rhodopsin and control (actin) were... Figure 13 Report A.

[0192] Figure 13 Figure B shows the quantification of RHO protein normalized relative to actin used as a control. The figure shows the mean ± SEM from three independent experiments.

[0193] The results showed that RHO-P23H protein degradation was faster in BDNF-treated cells compared to cells treated with the carrier. These data suggest that the reduction of RHO-P23H protein in BDNF-treated 661W RHO-P23H cells may be dependent on faster RHO degradation.

[0194] In addition, to investigate the possibility that BDNF can clear RHO-P23H accumulation through proteasome activity, 661W RHO-P23H cells were treated with the proteasome inhibitor bortezomib (BORT) for 24 hours 24 hours after transfection.

[0195] During the last 6 hours of bortezomib treatment, 661W RHO-P23H cells were cultured in the presence of BDNF or a carrier solution and subjected to Western blot analysis of rhodopsin.

[0196] like Figure 13 As shown in Figure C, RHO-P23H was significantly reduced after BDNF treatment compared to cells treated with the load (“BORT + VEH”).

[0197] It is noteworthy that the quantification of rhodopsin in 661W RHO-P23H cells treated with BORT and BDNF solutions was very similar to that in 661W WT wild-type cells treated with BDNF.

[0198] Given that BDNF has shown effectiveness in clearing P23H rhodopsin even in the presence of proteasome inhibitors, these data confirm that BDNF treatment plays a specific role in activating the degradation of RHO-P23H in lysosomes.

Claims

1. Brain-derived neurotrophic factor (BDNF) for the treatment of rhodopsin-mediated retinitis pigmentosa in subjects, wherein the BDNF is administered as a protein.

2. The BDNF of claim 1, wherein the rhodopsin-mediated retinitis pigmentosa is rhodopsin-mediated autosomal dominant retinitis pigmentosa.

3. The BDNF of claim 2, wherein the rhodopsin-mediated autosomal dominant retinitis pigmentosa is associated with type I rhodopsin mutations.

4. The BDNF of claim 3, wherein the type I rhodopsin mutation is selected from one of the following mutations: L328P, T342M, Q344R, Q344P, Q344ter, V345L, V345M, A346P, P347A, P347R, P347Q, P347L, P347S, P347T, ter349Q, ter349E.

5. The BDNF of claim 2, wherein the rhodopsin-mediated autosomal dominant retinitis pigmentosa is associated with type II rhodopsin mutations.

6. The BDNF of claim 5, wherein the type II rhodopsin mutation is selected from one of the following mutations: N15S, T17M, V20G, P23A, P23H, P23L, Q28H, G51R, G51V, P53R, T58R, T58M, V87D, G89D, G106R, G106W, C110F, C110R, C110S, C110Y, E113K, L125R, W161R, A164E, A164V, C167R, C167W, P171 Q, P171L, P171S, Y178N, Y178D, Y178C, E181K, G182S, G182V, C185R, C187G, C187Y, G188R, G188E, D190N, D190G, D190Y, H211R, H211P, C222R, P267R, P267L, S270R, K296N, K296E, and K296M.

7. The BDNF of claim 5, wherein the type II rhodopsin mutation is P23H.

8. The BDNF of claims 1 to 7, wherein the BDNF is applied to the object via intravitreal injection.

9. The BDNF of the application according to claims 1 to 8, wherein the BDNF is a human BDNF, preferably a recombinant human BDNF.

10. The BDNF of claims 1 to 9, wherein the BDNF is administered to the subject by intravitreal injection in an amount of 10 μg to 500 μg per eye, preferably 15 μg to 250 μg, more preferably 18 μg to 50 μg, and even more preferably 20 μg.

11. An ophthalmic composition comprising BDNF and at least one ophthalmologically acceptable excipient for treating rhodopsin-mediated retinitis pigmentosa in a subject, wherein the BDNF is administered as a protein.

12. The ophthalmic composition according to claim 11, wherein the BDNF is human BDNF, preferably recombinant human BDNF.

13. The ophthalmic composition according to claim 11 or 12, wherein it is a liquid composition.

14. The ophthalmic composition according to claim 13, wherein BDNF is contained in the composition at a concentration of 0.2 μg / μl to 10 μg / μl, preferably 0.3 μg / μl to 5 μg / μl, more preferably 0.35 μg / μl to 1 μg / μl, and even more preferably 0.4 μg / μl.

15. The ophthalmic composition of claims 11 to 14, wherein the ophthalmic composition is administered to the subject via intravitreal injection.

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