CN118717946A - Application of a chimeric protein in the treatment of optic nerve degenerative diseases - Google Patents

Abstract

The invention provides application of chimeric protein in treating optic nerve degeneration diseases, in particular relates to application of chimeric protein and encoding nucleic acid thereof in preparing a composition or preparation for preventing and/or treating optic nerve degeneration diseases. The chimeric protein is a GPR protein mutant that replaces and mutates one or more transmembrane regions of a wild-type GPR protein corresponding to accession No. AF349993_1 selected from the group consisting of: a transmembrane region No. 4-5; and/or transmembrane region No. 4-6; and/or transmembrane region No. 1-2, the chimeric proteins of the present invention having a specific structure are useful for preventing and/or treating an optic neurodegenerative disease (such as glaucoma).

Description

Application of a chimeric protein in treating optic nerve degenerative diseases

Technical Field

The present invention relates to the field of biotechnology, and in particular to application of a chimeric protein in treating optic nerve degenerative diseases.

Background Art

Light therapy was used to treat trauma and skin diseases as early as ancient Egypt and India. Modern light therapy is widely used to treat skin diseases such as lupus vulgaris, psoriasis, vitiligo, etc., as well as neurological diseases such as multiple sclerosis and retinopathy. In recent years, near-infrared (NIR) has been used to treat Alzheimer's disease and Parkinson's disease. Near-infrared irradiation can increase the content of adenosine triphosphate (ATP), reduce the deposition of β-amyloid protein, and alleviate the movement disorder of Parkinson's disease and the cognitive disorder of Alzheimer's disease. However, near-infrared (NIR) therapy is still in the preclinical stage and has limited effect on advanced neurodegenerative diseases. In recent years, with the development of optogenetics, our understanding of traditional light therapy has been broadened. Optogenetics has been successfully used to treat visual disorders and neuropsychiatric diseases in recent years.

The optic nerve is a nerve fiber of the retina and is part of the central nervous system. The visual information received by the retina is transmitted to the brain through the optic nerve. Optic nerve diseases mainly include optic neuritis, optic atrophy, ischemic optic disc disease, and papilledema. Glaucoma is a group of diseases characterized by optic disc atrophy and depression, visual field defects, and decreased vision. Pathological increase in intraocular pressure and insufficient blood supply to the optic nerve are the primary risk factors for the onset of glaucoma. The tolerance of the optic nerve to pressure damage is also related to the occurrence and development of glaucoma.

Mitochondria are endosymbiotic organelles in eukaryotic cells that are responsible for energy metabolism, heat production, cell death, ion and metabolic substrate homeostasis. The respiratory chain complex on the inner membrane of mitochondria produces a transmembrane proton gradient. The energy stored in the form of proton electrochemical gradient drives ATP synthase to catalyze ADP and Pi to synthesize ATP, and plays an important role in maintaining the resting potential of neurons, muscle contraction and protein synthesis. Mitochondrial function defects are associated with many neurodegenerative system diseases, diabetes, cancer, and retinal degeneration. As the largest complex in the electron transport chain (containing 45 to 64 subunits), defects in mitochondrial complex I account for 30% of mitochondrial diseases.

However, there are still few reports on the treatment of optic nerve degeneration diseases caused by mitochondrial defects. Therefore, there is an urgent need in the art to develop a new method for treating optic nerve degeneration diseases.

Summary of the invention

The present invention provides a new method for treating optic nerve degenerative diseases.

The first aspect of the present invention provides a chimeric protein, or a polynucleotide encoding it, or a promoter thereof, for preparing a composition or a preparation for preventing and/or treating optic nerve degenerative diseases, wherein the chimeric protein is a GPR protein mutant, wherein the GPR protein mutant has one or more transmembrane regions selected from the group consisting of:

Transmembrane domains 4-5; and/or

Transmembrane regions 4-6; and/or

Transmembrane regions 1-2.

In another preferred embodiment, in the GPR protein mutant, the transmembrane region 4-5 corresponding to the accession number AF349993_1 of the wild-type GPR protein is replaced by the transmembrane region 4-5 of the APR protein.

In another preferred embodiment, the GPR protein mutant has transmembrane regions 4 to 6 corresponding to accession number AF349993_1 of the wild-type GPR protein replaced by transmembrane regions 4 to 6 of the APR protein.

In another preferred embodiment, in the GPR protein mutant, the transmembrane regions 1-2 corresponding to the accession number AF349993_1 of the wild-type GPR protein are replaced by the transmembrane regions 1-2 of the APR protein.

In another preferred embodiment, the GPR protein mutant is substituted at positions 114-165 of the wild-type GPR protein corresponding to transmembrane regions 4-5 with accession number AF349993_1.

In another preferred embodiment, the GPR protein mutant undergoes substitution at positions 114-195 of the wild-type GPR protein corresponding to transmembrane regions 4-6 with accession number AF349993_1.

In another preferred embodiment, the GPR protein mutant undergoes substitution at positions 33-91 of the wild-type GPR protein corresponding to transmembrane regions 1-2 with accession number AF349993_1.

In another preferred example, the 4th to 5th transmembrane regions of the APR protein correspond to positions 120-170 of the protein sequence with accession number WP_014952819.

In another preferred example, the 4th to 6th transmembrane regions of the APR protein correspond to positions 120-201 of the protein sequence with accession number WP_014952819.

In another preferred example, the 1st to 2nd transmembrane regions of the APR protein correspond to positions 38-96 of the protein sequence with accession number WP_014952819.

In another preferred embodiment, the chimeric protein comprises the structure of formula I from the N-terminus to the C-terminus:

Z1-Z2-Z3(I)

Wherein, Z1 is the first transmembrane region of GPR protein;

Z2 is the transmembrane region of the APR protein;

Z3 is the second transmembrane region of the GPR protein;

Wherein, each "-" is independently a connecting peptide or a peptide bond.

In another preferred example, the Z1 element corresponds to positions 1-114 of the protein sequence with accession number AF349993_1.

In another preferred example, the Z2 element corresponds to positions 120-170 or 120-201 of the protein sequence with accession number WP_014952819.

In another preferred example, the Z3 element corresponds to positions 166-249 or 196-249 of the protein sequence with accession number AF349993_1.

In another preferred embodiment, the chimeric protein comprises the structure of formula IV from the N-terminus to the C-terminus:

P0-P1-P2(IV)

In the formula,

P0 is a signal peptide of an optional GPR protein;

P1 is the transmembrane region of the APR protein;

P2 is the transmembrane region of the GPR protein;

Wherein, each "-" is independently a connecting peptide or a peptide bond.

In another preferred example, the P0 element corresponds to positions 1-32 of the protein sequence with accession number AF349993_1.

In another preferred example, the P1 element corresponds to positions 38-96 of the protein sequence with accession number WP_014952819.

In another preferred example, the P2 element corresponds to positions 92-249 of the protein sequence with accession number AF349993_1.

In another preferred embodiment, the chimeric protein is selected from the following group:

(A) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 or 3 or 5;

(B) a polypeptide having ≥80% homology (preferably, ≥90% homology; more preferably ≥95% homology; most preferably, ≥97% homology, such as 98% or more, 99% or more) with the amino acid sequence of SEQ ID NO: 1 or 3 or 5, and the polypeptide has mitochondrial localization and proton pump activity;

(C) A derivative polypeptide having mitochondrial localization and proton pump activity formed by substituting, deleting or adding 1-15 amino acid residues of the amino acid sequence shown in any one of SEQ ID NO: 1, 3 or 5.

In another preferred embodiment, the amino acid sequence of the chimeric protein is shown in SEQ ID NO: 1 or 3 or 5.

In another preferred embodiment, the chimeric protein includes a wild-type chimeric protein and a mutant chimeric protein.

In another preferred embodiment, the mutant chimeric protein is mutated in one or more core amino acids associated with mitochondrial localization and proton pump activity selected from the following group corresponding to SEQ ID NO.: 1 of the GPR protein mutant:

Glycine (G) at position 196; and/or

Glycine (G) at position 137.

In another preferred embodiment, the glycine (G) at position 196 is mutated to serine (S); and/or

The glycine (G) at position 137 was mutated to threonine (T).

In another preferred embodiment, the amino acid sequence of the mutant chimeric protein is shown in SEQ ID NO.:7.

In another preferred embodiment, the promoter is selected from the following group: a small molecule compound, a nucleic acid construct expressing a chimeric protein, or a combination thereof.

In another preferred embodiment, the nucleic acid construct for expressing the chimeric protein comprises a chimeric protein encoding polynucleotide and a vector for expressing the chimeric protein.

In another preferred embodiment, the vector includes a viral vector and a non-viral vector.

In another preferred embodiment, the viral vector is selected from the following group: an adeno-associated viral vector, a lentiviral vector, or a combination thereof.

In another preferred embodiment, the non-viral vector comprises a plasmid or a stem cell.

In another preferred embodiment, the chimeric protein has mitochondrial localization and proton pump activity.

In another preferred embodiment, the optic nerve degeneration disease includes primary optic nerve degeneration and secondary optic nerve degeneration.

In another preferred embodiment, the optic nerve degenerative disease is selected from the group consisting of diabetic eye disease, ischemic papillopathy, papillitis, papilledema, retinal choroiditis, retinitis pigmentosa, central retinal artery occlusion, glaucoma, or a combination thereof.

In another preferred embodiment, the composition or preparation is also used for one or more purposes selected from the following group:

(a) Improve visual acuity;

(b) Improve the survival rate of peripheral retinal ganglion cells;

(c) Alleviate glaucomatous retinal ganglion cell degeneration and visual loss;

(d) Reduce the high pressure inside the eye caused by glaucoma.

In another preferred embodiment, the chimeric protein is a light-activated protein.

In another preferred embodiment, the chimeric protein is activated using ambient light, an external light emitting diode (LED), a laser or other suitable light sources.

In another preferred embodiment, the light activation refers to continuous illumination with visible light (such as green light) with a wavelength of 400-700 nm, preferably 450-600 nm (such as 530 nm).

In another preferred embodiment, the light-activated illumination is 2000-5000 lux, preferably 3000-4000 lux (eg 3200 lux).

In another preferred embodiment, light activation is performed continuously for 2-24 hours, preferably 4-20 hours, more preferably 6-16 hours, and most preferably 8-15 hours (such as 12 hours) per day.

The second aspect of the present invention provides a pharmaceutical composition comprising:

(a1) A first active ingredient for preventing and/or treating optic nerve degenerative diseases, the first active ingredient comprising: a chimeric protein, or a polynucleotide encoding it, or a promoter thereof, wherein the chimeric protein is a GPR protein mutant, wherein the GPR protein mutant has one or more transmembrane regions corresponding to the accession number AF349993_1 of the wild-type GPR protein replaced:

Transmembrane domains 4-5; and/or

Transmembrane regions 4-6; and/or

Transmembrane regions 1-2;

(a2) optionally, a second active ingredient for preventing and/or treating optic nerve degenerative diseases, wherein the second active ingredient comprises: other drugs for preventing and/or treating optic nerve degenerative diseases; and

(b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the promoter is selected from the following group: a small molecule compound, a nucleic acid construct expressing a chimeric protein, or a combination thereof.

In another preferred embodiment, the nucleic acid construct for expressing the chimeric protein comprises a chimeric protein encoding polynucleotide and a vector for expressing the chimeric protein.

In another preferred embodiment, the vector includes a viral vector and a non-viral vector.

In another preferred embodiment, the viral vector is selected from the following group: an adeno-associated viral vector, a lentiviral vector, or a combination thereof.

In another preferred embodiment, in the pharmaceutical composition, the component (a1) accounts for 1-99wt%, preferably 10-90wt%, and more preferably 30-70wt% of the total weight of the pharmaceutical composition.

In another preferred embodiment, in the pharmaceutical composition, the component (a2) accounts for 1-99wt%, preferably 10-90wt%, and more preferably 30-70wt% of the total weight of the pharmaceutical composition.

In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.

In another preferred embodiment, the other drugs for preventing and/or treating optic nerve degenerative diseases include B vitamins, nerve nourishing drugs such as gangliosides or nerve growth factors.

In another preferred embodiment, the pharmaceutical composition may be a single compound or a mixture of multiple compounds.

In another preferred embodiment, the pharmaceutical composition is used to prepare a drug or preparation for treating or preventing optic nerve degenerative diseases.

In another preferred embodiment, the pharmaceutical dosage form is an oral or parenteral dosage form.

In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.

In another preferred embodiment, the non-oral dosage form is an injection or injection.

In another preferred embodiment, the total content of the active ingredient (a1) and the active ingredient (a2) is 1 to 99 wt %, more preferably 5 to 90 wt %, of the total weight of the composition.

The third aspect of the present invention provides a medicine kit, comprising:

(i) a first container, and an active ingredient (a1) chimeric protein, or a polynucleotide encoding it, or a promoter thereof, or a drug containing the active ingredient (a1) in the first container, wherein the chimeric protein is a GPR protein mutant, wherein the GPR protein mutant has one or more transmembrane regions corresponding to the wild-type GPR protein with accession number AF349993_1 selected from the following group replaced:

Transmembrane regions 4-5; and/or

Transmembrane regions 4-6; and/or

Transmembrane regions 1-2;

(ii) an optional second container, and the active ingredient (a2) in the second container, other drugs for preventing and/or treating optic nerve degenerative diseases, or drugs containing the active ingredient (a2).

In another preferred embodiment, the first container and the second container are the same or different containers.

In another preferred embodiment, the drug in the first container is a single-ingredient preparation containing a chimeric protein.

In another preferred embodiment, the drug in the second container is a single-ingredient preparation containing other drugs for preventing and/or treating optic nerve degenerative diseases.

In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

In another preferred embodiment, the kit further comprises instructions for administering the active ingredient (a1), or administering the active ingredient (a1) and the active ingredient (a2) in combination to prevent and/or treat optic nerve degenerative diseases.

In another preferred embodiment, the dosage form of the preparation containing the active ingredient (a1) chimeric protein or containing (a2) other drugs for preventing and/or treating optic nerve degenerative diseases includes capsules, tablets, suppositories, or intravenous injections.

The fourth aspect of the present invention provides a use of the pharmaceutical composition described in the second aspect of the present invention or the medicine kit described in the third aspect of the present invention for preparing a drug for preventing and/or treating optic nerve degenerative diseases.

A fifth aspect of the present invention provides a method for preventing and/or treating optic nerve degenerative diseases, comprising the steps of:

A chimeric protein, or a polynucleotide encoding it or a promoter thereof, or the pharmaceutical composition according to the second aspect of the present invention, or the drug kit according to the third aspect of the present invention is administered to a subject in need thereof, wherein the chimeric protein is a GPR protein mutant, and the GPR protein mutant has one or more transmembrane regions corresponding to the accession number AF349993_1 of the wild-type GPR protein replaced:

Transmembrane regions 4-5; and/or

Transmembrane regions 4-6; and/or

Transmembrane regions 1-2.

In another preferred embodiment, the administration includes oral administration and injection.

In another preferred embodiment, the subject includes a human or a non-human mammal.

In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

In another preferred embodiment, the chimeric protein is administered in the form of monomers and/or dimers.

In another preferred embodiment, the method is carried out under light.

In another preferred embodiment, the chimeric protein is administered 1-7 consecutive days per week, preferably 2-5 consecutive days per week, and more preferably 2-3 consecutive days per week.

In another preferred embodiment, the chimeric protein is administered for 1-20 weeks, preferably 2-12 weeks, and more preferably 4-8 weeks.

In another preferred embodiment, the administration of the chimeric protein comprises photoactivation.

In another preferred embodiment, the light activation includes activating the chimeric protein using ambient light, an external light emitting diode (LED), a laser or other suitable light sources.

In another preferred embodiment, the light activation is continuous illumination with visible light (such as green light) with a wavelength of 400-700 nm, preferably 450-600 nm (such as 530 nm).

In another preferred embodiment, the light-activated illumination is 2000-5000 lux, preferably 3000-4000 lux (eg 3200 lux).

In another preferred embodiment, the light activation is performed continuously for 2-24 hours per day, preferably, 4-20 hours, more preferably, 6-16 hours, and most preferably, 8-15 hours (such as 12 hours).

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the construction and verification of EcGAPR transgenic mice. (A) Schematic diagram of the construction of mt-EcGAPR transgenic mice using CRISPR-Cas9 technology. (B) Immunofluorescence staining results of hippocampal neurons in primary culture of mt-EcGAPR mice. mt-EcGAPR contains a myc tag at the N-terminus. Co-staining with myc antibody and mitochondrial inner membrane protein mitofilin showed that myc positive signals co-localized with mitofilin, suggesting that mt-EcGAPR can be correctly localized to mitochondria in mice.

Figure 2 shows the retinal sections of EcGAPR transgenic mice and the mouse glaucoma model established by silicone oil injection. (A) Immunofluorescence staining results of retinal sections of mt-EcGAPR transgenic mice and control mice. Green: GFP. Silver: PKC-α, a marker of retinal bipolar cells. Red: Brn3a, a marker of retinal ganglion cells. Blue: DAPI, marking cell nuclei. Scale bar, 25μm. (B) Schematic diagram of the mouse glaucoma model established by silicone oil injection. Top: OCT embedding image of normal mouse eyeball, bottom: OCT embedding image of mouse eyeball after silicone oil injection.

Figure 3 shows the pattern of light therapy and changes in intraocular pressure in glaucoma model mice (A) The mice were placed in a cage equipped with LED (~530nm, ~3200 lux). (B) The light pattern was 12 hours of light and 12 hours of darkness, and the light lasted for 8 weeks. (C) During the light treatment, the changes in intraocular pressure of glaucoma model mice, the numbers of EcGAPR mice from 1 to 8 weeks were 13, 12, 11, 11, 11, 10, 7, 7, 7. The numbers of control mice from 1 to 8 weeks were 13, 10, 10, 10, 10, 9, 6, 4, 4.

Figure 4 shows the changes in visual acuity during light therapy of glaucoma model mice (A) Visual acuity of mice was measured using optokinetic response. Four LCD screens were arranged in a square, with a camera on top to track the movement of mice. A black and white drifting grating with a contrast ratio of 100% was displayed on the monitor at a constant speed (12 degrees/second) clockwise (left eye) or counterclockwise (right eye). When the moving pattern stimulates most of the visual field of the model animal, a sense of self-motion is generated. In order to reduce the movement of the image formed on the retina, the animal's eyes, head and body will naturally move in the direction of eye gaze (that is, the direction of object movement). The movement of the head or body is called compensatory head movement. (B) Changes in visual acuity of glaucoma model mice during light exposure. The numbers of EcGAPR mice from 1 to 8 weeks were 20, 18, 11, 11, 9, 8, 8, and 6, respectively. The numbers of control mice from 1 to 8 weeks were 22, 18, 11, 11, 9, 8, 5, and 5, respectively. T test, p<0.0001.

Figure 5 shows the changes in the number of retinal ganglion cells during light therapy in glaucoma model mice (A) Brn3a labeled retinal ganglion cells, and the number of retinal ganglion cells in control mice decreased significantly after 1 and 2 weeks of light exposure to model glaucoma. (B) The number of retinal ganglion cells in EcGAPR mice after 1 and 2 weeks of light exposure to model glaucoma did not change significantly. (C) Compared with the contralateral eyes (not injected with silicone oil), the percentage of surviving retinal ganglion cells on the silicone oil-injected side. The number of EcGAPR mice at 1-2 weeks was 6 and 4, respectively, and the number of control mice at 1-2 weeks was 4 and 5. t-test, 1 week and 2 weeks p = 0.0073 and 0.0001. (D) There was no significant difference in the number of retinal ganglion cells in the contralateral eyes (not injected with silicone oil) between the two groups.

DETAILED DESCRIPTION

Through extensive and in-depth research, the inventors first discovered that chimeric proteins with a specific structure can effectively prevent and/or treat optic nerve degenerative diseases (such as glaucoma). On this basis, the inventors completed the present invention.

the term

GPR (Green proteorhodopsin) gene and protein

In the present invention, the proton pump rhodopsin GPR can be derived from humans, non-human mammals or bacteria. A representative GPR gene and the amino acid sequence of the GPR protein encoded by it are shown in AF349993_1 (genbank accession number), which is derived from marine proteus and encodes a membrane protein with 249 amino acids, which has light proton pump activity.

APR (alpha proteorhodopsin) gene and protein

In the present invention, the proton pump rhodopsin APR can be derived from humans, non-human mammals or bacteria. A representative APR gene and the amino acid sequence of the APR protein encoded by it are shown in WP_014952819 (genbank accession number), which is derived from marine proteus and encodes a membrane protein with 255 amino acids, which has light proton pump activity.

Protein or polypeptide of the present invention, and polynucleotide encoding the same

In the present invention, "chimeric protein", "EcGAPR", "protein of the present invention", "chimeric protein of the present invention", "polypeptide of the present invention", and "chimeric polypeptide" are used interchangeably, and all refer to GPR protein mutants, wherein transmembrane regions 4-5 in the GPR protein mutant are replaced by transmembrane regions 4-5 of APR protein, or transmembrane regions 4-6 in the GPR protein mutant are replaced by transmembrane regions 4-6 of APR protein; or transmembrane regions 1-2 in the GPR protein mutant are replaced by transmembrane regions 1-2 of APR protein. In a preferred embodiment, the chimeric protein of the present invention is as shown in Formula I or Formula IV. In other preferred embodiments, the chimeric protein of the present invention is a polypeptide having an amino acid sequence shown in SEQ ID NO: 1 or 3 or 5 or a variant polypeptide thereof.

In particular, the chimeric protein of the present invention includes wild-type and mutant chimeric proteins, wherein the mutant chimeric protein refers to a protein produced by a point mutation at a position corresponding to the 196th glycine (G) and/or the 137th glycine (G) of SEQ ID NO.: 1 on the GPR protein mutant. The above two sites are core amino acid sites related to mitochondrial localization and proton pump activity. The "corresponding position" refers to a site corresponding to the 196th glycine (G) and/or the 137th glycine (G) of SEQ ID NO.: 1 in a sequence alignment of homologous proteins. The mutation retains or enhances the mitochondrial localization and proton pump activity of the mutant chimeric protein. Preferably, the mutation is serine (S) or threonine (T). In a preferred embodiment, the mutant chimeric protein is a polypeptide having an amino acid sequence shown in SEQ ID NO: 7 or a variant polypeptide thereof.

As used herein, "isolated" means that a substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist with them in their natural state.

As used herein, "isolated chimeric protein or polypeptide" means that the chimeric protein of the present invention is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated therewith. Those skilled in the art can purify the chimeric protein or polypeptide of the present invention using standard protein purification techniques. Substantially pure protein or polypeptide can produce a single major band on a non-reducing polyacrylamide gel. The purity of the chimeric protein or polypeptide of the present invention can be analyzed by amino acid sequence.

The polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptide of the present invention can be a naturally purified product, or a chemically synthesized product, or produced using recombinant technology from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects and mammalian cells). Depending on the host used in the recombinant production scheme, the polypeptide of the present invention can be glycosylated, or can be non-glycosylated. The polypeptide of the present invention may also include or not include an initial methionine residue.

The present invention also includes fragments, derivatives and analogs of chimeric proteins. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the chimeric protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a signal peptide sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with an antigen IgG fragment). According to the teachings herein, these fragments, derivatives and analogs belong to the well-known scope of those skilled in the art.

The term "chimeric protein" also includes variant forms or variant polypeptides with the same function as the chimeric protein. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and/or substitutions, and the addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of chimeric proteins.

The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with chimeric polypeptide DNA under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against chimeric polypeptides. The present invention also provides other polypeptides, such as fusion proteins formed by chimeric polypeptides or fragments thereof and other polypeptides. In addition to almost full-length polypeptides, the present invention also includes soluble fragments of chimeric polypeptides. Generally, the fragment has at least about 10 consecutive amino acids of the chimeric polypeptide sequence, usually at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

The invention also provides analogs of chimeric proteins or polypeptides. The difference between these analogs and chimeric polypeptides may be differences in amino acid sequence, or differences in modification forms that do not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modifications (usually without changing the primary structure) include: chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.

In the present invention, "conservative variant polypeptides of chimeric proteins" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the chimeric protein. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

Table I

In the present invention, the "coding gene", "coding nucleic acid" and "coding polynucleotide" of the chimeric protein are used interchangeably, and all refer to the polynucleotide sequence that can produce the chimeric protein of the present invention through transcription and/or translation. The coding polynucleotide of the present invention can be in the form of DNA or RNA, can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand. The DNA form can be cDNA, genomic DNA or artificially synthesized DNA.

The polynucleotide encoding the mature polypeptide of the chimeric protein includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

Preparation of the protein of the present invention

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably purified to homogeneity.

The chimeric nucleotide full-length sequence or fragments thereof of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each amplification in the correct order.

Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

The method of amplifying DNA/RNA using PCR technology (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-rapid amplification of cDNA ends) can be preferably used. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized by conventional methods. The amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

The present invention also relates to vectors comprising the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors or chimeric protein coding sequences of the present invention, and methods for producing the polypeptides of the present invention by recombinant technology.

The polynucleotide sequences of the present invention can be used to express or produce recombinant chimeric polypeptides by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally, the following steps are involved:

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a chimeric polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2) Host cells cultured in a suitable culture medium;

(3) Isolate and purify proteins from culture medium or cells.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

Peptide linker

The chimeric protein of the present invention may optionally contain a peptide linker. The size and complexity of the peptide linker may affect the activity of the protein. Generally, the peptide linker should have sufficient length and flexibility to ensure that the two connected proteins have sufficient spatial freedom to perform their functions. At the same time, the formation of α-helix or β-fold in the peptide linker should be avoided to affect the stability of the chimeric protein.

The length of the connecting peptide is generally 0-10 amino acids, preferably 1-5 amino acids.

Pharmaceutical compositions and methods of administration

The present invention also provides a composition comprising an effective amount of the chimeric protein of the present invention and a pharmaceutically acceptable carrier. Optionally, the composition may also contain other drugs for preventing and/or treating optic nerve degeneration diseases. Generally, the chimeric protein of the present invention and other drugs for preventing and/or treating optic nerve degeneration diseases can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably, the pH is about 6-8.

As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce function or activity in humans and/or animals and can be accepted by humans and/or animals, such as 0.001-99wt%; preferably 0.01-95wt%; more preferably, 0.1-90wt%.

As used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical composition of the present invention contains a safe and effective amount of the chimeric protein of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be prepared as a sustained-release preparation.

The effective amount of the chimeric protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the chimeric protein of the present invention and other drugs for preventing and/or treating optic nerve degenerative diseases, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For glaucoma patients, generally, when the chimeric protein of the present invention is administered at a dose of about 0.5 mg-5 mg/kg body weight (preferably 2 mg-4 mg/kg body weight) per day, a satisfactory effect can be obtained. For example, depending on the urgency of the treatment condition, several divided doses may be administered per day, or the dose may be reduced proportionally.

Retinal Degenerative Diseases

Optic neurodegenerative disease refers to visual impairment caused by atrophy and inflammation of the retinal nerve fibers, leading to decreased or loss of function.

In the present invention, retinal degenerative diseases include glaucoma, optic neuritis, optic atrophy, ischemic optic disc disease, and papilledema.

Glaucoma is a common progressive optic nerve degeneration disease that often leads to irreversible blindness. Previous studies have shown that abnormal mitochondrial function is highly correlated with glaucoma. High intraocular pressure is one of the risk factors for glaucoma, so high intraocular pressure is often used to induce glaucoma models.

The main advantages of the present invention include:

(1) The present invention first discovered that a chimeric protein with a specific structure can effectively prevent and/or treat optic nerve degenerative diseases (such as glaucoma).

(2) The present invention first discovered that the chimeric protein of the present invention can also protect retinal ganglion cell degeneration and visual loss caused by drug-induced glaucoma.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

Unless otherwise specified, the reagents and materials in the examples of the present invention are all commercially available products.

Materials and Methods

1. EcGAPR transgenic mice

Screening of mutant chimeric protein EcGAPR and its specific structure as shown in Chinese patent application CN

The amino acid sequence of the mutant chimeric protein EcGAPR obtained by screening is shown in SEQ ID NO.7 (corresponding to SEQ ID NO.: 2 of CN 201810456360.0), and its nucleotide sequence is shown in SEQ ID NO.8 (corresponding to SEQ ID NO.3 of CN 201810456360.0).

The CAG-mt-EcGAPR-2A-EGFP-WPRE-polyA gene was inserted into the Rosa26 site of the genome using CRISPR/Cas9 technology. The gRNA and Cas9 mRNA were obtained by in vitro transcription. The 5' homologous recombination arm, 3' homologous recombination arm and CAG-mt-EcGAPR-2A-EGFP-WPRE-polyA were constructed into the donor plasmid using homologous recombination. The gRNA and Cas9 mRNA and the donor plasmid were co-injected into mouse fertilized eggs to obtain F0 generation mice. The F0 generation mice were hybridized with wild-type mice to obtain F1 generation transgenic mice. In the CAG-mt-EcGAPR-2A-EGFP-WPRE-polyA gene, CAG is the promoter, mt is the mitochondrial inner membrane protein cytochrome c oxidase VIII signal peptide sequence repeated 4 times, 2A is the 2A peptide, and WPRE is a post-transcriptional regulatory element.

2. Glaucoma Modeling

Mice were anesthetized with 1.25% Avertin injected intraperitoneally and placed in the left lateral position 5-10 minutes after injection. A drop of sodium hyaluronate was placed on the right cornea to keep it moist, and a 32G x 4mm needle was used to pierce the cornea to the anterior chamber with minimal damage close to the superior temporal lobe. When the aqueous humor gushed out, silicone oil was injected into the anterior chamber. The injection was stopped when the silicone oil covered most of the iris. Levofloxacin hydrochloride eye ointment (0.015g in 5g) was applied to the injection area to prevent infection.

3. Intraocular pressure measurement

The intraocular pressure of mice was measured weekly using a tonometer. During the measurement, the mice were anesthetized with 1.25% Avertin injected intraperitoneally, and the intraocular pressure was measured 15 minutes later. The average of 3 consecutive values was taken as the measured value each time, and the measurement was performed every 5 minutes.

4. Visual acuity test

The visual acuity of mice was measured by optokinetic response (visual acuity refers to the feeling of self-motion when a moving pattern stimulates most of the visual field of the model animal, that is, in order to reduce the movement of the image formed on the retina, the eyes, head and body of the model animal will naturally move in the direction of eye gaze (that is, the direction of object movement). The movement of the head or body is called compensatory head movement, and the movement of the eyes is called compensatory eye movement response). Four LCD screens were arranged in a square, and a camera was placed on the top to track the movement of the mice. A black and white drifting grating with a contrast of 100% was displayed on the monitor at a constant speed (12 degrees/second) clockwise (left eye) or counterclockwise (right eye). Before the test, the mice were adapted for 1 day. The mice were placed on the central base of the four screens. At the beginning, the screen displayed gray. When the mouse stopped moving, the gray background was replaced by a slow drifting grating. When the tracking behavior was observed, the frequency was increased until the maximum frequency of the response was produced. The visual acuity of the silicone oil injected eye was counted and compared with the contralateral side.

5. Immunostaining

Immunostaining of cells: The cells cultured on the coverslips were washed 3 times with PBS and fixed with 4% FSB (4% PFA, 4% sucrose in 1x PBS). The cells were blocked with 5% blocking solution (0.5% Triton X-100, 5% goat serum in PBS). Then, the cells were incubated with primary antibody for 30 minutes, washed 3 times with PBS, and then incubated with secondary antibody in the dark for 30 minutes. After incubation, the cells were washed 3 times with PBS and photographed under a microscope.

Immunostaining of the complete retina: The mouse was perfused with saline and 4% PFA in sequence, and the eyeballs were removed and fixed in 4% PFA for 30 minutes. The retina was dissected and fixed overnight in 4% PFA. After washing with PBS, it was treated in 0.5% TritonX-100 for 1 hour. After blocking with 10% DST for 2 hours, it was incubated with the primary antibody at 4°C for 30-36 hours. After washing with PBS, it was incubated with the secondary antibody at room temperature in the dark for 2.5 hours. After incubation, the residual secondary antibody was washed with PBS, dried, and sealed with AQUA-MOUNT, and observed under a microscope.

Immunostaining of retinal sections: After the retina was removed, it was dehydrated with 4℃.10%, 20%, and 30% sucrose in turn, embedded with OCT, and frozen at -80℃. Slices were sliced with a slicer, with a thickness of 14μm. The slices were washed with PBS 3 times, 15 minutes each time. 0.05% TritonX-100 was treated for 30 minutes, and 10% DST was blocked at room temperature for 1 hour. Incubated with primary antibody at 4℃ for 20-24 hours, washed with PBS 3 times, 15 minutes each time, and then incubated with secondary antibody at room temperature in the dark for 2.5 hours. Washed with PBS 3 times, 15 minutes each time, then the slices were dried, sealed with AQUA-MOUNT, and observed under a microscope.

In the present invention, the molecular screening of proton pump rhodopsin and the construction and specific structure of the chimeric protein and mutant chimeric protein (ie, EcGAPR (enhanced chimeric GPR and APR)) of the present invention are as described in Chinese patent application CN 201810456360.0.

A representative amino acid sequence of a chimeric protein in which transmembrane regions 4-5 of GPR are replaced by the corresponding transmembrane regions of APR is shown in SEQ ID NO.: 1 (corresponding to SEQ ID NO.: 1 of CN 201810456360.0), and the nucleotide sequence is shown in SEQ ID NO.: 2 (corresponding to SEQ ID NO.: 4 of CN 201810456360.0), and a representative amino acid sequence of a chimeric protein in which transmembrane regions 4-6 of GPR are replaced by the corresponding transmembrane regions of APR is shown in SEQ ID NO.: 3 (corresponding to SEQ ID NO.: 5 of CN 201810456360.0), and the nucleotide sequence is shown in SEQ ID NO.: 4 (corresponding to SEQ ID NO.:

201810456360.0: SEQ ID NO.: 7), a representative amino acid sequence of a chimeric protein in which transmembrane regions 1-2 of GPR are replaced by the corresponding transmembrane regions of APR is shown in SEQ ID NO.: 5 (corresponding to SEQ ID NO.: 6 of CN201810456360.0), and the nucleotide sequence is shown in SEQ ID NO.: 6 (corresponding to SEQ ID NO.: 8 of CN201810456360.0), and a representative amino acid sequence of a mutant chimeric protein is shown in SEQ ID NO. 7 (i.e., EcGAPR, corresponding to SEQ ID NO.: 2 of CN 201810456360.0), and its nucleotide sequence is shown in SEQ ID NO. 8 (corresponding to SEQ ID NO. 3 of CN 201810456360.0).

Experimental Results

EcGAPR is a rhodopsin that can efficiently locate to mitochondria and maintain proton pump function, which is screened from existing proton pump rhodopsin in nature and modified by genetic engineering. The structure is described in Chinese patent application CN201810456360.0. Crisper/cas9 technology was used to construct transgenic mice (Figure 1A-B) that stably and widely express mitochondrial-localized EcGAPR (mt-EcGAPR) throughout the body. The transgenic mice have normal birth and mortality rates, and there are no significant differences in phenotype, suggesting that overexpression of mt-EcGAPR does not cause cytotoxicity. The results of primary cell culture (Figure 1B) show that in mt-EcGAPR transgenic mice, mt-EcGAPR can still maintain mitochondrial localization. Figure 2A shows that mt-EcGAPR is expressed normally in the retinal tissue of transgenic mice and has no effect on the structure of the mouse retina.

Glaucoma is a common progressive retinal degenerative disease that often leads to irreversible blindness. Previous studies have shown that abnormal mitochondrial function is highly correlated with glaucoma. High intraocular pressure is one of the risk factors for glaucoma, so high intraocular pressure is often used to induce glaucoma models. One method is to use silicone oil injection to cause intraocular hypertension and degeneration of retinal ganglion cells to induce a glaucoma model. A glaucoma model was constructed in mice overexpressing mt-EcGAPR (Figure 2B). Light therapy was given to glaucoma model mice, and changes in intraocular pressure were detected. The results showed that in the sixth week of injection, there was a significant difference in intraocular pressure between mt-EcGAPR and wild-type control groups, and the intraocular pressure of mice overexpressing mt-EcGAPR was lower, suggesting that mt-EcGAPR can reduce intraocular pressure and relieve glaucoma symptoms (Figure 3A-C). Light was given to glaucoma model mice, and the visual acuity of the mice was checked weekly (Figure 4A-B). The visual acuity of the mt-EcGAPR mice exposed to light was more preserved than that of wild-type mice. In the first and second weeks, the visual acuity of the mt-EcGAPR mice exposed to light decreased to 79% and 71%, respectively, while that of the wild-type mice decreased to 47% and 38%, respectively (Figure 4B). The changes in the number of retinal ganglion cells during light treatment in glaucoma model mice were examined (Figure 5A-D). Immunohistochemistry showed that 50% and 45% of peripheral retinal ganglion cells survived in the wild-type mice in the first and second weeks, respectively, while 83% and 82% of peripheral retinal ganglion cells survived in the mt-EcGAPR mice exposed to light, respectively (Figure 5C-D). There was no significant difference in the number of retinal ganglion cells on the contralateral side that was not injected. Therefore, optogenetic activation of mt-EcGAPR-targeted mitochondria can significantly alleviate glaucomatous retinal ganglion cell degeneration and visual loss. Combined with the results of Figure 3 above (in the sixth week after injection, there was a significant difference in intraocular pressure between the mt-EcGAPR and wild-type control groups), it suggests that mt-EcGAPR can prevent the immune response caused by massive cell death.

In addition, the inventors used other chimeric proteins mentioned in CN 201810456360.0, such as cGAPR, cGAPR46 and cGAPR12, and cGAPR46 and cGAPR12 (recorded in CN 201810456360.0 as follows: Among them, chimera 1457 (transmembrane regions 4-5 of GPR are replaced by the corresponding transmembrane regions of APR) has the highest weighted index and is named cGAPR (chimeric GPR and APR), the amino acid sequence is shown in SEQ ID NO.: 1, and the nucleotide sequence is shown in SEQ ID NO.: 4, and chimera 1467 (transmembrane regions 4-6 of GPR are replaced by the corresponding transmembrane regions of APR) is named cGAPR46, the amino acid sequence is shown in SEQ ID NO.: 5, and the nucleotide sequence is shown in SEQ ID NO.:7, a double mutant of the chimera 0127 (transmembrane regions 1-2 of GPR were replaced by the corresponding transmembrane region of APR, named cGAPR12, with its amino acid sequence as shown in SEQ ID NO.:6, and its nucleotide sequence as shown in SEQ ID NO.:8) was experimented with and achieved effects similar to those of mt-EcGAPR, both of which could significantly reduce glaucomatous retinal ganglion cell degeneration and visual loss.

Discussion and Outlook

Mitochondria are very delicate organelles with limited capacity, especially the protein density on the inner membrane is extremely high. Overexpression of proteins in mitochondria is likely to cause changes in mitochondrial morphology and function. Therefore, in order for mitochondrial light-activated rhodopsin to function, rhodopsin must meet the following criteria: (1) mitochondria maintain normal morphology and membrane voltage; (2) rhodopsin has proton pump activity in mammalian cells; (3) rhodopsin should maintain mitochondrial localization in different systems and not leak expression on the cell membrane; (4) rhodopsin has a suitable reversal voltage to prevent the mitochondrial membrane from hyperpolarizing and producing excessive reactive oxygen species that damage cells. EcGAPR screened by genetic engineering methods meets all of the above criteria.

Studies have shown that the occurrence of glaucoma is related to mitochondrial dysfunction. Increased intraocular pressure leads to insufficient blood supply to the retina, resulting in impaired mitochondrial function. The incidence and progression of glaucoma increase with age, which is related to the structural and functional degeneration of mitochondria with age. For example, the ability of oxidative phosphorylation is weakened, the production of reactive oxygen species increases, and the mutation of mitochondrial DNA increases. Because the optic nerve is rich in mitochondria, it may be particularly sensitive to these changes, and damage to the optic nerve will affect visual function.

EcGAPR transgenic mice show a certain protective effect against glaucoma induced by high intraocular pressure under light. EcGAPR can provide proton motive force to mitochondria in the presence of light when mitochondrial respiratory chain proteins are damaged to improve the energy deficiency of cells. In the state of poor nutrition in the environment, light is used to provide cells with the energy needed for growth. The pathogenesis of diabetic eye disease and glaucoma is similar. Both are caused by microvascular circulation disorders that cause insufficient mitochondrial metabolic function, resulting in retinal damage. Glaucoma is an acute disease, while diabetic eye disease is a chronic disease. Therefore, in theory, EcGAPR's intervention and compensation function for mitochondria should also be applicable to diabetic eye disease and some other retinal/optic nerve degenerative diseases (such as ischemic optic disc disease, papillitis, papilledema, retinal choroiditis, retinitis pigmentosa, central retinal artery occlusion, etc.).

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (10)

1. Use of a chimeric protein, or a polynucleotide encoding the same, or an enhancer thereof, for the preparation of a composition or formulation for the prevention and/or treatment of an optic neurodegenerative disease, said chimeric protein being a GPR protein mutant that replaces one or more transmembrane regions of a wild-type GPR protein corresponding to accession No. AF349993_1 selected from the group consisting of:

A transmembrane region No. 4-5; and/or

A transmembrane region No. 4-6; and/or

Transmembrane region No. 1-2.

2. The use according to claim 1, wherein the chimeric protein comprises the structure of formula I from N-terminus to C-terminus:

Z1-Z2-Z3(I)

Wherein Z1 is the first transmembrane region of GPR protein;

Z2 is the transmembrane region of the APR protein;

Z3 is the second transmembrane region of GPR protein;

wherein each "-" is independently a connecting peptide or peptide bond.

3. The use according to claim 2, wherein the Z1 element corresponds to positions 1-114 of the protein sequence with accession number AF349993 _1.

4. The use according to claim 2, wherein the Z2 element corresponds to position 120-170 or 120-201 of the protein sequence under accession number wp_ 014952819.

5. The use according to claim 2, wherein the Z3 element corresponds to positions 166-249 or 196-249 of the protein sequence under accession No. AF349993 _1.

6. The use according to claim 1, wherein the chimeric protein is selected from the group consisting of:

(A) A polypeptide having the amino acid sequence shown in SEQ ID NO.1 or 3 or 5;

(B) A polypeptide having 80% or more homology (preferably 90% or more homology; preferably 95% or more homology; most preferably 97% or more homology, such as 98% or more, 99% or more) to the amino acid sequence shown in SEQ ID NO 1 or 3 or 5, and having mitochondrial localization and proton pump activity;

(C) And (3) the derivative polypeptide which is formed by substituting, deleting or adding 1-15 amino acid residues into the amino acid sequence shown in any one of SEQ ID NO 1, 3 or 5 and has mitochondrial localization and proton pump activity.

7. The use according to claim 1, wherein the neurodegenerative disorder comprises primary optic neurodegeneration, secondary optic neurodegeneration.

8. A pharmaceutical composition, in the form of a capsule, characterized by comprising the following steps:

(a1) A first active ingredient for use in the prevention and/or treatment of an optic neurodegenerative disease, the first active ingredient comprising: a chimeric protein, or a polynucleotide encoding the same, or a promoter thereof, which is a GPR protein mutant that replaces one or more transmembrane regions of a wild-type GPR protein corresponding to accession No. AF349993_1 selected from the group consisting of:

A transmembrane region No. 4-5; and/or

A transmembrane region No. 4-6; and/or

A transmembrane region 1-2;

(a2) Optionally, a second active ingredient for preventing and/or treating an optic neurodegenerative disease, the second active ingredient comprising: other drugs for preventing and/or treating optic neurodegenerative diseases; and

(B) A pharmaceutically acceptable carrier.

9. A medicine box, which comprises a medicine box body, characterized by comprising the following steps:

(i) A first container, and a chimeric protein of active ingredient (a 1), or a polynucleotide encoding the same, or an enhancer thereof, or a medicament containing active ingredient (a 1) located in the first container, wherein the chimeric protein is a GPR protein mutant that replaces one or more transmembrane regions of a wild-type GPR protein corresponding to accession AF349993_1 selected from the group consisting of:

A transmembrane region No. 4-5; and/or

A transmembrane region No. 4-6; and/or

A transmembrane region 1-2;

(ii) An optional second container, and an active ingredient (a 2) in the second container, other medicament for preventing and/or treating an optic neurodegenerative disease, or medicament containing the active ingredient (a 2).

10. Use of a pharmaceutical composition according to claim 8 or a kit according to claim 9 for the preparation of a medicament for the prevention and/or treatment of an optic neurodegenerative disease.