CN111705069A

CN111705069A - Multi-neurotrophic factor combined expression vector and application thereof

Info

Publication number: CN111705069A
Application number: CN202010563817.5A
Authority: CN
Inventors: 李斌; 刘婷
Original assignee: Wuhan Niufusi Biological Technology Co ltd
Current assignee: Wuhan Niufusi Biological Technology Co ltd; Wuhan Neurophth Biotechnology Ltd Co
Priority date: 2020-06-19
Filing date: 2020-06-19
Publication date: 2020-09-25

Abstract

The present application relates to a viral vector, characterized in that it comprises a first nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein; a second nucleotide sequence encoding an insulin-like growth factor IGF-1 protein; and a third nucleotide sequence encoding osteopontin OPN.

Description

Multi-neurotrophic factor combined expression vector and application thereof

Technical Field

The application relates to the field of biomedicine, in particular to a multi-neurotrophic factor combined expression vector and application thereof.

Background

Glaucoma (glaucoma) is a group of eye diseases with characteristic optic nerve damage and visual function damage, and is a pathological change caused by increased intraocular pressure mainly due to the obstruction of the aqueous humor circulation pathway, but there are some patients presenting normal tension glaucoma. Currently, glaucoma is the second eye disease leading to vision loss worldwide.

The primary treatment for glaucoma at this stage is to lower intraocular pressure in an attempt to slow the progression of optic nerve damage. However, even if the intraocular pressure of some patients is reduced to a normal level or lower, some glaucoma patients are blinded by one eye, and 9% of the patients are even blinded by both eyes. Therefore, the treatment of acute or chronic optic nerve injury related diseases such as glaucoma remains a far from unmet medical need, and there is an urgent need for drugs that can help patients maintain, even improve, visual function.

Disclosure of Invention

The present application provides a recombinant nucleic acid comprising: a first nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein; a second nucleotide sequence encoding an insulin-like growth factor IGF-1 protein; and a third nucleotide sequence encoding osteopontin OPN.

In certain embodiments, the sequence of the ciliary neurotrophic factor CNTF protein comprises a protein sequence selected from the group consisting of:

a) the protein sequence is similar to SEQ ID NO:7 are the same;

b) the protein sequence is similar to SEQ ID NO:7 has more than or equal to 98 percent of sameness;

c) the protein sequence is similar to SEQ ID NO:7 has the same quality of more than or equal to 95 percent;

d) the protein sequence is similar to SEQ ID NO:7 has more than or equal to 90 percent of sameness; and

e) the protein sequence is similar to SEQ ID NO:7 has more than or equal to 80 percent of sameness.

In certain embodiments, the first nucleotide sequence comprises a nucleotide sequence selected from the group consisting of seq id no:

a) the first nucleotide sequence is identical to SEQ ID NO:1 are the same;

b) the first nucleotide sequence is identical to SEQ ID NO:1 has more than or equal to 98 percent of sameness;

c) the first nucleotide sequence is identical to SEQ ID NO:1 has the same quality of more than or equal to 95 percent;

d) the first nucleotide sequence is identical to SEQ ID NO:1 has more than or equal to 90 percent of sameness; and

e) the first nucleotide sequence is identical to SEQ ID NO:1 has more than or equal to 80 percent of sameness.

In certain embodiments, the sequence of the insulin-like growth factor IGF-1 protein comprises a protein sequence selected from the group consisting of:

a) the protein sequence is similar to SEQ ID NO:8 are the same;

b) the protein sequence is similar to SEQ ID NO:8 has more than or equal to 98 percent of sameness;

c) the protein sequence is similar to SEQ ID NO:8 has the same quality of more than or equal to 95 percent;

d) the protein sequence is similar to SEQ ID NO:8 has more than or equal to 90 percent of sameness; and

e) the protein sequence is similar to SEQ ID NO:8 has more than or equal to 80 percent of sameness.

In some embodiments, the second nucleotide sequence comprises a nucleotide sequence selected from the group consisting of seq id no:

a) and the second nucleotide sequence is identical to SEQ ID NO:2 are the same;

b) and the second nucleotide sequence is identical to SEQ ID NO:2 has more than or equal to 98 percent of sameness;

c) and the second nucleotide sequence is identical to SEQ ID NO:2 has the same quality of more than or equal to 95 percent;

d) and the second nucleotide sequence is identical to SEQ ID NO:2 has more than or equal to 90 percent of sameness; and

e) and the second nucleotide sequence is identical to SEQ ID NO:2 has more than or equal to 80 percent of sameness.

In certain embodiments, the sequence of osteopontin OPN comprises a protein sequence selected from the group consisting of:

a) the protein sequence is similar to SEQ ID NO:9 are the same;

b) the protein sequence is similar to SEQ ID NO:9 has more than or equal to 98 percent of sameness;

c) the protein sequence is similar to SEQ ID NO:9 has the same quality of more than or equal to 95 percent;

d) the protein sequence is similar to SEQ ID NO:9 has more than or equal to 90 percent of sameness; and

e) the protein sequence is similar to SEQ ID NO:9 has more than or equal to 80 percent of sameness.

In certain embodiments, the third nucleotide sequence comprises a nucleotide sequence selected from the group consisting of seq id no:

a) and the third nucleotide sequence is similar to SEQ ID NO:3 are the same;

b) and the third nucleotide sequence is similar to SEQ ID NO:3 has more than or equal to 98 percent of sameness;

c) and the third nucleotide sequence is similar to SEQ ID NO:3 has the same quality of more than or equal to 95 percent;

d) and the third nucleotide sequence is similar to SEQ ID NO:3 has more than or equal to 90 percent of sameness; and

e) and the third nucleotide sequence is similar to SEQ ID NO:3 has more than or equal to 80 percent of sameness.

In certain embodiments, a self-cleaving polypeptide-encoding nucleotide sequence of different origin is included between two adjacent such nucleotide sequences to reduce the probability of homologous recombination.

In certain embodiments, the self-cleaving polypeptide is a 2A peptide, preferably, the self-cleaving polypeptide is a P2A peptide (SEQ ID NO:10) and a T2A peptide (SEQ ID NO:11), respectively.

In certain embodiments, the nucleotide sequence encoding the self-cleaving polypeptide and the nucleotide sequence are directly linked without an intervening additional nucleotide sequence.

In certain embodiments, the three nucleotide sequences are in the order of the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence, in tandem, in the 5 'to 3' direction.

In certain embodiments, the recombinant nucleic acid is selected from the group consisting of nucleotide sequences of seq id no:

a) the nucleotide sequence is similar to SEQ ID NO:6 are the same;

b) the nucleotide sequence is similar to SEQ ID NO:6 has more than or equal to 98 percent of sameness;

c) the nucleotide sequence is similar to SEQ ID NO:6 has more than or equal to 95 percent of sameness;

d) the nucleotide sequence is similar to SEQ ID NO:6 has more than or equal to 90 percent of sameness; and

e) the nucleotide sequence is similar to SEQ ID NO:6 has more than or equal to 80 percent of sameness.

In another aspect, the present application provides a vector comprising a recombinant nucleic acid as described herein.

In certain embodiments, the AAV inverted terminal repeat ITRs are included in the recombinant nucleic acid.

In certain embodiments, the vector is selected from adeno-associated viral vectors.

In certain embodiments, the adeno-associated virus is selected from AAV2, AAV5, AAV7, or AAV8, or a combination thereof.

In certain embodiments, the nucleotide sequence further comprises a promoter or enhancer, preferably the promoter is selected from CMV, CAG or SYN.

In certain embodiments, the vectors encode the ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1, and osteopontin OPN that are capable of independent presence as non-fusion proteins upon expression.

In another aspect, the present application provides a vector as described herein for use in treating a disease associated with optic nerve injury.

In certain embodiments, the optic nerve injury-related disorder is acute or chronic optic nerve injury.

In certain embodiments, the optic nerve injury-related disease is autosomal Dominant Optic Atrophy (DOA), ischemic optic neuropathy, Leber's Hereditary Optic Neuropathy (LHON), or glaucoma.

In certain embodiments, the disease associated with optic nerve damage is glaucoma.

In another aspect, the present application provides the use of a vector as described herein in the manufacture of a medicament for the treatment of a disease associated with optic nerve injury.

In another aspect, the present application provides a pharmaceutical formulation comprising a carrier as described herein, and a pharmaceutically acceptable excipient.

In certain embodiments, the pharmaceutical formulation is a liquid formulation.

In another aspect, the present application provides a pharmaceutical formulation as described herein for use in the treatment of a disease associated with optic nerve damage.

In certain embodiments, the pharmaceutical formulation causes long-term high expression of the ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1, and osteopontin OPN in retinal cells.

In certain embodiments, the pharmaceutical formulation is capable of long-acting treatment of a disease associated with optic nerve injury.

In certain embodiments, the pharmaceutical preparation increases the survival rate of a ganglion cell and/or its axon.

In certain embodiments, the pharmaceutical formulation prevents or delays the apoptosis of ganglion cells.

In certain embodiments, the optic nerve injury-related disorder is acute or chronic optic nerve injury resulting in damage and/or death of the optic nerve and its ganglion cells.

In certain embodiments, the pharmaceutical formulation is injected intraocularly.

In certain embodiments, the intraocular injection is a vitreous cavity injection.

In certain embodiments, intraocular injection of the pharmaceutical formulation does not cause a significant inflammatory response or other complications.

In another aspect, the present application provides the use of a pharmaceutical formulation as described herein in the manufacture of a medicament for the treatment of a disease associated with optic nerve damage.

In another aspect, the present application provides a pharmaceutical formulation as described herein for use in treating a disease associated with optic nerve injury.

In another aspect, the present application provides a method of treating an ocular disease comprising administering to a patient two or more carriers or pharmaceutical formulations thereof, said two or more carriers comprising:

a) a first vector, said first vector comprising a first recombinant nucleic acid, said first recombinant nucleic acid comprising a nucleotide sequence encoding osteopontin OPN; and

b) a second vector, said second vector comprising a second recombinant nucleic acid, said second recombinant nucleic acid comprising a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein, or a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein.

In certain embodiments, the first vector or the second vector further comprises a third recombinant nucleic acid comprising a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein, or a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein.

In certain embodiments, the two or more vectors comprise a third vector comprising a third recombinant nucleic acid.

In certain embodiments, the second recombinant nucleic acid comprises a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein and the third recombinant nucleic acid comprises a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein.

In certain embodiments, the second recombinant nucleic acid comprises a nucleotide sequence encoding insulin-like growth factor IGF-1 protein and the third recombinant nucleic acid comprises a nucleotide sequence encoding ciliary neurotrophic factor CNTF protein.

In another aspect, the present application provides a method of treating an ocular disease comprising administering to a patient a vector described herein or a pharmaceutical formulation described herein.

In certain embodiments, the ocular disease is a disease associated with optic nerve injury.

In certain embodiments, the optic nerve injury-related disease is autosomal Dominant Optic Atrophy (DOA), Leber's Hereditary Optic Neuropathy (LHON), ischemic optic neuropathy, or glaucoma; preferably, the disease associated with optic nerve damage is glaucoma.

In certain embodiments, the carrier or pharmaceutical formulation is injected intraocularly.

In certain embodiments, the carrier or pharmaceutical formulation is capable of causing long-term high expression of the ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1 and osteopontin OPN in retinal cells.

In certain embodiments, the carrier or pharmaceutical formulation is capable of long-acting treatment of a disease associated with optic nerve injury.

In certain embodiments, the carrier or pharmaceutical agent increases the survival rate of the ganglion cells and/or their axons.

In certain embodiments, the carrier or pharmaceutical formulation prevents or delays ganglion cell apoptosis.

In certain embodiments, the carrier or pharmaceutical formulation prevents or delays a decrease in the thickness of the retinal ganglion cell layer.

In certain embodiments, the carrier or pharmaceutical formulation prevents or delays a decrease in the thickness of the retinal nerve fiber layer.

In certain embodiments, the carrier or pharmaceutical formulation prevents or delays the progression of abnormal visual field conditions.

In certain embodiments, the method does not cause a significant inflammatory response or other complications in the eye.

The carrier described herein has at least one benefit selected from the group consisting of: 1) high levels of expression of the ciliary neurotrophic factor CNTF protein, the insulin-like growth factor IGF-1 and osteopontin OPN in retinal ganglion cells and axons; 2) protecting optic nerve survival and repair, and promoting optic nerve regeneration; 3) remarkably improving the abnormal visual field, or preventing or delaying the deterioration of the abnormal visual field; 4) increasing or maintaining the thickness of the retinal ganglion cell layer; preventing or delaying the reduction in retinal ganglion cell layer thickness; 5) increasing or maintaining the thickness of the retinal nerve fiber layer; preventing or retarding a decrease in the thickness of the retinal nerve fiber layer; and 6) treating optic nerve damage-related diseases (e.g., glaucoma, DOA, LHON, and ischemic optic neuropathy), improving the patient's visual function.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIG. 1 shows a vector construction scheme of the present application.

FIG. 2 shows mRNA expression levels of genes encoding ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1 and osteopontin OPN according to the present application.

FIG. 3 shows the increase in intraocular pressure in the DBA/2J mouse model of chronic ocular hypertension compared to C57/BL6 mice.

FIG. 4a shows an immunoblot of the expression of the ciliary neurotrophic factor CNTF protein;

FIG. 4b shows an immunoblot profile of the expression of insulin-like growth factor IGF-1;

FIG. 4c shows an immunoblot of osteopontin OPN expression.

FIG. 5a shows the result of TUJ1 staining of the retinas of normal group (month 6 group of DBA/2J mice);

FIG. 5b shows TUJ1 staining of mouse retinas from the blank group (PBS group);

FIG. 5c shows TUJ1 staining of experimental group A (rAAV-CNTF group) mouse retina;

FIG. 5d shows TUJ1 staining of mouse retinas from Experimental group B (rAAV-CNTF/IGF 1/OPN group described herein);

FIG. 5e shows statistics of nodal cell (RGC) cell density per retinal area in normal group (DBA/2J mice month 6 group), blank group (PBS group), experimental group A (control rAAV-CNTF group) and experimental group B (rAAV-CNTF/IGF 1/OPN group described herein);

FIG. 5f shows a statistical plot of the cell density of the ganglion cells (RGCs) per retinal area in the normal group (month 6 group of DBA/2J mice), blank group (PBS group), experimental group A (control rAAV-CNTF group), and experimental group B (rAAV-CNTF/IGF 1/OPN group described herein), with statistical significance indicated by asterisks: denotes P <0.01, denotes P < 0.05.

FIG. 6 shows the results of optic nerve counts of normal group (month 6 group of DBA/2J mice), blank group (PBS group), experimental group A (control rAAV-CNTF group) and experimental group B (recombinant rAAV-CNTF/IGF1/OPN group described in the present application), with statistical significance indicated by asterisks: denotes P < 0.05.

FIG. 7 shows fundus photographs taken one month after intravitreal injection of rabbits in the blank (PBS) and infected (rAAV-CNTF/IGF 1/OPN groups described herein).

FIG. 8 shows H & E staining of retinal tissue sections one month after intravitreal injection of rabbits in blank (PBS) and infected (rAAV-CNTF/IGF 1/OPN as described herein).

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "ciliary neurotrophic factor CNTF" refers generally to ciliary neurophysifactor, a multifunctional cytokine. The natural ciliary neurotrophic factor CNTF contains 200 amino acids, the molecular weight is 23KDa, and the isoelectric point is about 5.8. The ciliary neurotrophic factor CNTF can promote the survival of in vivo neurons, prevent nerve injury and promote nerve fiber regeneration and cell differentiation. The ciliary neurotrophic factor CNTF can play a role as an endogenous neurotrophic factor activated after neuron damage, and plays a role in neuroprotection in the fields of retinal pigment degeneration (RP) and the like.

In the present application, the term "insulin-like growth factor IGF-1 protein" refers generally to insulin like growth factor, a hormone. The insulin-like growth factor IGF-1 protein can be encoded by the human IGF1 gene. Insulin-like growth factor IGF-1 protein primarily mediates Growth Hormone (GH) -stimulated somatic growth and may also mediate growth hormone-independent anabolic responses in cells and tissues. The molecular weight of the insulin-like growth factor IGF-1 protein is 7649, which can be synthesized by a variety of mesenchymal cells. Insulin-like growth factor IGF-1 protein may function by activating the insulin-like growth factor IGF-1 protein receptor.

In the present application, the term "osteopontin OPN" generally refers to osteopontin, or secreted phosphoprotein (SPP 1), which is a secreted glycoprotein. Osteopontin OPN can be widely involved in a variety of physiological and pathological processes in the body, such as: protecting cells and inhibiting apoptosis; regulating endothelial cell migration and chemotaxis of inflammatory cells, such as macrophage recruitment; improving the reconstruction and repair of damaged tissues; high levels of osteopontin OPN expression promote metastasis of cancer cells, such as breast and liver cancer. The human OPN gene expressed protein has 3 isomers of OPNa, OPNb and OPNc, and osteopontin OPN is cut by thrombin in vivo and divided into two sections, wherein one section at the C end can be directly combined with CD44v6 receptor, and the other section at the N end can be combined with integrin receptor.

In the present application, the term "recombinant nucleic acid" generally refers to an artificially synthesized nucleotide, deoxyribonucleotide or ribonucleotide, or an analog thereof.

In the present application, the term "AAV" generally refers to adenovirus itself or derivatives thereof. Adeno-associated virus (AAV) generally refers to a class of single-stranded DNA viruses belonging to the genus dependovirus, the family parvoviridae. The AAV genome may comprise Inverted Terminal Repeats (ITRs) and two Open Reading Frames (ORFs) at both ends of a DNA strand. The open reading frame may include rep and cap. Rep consists of multiple overlapping genes encoding Rep proteins required for the AAV life cycle, cap contains overlapping nucleotide sequences encoding capsid proteins, which may include VP1, VP2, and VP 3. The capsid proteins interact to form the capsid. AAV has many common serotypes, 100 virus variants. In the present application, the AAV capsid, ITRs and other selected AAV components are selected from any AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVAnc80, variants of any known or mentioned AAV or AAV yet to be discovered or variants or mixtures thereof.

In the present application, the term "optic nerve injury-related disease" generally refers to an ocular condition caused by optic nerve injury. The optic nerve damage often leads to optic nerve atrophy, eventually causing severe vision loss and even blindness. The optic nerve injury related disease may include optic neuritis, ischemic optic neuropathy, glaucoma, retinal vascular occlusion, or optic path occupying lesion.

In the present application, the term "DOA" generally refers to Autosomal Dominant Optic Atrophy (ADOA). The DOA is a chronically progressive optic nerve disease, considered to be the most common autosomal inherited optic neuropathy. The main clinical manifestations of DOA are visual deterioration, dyschromatopsia, visual field defects, etc., and the fundus changes to temporal disc pallor.

In the present application, the term "glaucoma" generally refers to a disease characterized by common atrophy and depression of the optic papilla, loss of visual field, and decreased vision. The main causes of impaired visual function in glaucoma are the progressive death of RGCs and optic nerve fiber loss. The primary treatment for glaucoma at this stage is to lower intraocular pressure in an attempt to slow the progression of optic nerve damage. Intraocular pressure lowering therapy is divided into two major categories, drug therapy and surgical therapy (including laser). The principle is to establish new dynamic balance of aqueous humor and to reduce intraocular pressure to a safe range by reducing the amount of aqueous humor produced or increasing the amount of aqueous humor outflow.

In the present application, the term "ischemic optic neuropathy" generally refers to a common acute optic neuropathy that can be classified as anterior (affecting the optic disc) and posterior (retrobulbar) lesions, as well as inflammatory and non-arterial lesions. The ischemic optic neuropathy can cause optic nerve ischemia and hypoxia, and damage to optic nerve is caused. The ischemic optic neuropathy can be caused by diseases such as hypertension, arteriosclerosis, temporal arteritis, carotid artery obstruction, diabetes or leukemia and polycythemia.

In the present application, the term "LHON" generally refers to Leber's hereditary optic neuropathy (Leber's clinical optic neuropathy), a maternally inherited disorder of optic neurodegeneration. LHON is clinically manifested as acute or subacute indolent visual deterioration of eyes simultaneously or successively, and may be accompanied by central visual field loss and dyschromatopsia. LHON is a rare disease, and is associated with mitochondria, and the G11778A mutation may be associated with the pathogenesis of LHON.

In the present application, the term "ganglion cells" refers generally to ganglion cells, which may be retinal ganglion cells. The ganglion cells receive electrical signals from the bipolar cells and transmit them to the optic nerve, which generates action potentials. The axon of the ganglion cells is optic nerve fiber, which is distributed on the surface of the retina in the eyeball and collected on the optic tract (optic nerve) papilla, and after emerging from the eyeball, the axon crosses the optic tract and ends at the lateral geniculate part.

In this application, the term "axon" generally refers to the axon of retinal ganglion cells. The cell bodies of the axons and retinal ganglion cells may constitute the optic nerve, which belongs to the central nervous system.

In the present application, the term "long-lasting" generally refers to a relatively long period of expression in retinal cells. For example, the expression level of the ciliary neurotrophic factor CNTF protein, the insulin-like growth factor IGF-1 protein, and/or the osteopontin OPN in the cells of subjects in and/or ex vivo or in vitro of treatment groups administered with the vectors and/or pharmaceutical formulations described herein can be maintained at significantly higher levels for a period of at least one week, at least one month, at least three months, or at least six months compared to a blank control group not administered with the vectors and/or pharmaceutical formulations described herein. For another example, the expression level of the ciliary neurotrophic factor CNTF protein, the insulin-like growth factor IGF-1 protein, and/or the osteopontin OPN in the subject of the treatment group administered the vector and/or pharmaceutical formulation described herein can be maintained at significantly higher levels for a period of at least one week, at least one month, at least three months, or at least six months in vivo and/or in vitro in cells thereof compared to a control group administered the vector and/or pharmaceutical formulation alone.

In the present application, the term "high expression" generally means that the expression amount in retinal cells is relatively high. For example, the expression level of the ciliary neurotrophic factor CNTF protein, the insulin-like growth factor IGF-1 protein, and/or the osteopontin OPN can be significantly increased in vivo and/or in vitro cells of subjects of the treatment group administered with the vector and/or pharmaceutical formulation described herein, compared to a blank control group not administered with the vector and/or pharmaceutical formulation described herein. For another example, the expression level of the ciliary neurotrophic factor CNTF protein, the insulin-like growth factor IGF-1 protein and/or the osteopontin OPN can be significantly increased in vivo and/or in vitro cells of subjects administered with the treatment group of the vectors and/or pharmaceutical formulations described herein compared to a control group administered with the ciliary neurotrophic factor CNTF protein, the insulin-like growth factor IGF-1 or the osteopontin OPN alone.

In the present application, the term "inflammatory response" generally refers to an inflammatory response occurring in the eye. The inflammatory response may include endophthalmitis (e.g., suppurative endophthalmitis), keratitis, vitreoretinitis, or anterior segment toxicity syndrome. The inflammatory response can lead to symptoms such as hypopyon, ocular pain, vision loss, photophobia, and the like.

In the present application, the term "complications" generally refers to complications arising from the inflammatory response described herein. For example, the complications may include cataracts; chronic cystoid macular edema; pupil deformation and posterior adhesion; secondary glaucoma; concurrent cataracts and/or eyeball atrophy.

In the present application, the term "self-cleaving peptides", also known as 2A peptides (2A self-cleaving peptides), generally refers to a class of peptide fragments 18-22 amino acid residues in length that induce self-cleavage of recombinant proteins containing the 2A peptide within cells. The 2A peptide is typically derived from the 2A region of the viral genome. In genetic engineering, the 2A peptide can divide a peptide chain translated from one Open Reading Frame (ORF) into several independent peptide chains. 2A peptides may include P2A, E2A, F2A, T2A, all of which are named for the virus of origin.

In this application, the term "alleviating" refers to reducing, diminishing or delaying a condition, disease, disorder or phenotype. The condition, disease, disorder or phenotype may include subjective perception by the subject, such as pain, dizziness or other physiological disorder, or medically detectable indication, such as a disease condition detected by medical testing means.

In the present application, the term "treatment" generally refers to clinical intervention to alter the natural course of the treated individual or cell in the course of clinical pathology. May include improving the disease state, eliminating the lesion, or improving prognosis.

In the present application, the term "comprising" or "comprises" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the stated value.

Detailed Description

Recombinant nucleic acid molecules

In one aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule may comprise a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein. The ciliary neurotrophic factor CNTF protein can comprise an amino acid sequence shown in SEQ ID NO. 7.

For example, the ciliary neurotrophic factor CNTF protein may comprise an amino acid sequence which is at least 80% homologous to the amino acid sequence shown in SEQ ID No. 7, e.g. at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to any of the amino acid sequences described herein, and the administration of said amino acid sequence in combination with insulin-like growth factor IGF-1 and osteopontin OPN as described herein may be capable of treating a disease associated with optic nerve damage.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein may comprise the nucleotide sequence shown in SEQ ID NO. 1.

For example, the nucleotide sequence encoding the CNTF protein may comprise a nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID No. 1, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with any one of the nucleotide sequences described herein, and the administration of the nucleotide sequence in combination with insulin-like growth factor IGF-1 and osteopontin OPN can treat optic nerve injury related diseases.

In another aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule may comprise a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein. The insulin-like growth factor IGF-1 protein may comprise the amino acid sequence set forth in SEQ ID NO. 8.

For example, the insulin-like growth factor IGF-1 protein may comprise an amino acid sequence that is at least 80% homologous to the amino acid sequence set forth in SEQ ID No. 8, e.g., any one of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous thereto, and which when administered in combination with the ciliary neurotrophic factor CNTF protein and the osteopontin OPN described herein is capable of treating a disease associated with optic nerve injury.

For example, the nucleotide sequence encoding insulin-like growth factor IGF-1 protein may comprise the nucleotide sequence set forth in SEQ ID NO. 2.

For example, the nucleotide sequence encoding insulin-like growth factor IGF-1 protein may comprise a nucleotide sequence that is at least 80% homologous to the nucleotide sequence depicted in SEQ id No. 2, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to any one of the nucleotide sequences, and the administration of the nucleotide sequence in combination with the ciliary neurotrophic factor CNTF protein and the osteopontin OPN described herein is capable of treating an optic nerve injury related disease.

In another aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule may comprise a nucleotide sequence encoding osteopontin OPN. The osteopontin OPN can comprise an amino acid sequence shown in SEQ ID NO. 9.

For example, the osteopontin OPN may comprise an amino acid sequence that is at least 80% homologous to the amino acid sequence set forth in SEQ ID NO. 9, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to any one of the amino acid sequences, and the administration of the amino acid sequence in combination with the ciliary neurotrophic factor CNTF protein and the insulin-like growth factor IGF-1 protein described herein is capable of treating diseases associated with optic nerve injury.

For example, the nucleotide sequence encoding osteopontin OPN may comprise the nucleotide sequence set forth in SEQ ID NO. 3.

For example, the nucleotide sequence encoding osteopontin OPN may comprise a nucleotide sequence that is at least 80% homologous to the nucleotide sequence set forth in SEQ ID No. 3, such as any one of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the nucleotide sequence, and the administration of the nucleotide sequence in combination with the ciliary neurotrophic factor CNTF protein and the insulin-like growth factor IGF-1 protein as described herein is capable of treating an optic nerve injury related disease.

In another aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule may comprise a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein and a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein. The ciliary neurotrophic factor CNTF protein may comprise an amino acid sequence shown in SEQ ID NO. 7, and the insulin-like growth factor IGF-1 protein may comprise an amino acid sequence shown in SEQ ID NO. 8.

For example, the ciliary neurotrophic factor CNTF protein may comprise an amino acid sequence that is at least 80% homologous, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the amino acid sequence shown in SEQ ID NO. 7, and the insulin-like growth factor IGF-1 protein may comprise an amino acid sequence that is at least 80% homologous, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the amino acid sequence shown in SEQ ID NO. 8, and the amino acid sequence may be administered in combination with an osteopontin OPN as described herein to treat an optic nerve injury related disorder And (6) treating the disease.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein may comprise the nucleotide sequence shown in SEQ ID NO. 1, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may comprise the nucleotide sequence shown in SEQ ID NO. 2.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein may comprise a nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO. 1, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous with any one of the nucleotide sequences shown in SEQ ID NO. 1, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may comprise a nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO. 2, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous with any one of the nucleotide sequences, and the nucleotide sequence can be used for treating optic nerve injury related diseases by being combined with osteopontin OPN.

In another aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule may comprise a nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and a nucleotide sequence encoding the osteopontin OPN. The ciliary neurotrophic factor CNTF protein can comprise an amino acid sequence shown in SEQ ID NO. 7, and the osteopontin OPN can comprise an amino acid sequence shown in SEQ ID NO. 9.

For example, the ciliary neurotrophic factor CNTF protein may comprise an amino acid sequence that is at least 80% homologous, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the amino acid sequence shown in SEQ ID NO. 7, and the osteopontin OPN may comprise an amino acid sequence that is at least 80% homologous, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the amino acid sequence shown in SEQ ID NO. 9, and the amino acid sequence may be capable of treating an optic nerve injury-related disease when administered in combination with an insulin-like growth factor IGF-1 protein as described herein And (6) treating the disease.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein may comprise the nucleotide sequence shown in SEQ ID NO. 1, and the nucleotide sequence encoding the osteopontin OPN may comprise the nucleotide sequence shown in SEQ ID NO. 3. .

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein may comprise a nucleotide sequence having at least 80% homology to the nucleotide sequence shown in SEQ ID NO. 1, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to any one of the nucleotide sequences shown in SEQ ID NO. 1, and the nucleotide sequence of osteopontin OPN may comprise a nucleotide sequence having at least 80% homology to the nucleotide sequence shown in SEQ ID NO. 3, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to any one of the nucleotide sequences shown in SEQ ID NO. 3, and administered in combination with the insulin-like growth factor IGF-1 protein as described herein Can be used for treating optic nerve injury related diseases.

In another aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule may comprise a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein and a nucleotide sequence encoding osteopontin OPN. The insulin-like growth factor IGF-1 protein may comprise the amino acid sequence shown in SEQ ID NO. 8 and the osteopontin OPN may comprise the amino acid sequence shown in SEQ ID NO. 9.

For example, the insulin-like growth factor IGF-1 protein may comprise an amino acid sequence that is at least 80% homologous to the amino acid sequence depicted in SEQ ID No. 8, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to any one of the amino acid sequences depicted in SEQ ID No. 8, and the osteopontin OPN may comprise an amino acid sequence that is at least 80% homologous to the amino acid sequence depicted in SEQ ID No. 9, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to any one of the amino acid sequences, and the amino acid sequences, when administered in combination with the ciliary neurotrophic factor CNTF protein described herein, are capable of And (6) treating the disease.

For example, the nucleotide sequence encoding insulin-like growth factor IGF-1 protein may comprise the nucleotide sequence set forth in SEQ ID NO. 2, and the nucleotide sequence encoding osteopontin OPN may comprise the nucleotide sequence set forth in SEQ ID NO. 3.

For example, the polynucleotide encoding insulin-like growth factor IGF-1 protein may comprise a nucleotide sequence that is at least 80% homologous to the nucleotide sequence set forth in SEQ ID NO. 2, e.g., any one of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, and the polynucleotide of osteopontin OPN may comprise a nucleotide sequence that is at least 80% homologous to the nucleotide sequence set forth in SEQ ID NO. 3, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, and the nucleotide sequence and the ciliary neurotrophic factor CNTF protein can be used for treating the optic nerve injury related diseases by being combined with.

In another aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule may comprise a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein, a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein and a nucleotide sequence encoding osteopontin OPN. The ciliary neurotrophic factor CNTF protein may have an amino acid sequence shown in SEQ ID NO. 7, the insulin-like growth factor IGF-1 protein may have an amino acid sequence shown in SEQ ID NO. 8, and the osteopontin OPN may have an amino acid sequence shown in SEQ ID NO. 9.

For example, the ciliary neurotrophic factor CNTF protein may comprise an amino acid sequence that is at least 80% homologous, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the amino acid sequence shown in SEQ ID NO. 7, the insulin-like growth factor IGF-1 protein may comprise an amino acid sequence that is at least 80% homologous, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the amino acid sequence shown in SEQ ID NO. 8, and the osteopontin OPN may comprise an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 9 An amino acid sequence, e.g., any one that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, and which amino acid sequence, when administered in combination, is capable of treating a disease associated with optic nerve injury.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein may comprise the nucleotide sequence shown in SEQ ID NO. 1, the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may comprise the nucleotide sequence shown in SEQ ID NO. 2, and the nucleotide sequence encoding the osteopontin OPN may comprise the nucleotide sequence shown in SEQ ID NO. 3.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein may comprise a nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO. 1, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with any one of the nucleotide sequences shown in SEQ ID NO. 1, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may comprise a nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO. 2, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with any one of the nucleotide sequences shown in SEQ ID NO. 2, and the nucleotide sequence of osteopontin OPN may comprise a nucleotide sequence at least 80% homologous to the nucleotide sequence shown in SEQ ID NO. 3, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to any one of the nucleotide sequences, and the combined administration of the nucleotide sequences can treat the optic nerve injury related diseases.

In another aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule may comprise the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein. The nucleotide sequence coding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence coding the insulin-like growth factor IGF-1 protein can be directly or indirectly connected.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may be linked by a nucleotide sequence encoding a self-cleaving polypeptide. For example, the self-cleaving polypeptide may comprise a polypeptide sequence selected from the group consisting of:

T2A peptide (GSG) EGRGSLLTCGDVEENPGP;

P2A peptide (GSG) ATNFSLLKQAGDVEENPGP;

E2A peptide (GSG) QCTNYALLKLAGDVESNPGP;

the F2A peptide (GSG) VKQTLNFDLLKLAGDVESNPGP. Wherein GSG (Gly-Ser-Gly, glycine, serine, glycine sequence) is an optional N-terminal linker sequence. In certain embodiments, the self-cleaving polypeptide may not comprise a GSG linker sequence. For example, the self-cleaving polypeptide can be a P2A peptide and/or a T2A peptide. For example, the self-cleaving polypeptide may comprise an amino acid sequence as set forth in any one of SEQ ID Nos. 10-11. For example, the self-cleaving polypeptide may comprise the P2A amino acid sequence set forth in SEQ ID NO. 10. For example, the self-cleaving polypeptide may comprise the T2A amino acid sequence set forth in SEQ ID NO. 11.

For example, the self-cleaving polypeptide may comprise an amino acid sequence that is at least 80% homologous to the amino acid sequence set forth in SEQ ID Nos. 10-11, e.g., any one of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, and is capable of effecting efficient intracellular self-cleavage of the linked ciliary neurotrophic factor CNTF protein and insulin-like growth factor IGF-1 protein.

For example, the nucleotide sequence encoding the self-cleaving polypeptide may comprise a nucleotide sequence set forth in any one of SEQ ID NOs 4-5. For example, the nucleotide sequence encoding a self-cleaving polypeptide may comprise the nucleotide sequence of P2A set forth in SEQ ID NO. 4. For example, the nucleotide sequence encoding a self-cleaving polypeptide may comprise the nucleotide sequence of T2A set forth in SEQ ID NO. 5.

For example, the nucleotide sequence encoding the self-cleaving polypeptide may comprise a nucleotide sequence that is at least 80% homologous, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, to the nucleotide sequence set forth in any one of SEQ ID NOs 4-5, and the self-cleaving polypeptide is capable of effecting highly efficient self-cleavage of the linked ciliary neurotrophic factor CNTF protein and insulin-like growth factor IGF-1 protein in a cell.

In another aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule can comprise the nucleotide sequence coding for the ciliary neurotrophic factor CNTF protein and the nucleotide sequence coding for the osteopontin OPN. The nucleotide sequence coding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence coding the osteopontin OPN can be directly or indirectly connected.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the osteopontin OPN can be connected through a nucleotide sequence encoding a self-cleaving polypeptide. For example, the self-cleaving polypeptide may comprise a polypeptide sequence selected from the group consisting of:

T2A peptide (GSG) EGRGSLLTCGDVEENPGP;

P2A peptide (GSG) ATNFSLLKQAGDVEENPGP;

E2A peptide (GSG) QCTNYALLKLAGDVESNPGP;

For example, the self-cleaving polypeptide may comprise an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO 10-11, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with any one of the amino acid sequences, which is capable of achieving efficient intracellular self-cleavage of the linked ciliary neurotrophic factor CNTF protein and osteopontin OPN.

For example, the nucleotide sequence encoding the self-cleaving polypeptide may comprise a nucleotide sequence having at least 80% homology with the nucleotide sequence set forth in any one of SEQ ID nos. 4-5, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with any one of the nucleotide sequences, which is capable of achieving efficient intracellular self-cleavage of the linked ciliary neurotrophic factor CNTF protein and osteopontin OPN.

In another aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule may comprise the nucleotide sequence encoding insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding osteopontin OPN. The nucleotide sequence encoding insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding osteopontin OPN may be linked directly or indirectly.

For example, the nucleotide sequence encoding insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding osteopontin OPN may be linked by a nucleotide sequence encoding a self-cleaving polypeptide. For example, the self-cleaving polypeptide may comprise a polypeptide sequence selected from the group consisting of:

T2A peptide (GSG) EGRGSLLTCGDVEENPGP;

P2A peptide (GSG) ATNFSLLKQAGDVEENPGP;

E2A peptide (GSG) QCTNYALLKLAGDVESNPGP;

the F2A peptide (GSG) VKQTLNFDLLKLAGDVESNPGP. Wherein GSG (Gly-Ser-Gly, glycine, serine, glycine sequence) is an optional N-terminal linker sequence. In certain embodiments, the self-cleaving polypeptide does not comprise a GSG linker sequence. For example, the self-cleaving polypeptide can be a P2A peptide and/or a T2A peptide. For example, the self-cleaving polypeptide may comprise an amino acid sequence set forth in any one of SEQ ID NOs 10-11. For example, the self-cleaving polypeptide may comprise the P2A amino acid sequence set forth in SEQ ID NO. 10. For example, the self-cleaving polypeptide may comprise the T2A amino acid sequence set forth in SEQ ID NO. 11.

For example, the self-cleaving polypeptide may comprise an amino acid sequence that is at least 80% homologous to the amino acid sequence set forth in SEQ ID Nos. 10-11, e.g., any one of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, and is capable of achieving efficient intracellular self-cleavage of the linked insulin-like growth factor IGF-1 protein and osteopontin OPN.

For example, the nucleotide sequence encoding the self-cleaving polypeptide may comprise a nucleotide sequence that is at least 80% homologous, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, to the nucleotide sequence set forth in any one of SEQ ID NOs 4-5, which may be capable of achieving efficient intracellular self-cleavage of the linked insulin-like growth factor IGF-1 protein and osteopontin OPN.

In another aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule may comprise a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein, a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein and a nucleotide sequence encoding osteopontin OPN. The nucleotide sequence coding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence coding the insulin-like growth factor IGF-1 protein, and the nucleotide sequence coding the insulin-like growth factor IGF-1 protein and the nucleotide sequence coding the osteopontin OPN can be independently connected directly or indirectly.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding the osteopontin OPN may be independently linked by a nucleotide sequence encoding a self-cleaving polypeptide. For example, the self-cleaving polypeptide may comprise a polypeptide sequence selected from the group consisting of:

T2A peptide (GSG) EGRGSLLTCGDVEENPGP;

P2A peptide (GSG) ATNFSLLKQAGDVEENPGP;

E2A peptide (GSG) QCTNYALLKLAGDVESNPGP;

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may be linked by a splicing polypeptide of the amino acid sequence P2A shown in SEQ ID NO:10, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding the osteopontin OPN may be linked by a splicing polypeptide of the amino acid sequence P2A shown in SEQ ID NO: 10.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may be linked by a splicing polypeptide of the P2A amino acid sequence shown in SEQ ID NO:10, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding the osteopontin OPN may be linked by a splicing polypeptide of the T2A amino acid sequence shown in SEQ ID NO: 11.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may be linked by a splicing polypeptide of the amino acid sequence T2A shown in SEQ ID NO:11, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding the osteopontin OPN may be linked by a splicing polypeptide of the amino acid sequence P2A shown in SEQ ID NO: 10.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may be linked by a splicing polypeptide of the amino acid sequence T2A shown in SEQ ID NO:11, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding the osteopontin OPN may be linked by a splicing polypeptide of the amino acid sequence T2A shown in SEQ ID NO: 11.

For example, the self-cleaving polypeptide may comprise an amino acid sequence that is at least 80% homologous to the amino acid sequence set forth in SEQ ID Nos. 10-11, e.g., any one of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, and is capable of achieving efficient intracellular self-cleavage of the linked ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1 protein, and osteopontin OPN.

For example, the nucleotide sequence encoding the self-cleaving polypeptide may independently comprise a nucleotide sequence set forth in any one of SEQ ID NOs 4-5. For example, the nucleotide sequence encoding a self-cleaving polypeptide may comprise the nucleotide sequence of P2A set forth in SEQ ID NO. 4. For example, the nucleotide sequence encoding a self-cleaving polypeptide may comprise the nucleotide sequence of T2A set forth in SEQ ID NO. 5.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may be linked by a splicing polypeptide comprising the nucleotide sequence P2A shown in SEQ ID NO. 4, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding the osteopontin OPN may be linked by a splicing polypeptide comprising the nucleotide sequence P2A shown in SEQ ID NO. 4.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may be linked by a splicing polypeptide comprising the nucleotide sequence P2A shown in SEQ ID NO. 4, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding the osteopontin OPN may be linked by a splicing polypeptide comprising the nucleotide sequence T2A shown in SEQ ID NO. 5.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may be linked by a splicing polypeptide comprising the nucleotide sequence T2A shown in SEQ ID NO. 5, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding the osteopontin OPN may be linked by a splicing polypeptide comprising the nucleotide sequence P2A shown in SEQ ID NO. 4.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may be linked by a splicing polypeptide comprising the nucleotide sequence T2A shown in SEQ ID NO. 5, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein and the nucleotide sequence encoding the osteopontin OPN may be linked by a splicing polypeptide comprising the nucleotide sequence T2A shown in SEQ ID NO. 5.

For example, the nucleotide sequence encoding the self-cleaving polypeptide may comprise a nucleotide sequence that is at least 80% homologous, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, to the nucleotide sequence set forth in any one of SEQ ID NOs 4-5, and the self-cleaving polypeptide is capable of effecting highly efficient intracellular self-cleavage of the linked ciliary neurotrophic factor CNTF protein, the linked insulin-like growth factor IGF-1 protein, and the osteopontin OPN.

In another aspect, the present application provides a recombinant nucleic acid molecule. The recombinant nucleic acid molecule can sequentially comprise the nucleotide sequence coding the ciliary neurotrophic factor CNTF protein, the nucleotide sequence coding the self-splicing polypeptide P2A, the nucleotide sequence coding the insulin-like growth factor IGF-1 protein, the nucleotide sequence coding the self-splicing polypeptide T2A and the nucleotide sequence coding the osteopontin OPN from the 5 'end to the 3' end.

For example, the recombinant nucleic acid molecule can comprise the nucleotide sequence coding for the ciliary neurotrophic factor CNTF protein, the nucleotide sequence coding for the self-splicing polypeptide P2A, the nucleotide sequence coding for the insulin-like growth factor IGF-1 protein, the nucleotide sequence coding for the self-splicing polypeptide T2A and the nucleotide sequence coding for the osteopontin OPN in sequence from 5 'to 3', the ciliary neurotrophic factor CNTF protein may comprise SEQ ID NO:7, or a pharmaceutically acceptable salt thereof, wherein, the self-cleaving polypeptide P2A may comprise SEQ ID NO:10, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown in the specification, the insulin-like growth factor IGF-1 protein may comprise SEQ ID NO:8, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown in figure 8, the self-cleaving polypeptide T2A may comprise seq id NO:11, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence is shown as 11, and the osteopontin OPN may comprise SEQ ID NO:9, or a pharmaceutically acceptable salt thereof.

For example, the ciliary neurotrophic factor CNTF protein may comprise an amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 7, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and the self-cleaving polypeptide P2A may comprise an amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 10, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and the insulin-like growth factor IGF-1 protein may comprise an amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 8 An amino acid sequence, e.g.any amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the amino acid sequence shown in SEQ ID NO. 11, the self-cleaving polypeptide T2A may comprise an amino acid sequence which is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 11, e.g.any amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the amino acid sequence shown in SEQ ID NO. 11, and the osteopontin OPN may comprise an amino acid sequence which is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 9, e.g.g.g, At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to any one of the amino acid sequences, and the amino acid sequences are capable of treating a disease associated with optic nerve injury when administered in combination.

For example, the recombinant nucleic acid molecule may sequentially comprise, from 5 'end to 3' end, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein, the nucleotide sequence encoding the self-splicing polypeptide P2A, the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein, the nucleotide sequence encoding the self-splicing polypeptide T2A and the nucleotide sequence encoding the osteopontin OPN, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein may comprise the nucleotide sequence shown in SEQ ID NO:1, the nucleotide sequence encoding the self-splicing polypeptide P2A may comprise the nucleotide sequence shown in SEQ ID NO:4, the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may comprise the nucleotide sequence shown in SEQ ID NO:2, the nucleotide sequence encoding the self-splicing polypeptide T2A may comprise the nucleotide sequence shown in SEQ ID NO:5, and the nucleotide sequence encoding osteopontin OPN may comprise a nucleotide sequence shown in SEQ ID NO. 3, such as the nucleotide sequence of CNTF-2A-IGF1-2A-OPN shown in SEQ ID NO. 6 of the present application.

For example, the nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein may comprise a nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO. 1, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with any one of the nucleotide sequences shown in SEQ ID NO. 1, the nucleotide sequence encoding the self-splicing polypeptide P2A may comprise a nucleotide sequence having at least 80% homology with the nucleotide sequence shown in SEQ ID NO. 4, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with any one of the nucleotide sequences shown in SEQ ID NO. 4, and the nucleotide sequence encoding the insulin-like growth factor IGF-1 protein may comprise a nucleotide sequence having at least The nucleotide sequence shown as NO. 2 has a nucleotide sequence which is at least 80% homologous, such as any one of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, the nucleotide sequence encoding self-cleaving polypeptide T2A may comprise a nucleotide sequence which is at least 80% homologous to the nucleotide sequence shown as SEQ ID NO. 5, such as any one of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, and the nucleotide sequence of osteopontin OPN may comprise a nucleotide sequence which is at least 80% homologous to the nucleotide sequence shown as SEQ ID NO. 3, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and the nucleotide sequences are capable of treating an optic nerve injury-related disease when administered in combination.

For example, the nucleotide sequence of the nucleic acid molecule may comprise a nucleotide sequence that is at least 80% homologous to the nucleotide sequence set forth in SEQ ID No. 6, e.g., any one of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the nucleotide sequence set forth in SEQ ID No. 6, and the administration of the nucleotide sequence is capable of treating a disease associated with optic nerve damage.

Carrier

In one aspect, the present application provides a vector. The vector may comprise one or more open reading frames, which may comprise one or more of the recombinant nucleic acids described herein in their entirety and fragments thereof. For example, the vector may comprise one or more open reading frames expressing the sequences of the ciliary neurotrophic factor CNTF protein. For example, the vector may comprise one or more open reading frames expressing the sequence of insulin-like growth factor IGF-1. For example, the vector may comprise one or more open reading frames expressing the sequences of osteopontin OPN. For example, the vector may comprise one or more open reading frames expressing sequences of the ciliary neurotrophic factor CNTF protein, the insulin-like growth factor IGF-1, and/or the osteopontin OPN, and any combination thereof. For example, when one or more sequences expressing the ciliary neurotrophic factor CNTF protein, expressing the insulin-like growth factor IGF-1 and/or expressing osteopontin OPN are combined at will, the encoded ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1 and osteopontin OPN may exist independently as non-fusion proteins after expression.

For example, the vector may comprise a sequence expressing the ciliary neurotrophic factor CNTF protein and a sequence expressing the insulin-like growth factor IGF-1. For example, in the 5 'to 3' direction, a sequence expressing the ciliary neurotrophic factor CNTF protein and a sequence expressing the insulin-like growth factor IGF-1 may be included in this order. For example, from the 5 'end to the 3' end, the sequence expressing the insulin-like growth factor IGF-1 and the sequence expressing the ciliary neurotrophic factor CNTF protein may be included in this order.

For example, the vector may comprise a sequence expressing the ciliary neurotrophic factor CNTF protein and a sequence expressing the osteopontin OPN. For example, from the 5 'end to the 3' end, a sequence expressing the ciliary neurotrophic factor CNTF protein and a sequence expressing the osteopontin OPN may be included in this order. For example, from the 5 'end to the 3' end, the sequence expressing osteopontin OPN and the sequence expressing ciliary neurotrophic factor CNTF protein may be included in this order.

For example, the vector may comprise a sequence that expresses insulin-like growth factor IGF-1 and a sequence that expresses osteopontin OPN. For example, in the 5 'to 3' direction, the sequence expressing insulin-like growth factor IGF-1 and the sequence expressing osteopontin OPN may be included in this order. For example, the sequence expressing osteopontin OPN and the sequence expressing insulin-like growth factor IGF-1 may be included in sequence from 5 'to 3' in the direction.

For example, the vector may comprise a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the insulin-like growth factor IGF-1, and a sequence expressing the osteopontin OPN. For example, in the 5 'to 3' direction, a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the insulin-like growth factor IGF-1, and a sequence expressing the osteopontin OPN may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing osteopontin OPN, and a sequence expressing the insulin-like growth factor IGF-1 may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing insulin-like growth factor IGF-1, a sequence expressing ciliary neurotrophic factor CNTF protein, and a sequence expressing osteopontin OPN may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing insulin-like growth factor IGF-1, a sequence expressing osteopontin OPN, and a sequence expressing ciliary neurotrophic factor CNTF protein may be included in this order. For example, from the 5 'end to the 3' end, a sequence expressing osteopontin OPN, a sequence expressing ciliary neurotrophic factor CNTF protein, and a sequence expressing insulin-like growth factor IGF-1 may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing osteopontin OPN, a sequence expressing insulin-like growth factor IGF-1, and a sequence expressing ciliary neurotrophic factor CNTF protein may be included in this order.

In another aspect, the open reading frames may comprise one or more of the sequences expressing self-cleaving polypeptides described herein therebetween. For example, the self-cleaving polypeptide may comprise a polypeptide sequence selected from the group consisting of:

T2A peptide (GSG) EGRGSLLTCGDVEENPGP;

P2A peptide (GSG) ATNFSLLKQAGDVEENPGP;

E2A peptide (GSG) QCTNYALLKLAGDVESNPGP;

the F2A peptide (GSG) VKQTLNFDLLKLAGDVESNPGP. Wherein GSG (Gly-Ser-Gly, glycine, serine, glycine sequence) is an optional N-terminal linker sequence. In certain embodiments, the self-cleaving polypeptide does not comprise a GSG linker sequence. For example, the self-cleaving polypeptide can be a P2A peptide and/or a T2A peptide.

For example, the vector may comprise a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the self-cleaving polypeptide, and a sequence expressing the insulin-like growth factor IGF-1. For example, in the 5 'to 3' direction, a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the self-cleaving polypeptide and a sequence expressing the insulin-like growth factor IGF-1 may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing insulin-like growth factor IGF-1, a sequence expressing a self-splicing polypeptide, and a sequence expressing ciliary neurotrophic factor CNTF protein may be included in this order.

For example, the vector may comprise a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the self-cleaving polypeptide, and a sequence expressing the osteopontin OPN. For example, from the 5 'end to the 3' end, the sequence expressing the ciliary neurotrophic factor CNTF protein, the sequence expressing the self-splicing polypeptide and the sequence expressing the osteopontin OPN can be included in sequence. For example, from the 5 'end to the 3' end, the sequence expressing osteopontin OPN, the sequence expressing self-splicing polypeptide and the sequence expressing ciliary neurotrophic factor CNTF protein can be included in sequence.

For example, the vector may comprise a sequence that expresses insulin-like growth factor IGF-1, a sequence that expresses a self-cleaving polypeptide, and a sequence that expresses osteopontin OPN. For example, in the 5 'to 3' direction, the sequence may comprise, in order, a sequence expressing insulin-like growth factor IGF-1, a sequence expressing a self-cleaving polypeptide, and a sequence expressing osteopontin OPN. For example, in the 5 'to 3' direction, the sequence may comprise, in order, a sequence expressing osteopontin OPN, a sequence expressing self-cleaving polypeptide, and a sequence expressing insulin-like growth factor IGF-1.

For example, the vector may comprise a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the insulin-like growth factor IGF-1, a sequence expressing the osteopontin OPN, and a sequence expressing the self-cleaving polypeptide. For example, in the 5 'to 3' direction, a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the self-cleaving polypeptide, a sequence expressing the insulin-like growth factor IGF-1, a sequence expressing the self-cleaving polypeptide and a sequence expressing the osteopontin OPN may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the self-cleaving polypeptide, a sequence expressing the osteopontin OPN, a sequence expressing the self-cleaving polypeptide, and a sequence expressing the insulin-like growth factor IGF-1 may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing insulin-like growth factor IGF-1, a sequence expressing a self-splicing polypeptide, a sequence expressing ciliary neurotrophic factor CNTF protein, a sequence expressing a self-splicing polypeptide, and a sequence expressing osteopontin OPN may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing insulin-like growth factor IGF-1, a sequence expressing a self-splicing polypeptide, a sequence expressing osteopontin OPN, a sequence expressing a self-splicing polypeptide, and a sequence expressing ciliary neurotrophic factor CNTF protein may be included in this order. For example, from the 5 'end to the 3' end, a sequence expressing osteopontin OPN, a sequence expressing a self-splicing polypeptide, a sequence expressing ciliary neurotrophic factor CNTF protein, a sequence expressing a self-splicing polypeptide, and a sequence expressing insulin-like growth factor IGF-1 may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing osteopontin OPN, a sequence expressing a self-splicing polypeptide, a sequence expressing insulin-like growth factor IGF-1, a sequence expressing a self-splicing polypeptide, and a sequence expressing ciliary neurotrophic factor CNTF protein may be included in this order.

For example, in the 5 'to 3' direction, a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the self-splicing polypeptide P2A, a sequence expressing the insulin-like growth factor IGF-1, a sequence expressing the self-splicing polypeptide P2A and a sequence expressing the osteopontin OPN may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the self-splicing polypeptide P2A, a sequence expressing the insulin-like growth factor IGF-1, a sequence expressing the self-splicing polypeptide T2A and a sequence expressing the osteopontin OPN may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the self-splicing polypeptide T2A, a sequence expressing the insulin-like growth factor IGF-1, a sequence expressing the self-splicing polypeptide P2A and a sequence expressing the osteopontin OPN may be included in this order. For example, in the 5 'to 3' direction, a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the self-splicing polypeptide T2A, a sequence expressing the insulin-like growth factor IGF-1, a sequence expressing the self-splicing polypeptide T2A and a sequence expressing the osteopontin OPN may be included in this order.

In another aspect, the vector may comprise one or more AAV inverted terminal repeat ITRs. For example, the AAV inverted terminal repeat ITRs may be independently located at one or both ends of one or more of the open reading frames. For example, the AAV inverted terminal repeat ITRs may be located 5' to one or more of the open reading frames. For example, the AAV inverted terminal repeat ITRs may be located 3' to one or more of the open reading frames. For example, the AAV inverted terminal repeat ITRs may be located at the 5 'end and 3' end of one or more of the open reading frames.

For example, in the 5 'to 3' direction, an AAV inverted terminal repeat ITR, a sequence expressing the ciliary neurotrophic factor CNTF protein, a sequence expressing the self-splicing polypeptide P2A, a sequence expressing the insulin-like growth factor IGF-1, a sequence expressing the self-splicing polypeptide P2A, a sequence expressing the osteopontin OPN, and an AAV inverted terminal repeat ITR may be included in this order.

In another aspect, the vector may comprise one or more promoters. For example, one or more of the promoters may be CMV, CAG and/or the nerve-specific promoter SYN. For example, the promoter may be CMV.

For example, the one or more CMV can be located anywhere in the vector, and the CMV can initiate expression of a protein encoded in the open reading frame. For example, CMV can precede a sequence that expresses the ciliary neurotrophic factor CNTF protein. For example, CMV can precede the sequence of the splicing polypeptide P2A. For example, CMV can precede a sequence that expresses insulin-like growth factor IGF-1. For example, CMV can precede the sequence of the splicing polypeptide T2A. For example, CMV can precede the sequence expressing osteopontin OPN.

In another aspect, the vector may comprise one or more enhancers. For example, the one or more enhancers may be located anywhere in the vector, and the enhancer may enhance expression of the encoded protein in the open reading frame. For example, the enhancer may be located upstream and/or downstream from the initiation site.

In another aspect, the vector may be selected from adeno-associated viruses. For example, the serotype of the vector may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, and/or AAVAnc80, or variants and combinations thereof. For example, the adeno-associated virus can be selected from AAV2, AAV5, AAV7, and/or AAV8, or a combination thereof. For example, the adeno-associated virus can be selected from AAV 2.

For example, the AAV2 vector can be AAV2/2, AAV2/5, AAV2/8, or AAV 2/9. For example, the AAV2 vector can comprise pAAV-RC5-Amp, RC8-cap, AAV2/8, AAV-helper-Amp, AAV-helper.

Pharmaceutical preparation and pharmaceutical use

In one aspect, the present application provides the use of a pharmaceutical formulation in the manufacture of a medicament for the treatment of a disease associated with optic nerve injury. For example, the optic nerve injury-related disease may be acute or chronic optic nerve injury. For example, the optic nerve injury-related disease may be acute or chronic optic nerve injury resulting in damage and/or death of the optic nerve and its ganglion cells. For example, the disease associated with optic nerve damage can be autosomal Dominant Optic Atrophy (DOA), ischemic optic neuropathy, Leber's Hereditary Optic Neuropathy (LHON), or glaucoma. For example, the optic nerve injury-related disease may be glaucoma.

In one aspect, the present application provides a pharmaceutical formulation for use in treating a disease associated with optic nerve injury. For example, the optic nerve injury-related disease may be acute or chronic optic nerve injury. For example, the optic nerve injury-related disease may be acute or chronic optic nerve injury resulting in damage and/or death of the optic nerve and its ganglion cells. For example, the disease associated with optic nerve damage can be autosomal Dominant Optic Atrophy (DOA), ischemic optic neuropathy, Leber's Hereditary Optic Neuropathy (LHON), or glaucoma. For example, the optic nerve injury-related disease may be glaucoma.

In one aspect, the application provides a pharmaceutical formulation for use in treating a disease associated with optic nerve damage. For example, the optic nerve injury-related disease may be acute or chronic optic nerve injury. For example, the optic nerve injury-related disease may be acute or chronic optic nerve injury resulting in damage and/or death of the optic nerve and its ganglion cells. For example, the disease associated with optic nerve damage can be autosomal Dominant Optic Atrophy (DOA), ischemic optic neuropathy, Leber's Hereditary Optic Neuropathy (LHON), or glaucoma. For example, the optic nerve injury-related disease may be glaucoma.

For example, the pharmaceutical formulation may cause long-term high expression of the ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1 and/or osteopontin OPN in retinal cells. For example, the high expression may mean that the amount of protein expressed in a cell or a subject is 1.1 times or more, 1.3 times or more, 1.5 times or more, two times or more, three times or more, five times or more, ten times or more, fifty times or more, one hundred times or more, five hundred times or more, one thousand times or more, or one thousand times or more, which is not administered, after the pharmaceutical preparation of the present invention is administered. For example, the high expression may mean that after the pharmaceutical preparation of the present invention is administered, the expression level of the protein in the cell or the subject is 1.1 times or more, 1.3 times or more, 1.5 times or more, two times or more, three times or more, five times or more, ten times or more, fifty times or more, one hundred times or more, five hundred times or more, one thousand times or more, the expression level of the protein in the cell or the subject is 1.1 times or more, 1.3 times or more, 1.5 times or more, two times or more, three times or more, five times or more, ten. For example, the long-acting may refer to that the high expression level is maintained in a cell or a subject for at least one week, at least one month, at least three months, or at least six months after administration of the vector or pharmaceutical formulation of the present application as compared to administration of the ciliary neurotrophic factor CNTF protein vector alone.

For example, the pharmaceutical formulation may be used for long-acting treatment of diseases associated with optic nerve damage. For example, the long-acting can mean that, after administration of a vector or pharmaceutical formulation of the present application, the cell or subject maintains a therapeutic effect of the optic nerve injury-related disease for a period of at least one week, at least one month, at least three months, or at least six months longer than administration of a vector expressing the ciliary neurotrophic factor CNTF protein alone.

For example, the pharmaceutical preparation may increase the survival rate of ganglion cells and/or their axons. For example, the increase can refer to an increase in survival of ganglion cells and/or their axons of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 7-fold, or at least 10-fold after administration of a vector or pharmaceutical formulation of the present application as compared to non-administration. For example, the increase can refer to an increase in survival of ganglion cells and/or their axons of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 7-fold, or at least 10-fold after administration of a vector or pharmaceutical formulation of the present application as compared to administration of a ciliary neurotrophic factor CNTF protein vector alone. In the present application, the ganglion cells may comprise retinal ganglion cells.

For example, the pharmaceutical formulation may prevent or delay the apoptosis of ganglion cells. For example, the prevention can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, or at least 10 fold less of the ganglion cell apoptosis after administration of a vector or pharmaceutical formulation of the present application as compared to non-administration. For example, the prevention can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, or at least 10 fold less of ganglion cell apoptosis after administration of a vector or pharmaceutical formulation of the present application as compared to administration of a vector expressing ciliary neurotrophic factor CNTF protein alone. For example, the delay can be a delay in the onset of ganglion cell apoptosis of at least one week, at least one month, at least three months, or at least six months after administration of the vehicle or pharmaceutical formulation of the present application as compared to non-administration. For example, the delay can be a delay in the onset of ganglion cell apoptosis after administration of a vector or pharmaceutical formulation of the present application of at least one week, at least one month, at least three months, or at least six months, as compared to administration of a vector expressing the ciliary neurotrophic factor CNTF protein alone. In the present application, the ganglion cells may comprise retinal ganglion cells.

For example, the pharmaceutical formulation may prevent or delay the decrease in the thickness of the retinal ganglion cell layer. For example, the prevention can be a reduction in retinal ganglion cell layer thickness of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 7-fold, or at least 10-fold after administration of a vehicle or pharmaceutical formulation of the present application as compared to non-administration. For example, the prevention can be at least a 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, or at least 10 fold reduction in retinal ganglion cell layer thickness after administration of a vector or pharmaceutical formulation of the present application as compared to administration of a vector expressing ciliary neurotrophic factor CNTF protein alone. For example, the delay can be a delay in the reduction of retinal ganglion cell layer thickness of at least one week, at least one month, at least three months, or at least six months after administration of the vehicle or pharmaceutical formulation of the present application as compared to non-administration. For example, the delay can be a delay in the reduction of retinal ganglion cell layer thickness after administration of the vector or pharmaceutical formulation of the present application of at least one week, at least one month, at least three months, or at least six months, as compared to administration of the vector expressing the ciliary neurotrophic factor CNTF protein alone.

For example, the pharmaceutical formulation may prevent or delay the reduction of the thickness of the retinal nerve fiber layer. For example, the prevention can be a reduction in retinal nerve fiber layer thickness of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 7-fold, or at least 10-fold after administration of a vehicle or pharmaceutical formulation of the present application as compared to non-administration. For example, the prevention can be at least a 10%, at least a 15%, at least a 20%, at least a 25%, at least a 30%, at least a 35%, at least a 40%, at least a 45%, at least a 50%, at least a 55%, at least a 60%, at least a 65%, at least a 70%, at least a 75%, at least an 80%, at least an 85%, at least a 90%, at least a 95% or at least a 99% or at least a 1.5 fold, at least a 2 fold, at least a 2.5 fold, at least a 3 fold, at least a 3.5 fold, at least a 4 fold, at least a 4.5 fold, at least a 5 fold, at least a 7 fold or at least a 10 fold reduction in thickness of the retinal nerve fiber layer after administration of the vehicle or pharmaceutical formulation of the present application. For example, the delay can be a delay in the reduction of retinal nerve fiber layer thickness after administration of the vehicle or pharmaceutical formulation of the present application of at least one week, at least one month, at least three months, or at least six months compared to no administration. For example, the delay may be a delay in the reduction of the thickness of the retinal nerve fiber layer after administration of the vehicle or pharmaceutical formulation of the present application of at least one week, at least one month, at least three months, or at least six months compared to administration of the ciliary neurotrophic factor CNTF protein vector alone.

For example, the pharmaceutical formulation may prevent or delay the progression of abnormal visual field conditions. For example, the prevention can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, or at least 10 fold less worsening of a visual field abnormality after administration of a vector or pharmaceutical formulation of the present application as compared to non-administration. For example, the prevention can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, or at least 10 fold less deterioration of the aberrant visual field as compared to administration of the ciliary neurotrophic factor CNTF protein vector alone following administration of a vector or pharmaceutical formulation of the present application. For example, the delay can be a delay in worsening abnormal visual field condition of at least one week, at least one month, at least three months, or at least six months after administration of the vehicle or pharmaceutical formulation of the present application as compared to non-administration. For example, the delay may be a delay in worsening of a visual field abnormality after administration of a vector or pharmaceutical formulation of the present application, of at least one week, at least one month, at least three months, or at least six months, as compared to administration of a vector expressing the ciliary neurotrophic factor CNTF protein alone.

For example, the pharmaceutical formulation may not cause significant inflammatory reactions or other complications when injected intraocularly. For example, the inflammatory response or other complication may be conjunctival congestion, increased ocular secretions, and/or endophthalmitis.

For example, the pharmaceutical formulation may comprise one or more recombinant nucleic acids of the present application or two or more vectors of the present application, and a pharmaceutically acceptable excipient. For example, the formulation may be a liquid formulation.

For example, the pharmaceutical formulation may comprise one or more carriers of the present application. For example, a first vector may comprise a first recombinant nucleic acid which may comprise a nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein, and a second vector may comprise a second recombinant nucleic acid which may comprise a nucleotide sequence encoding the insulin-like growth factor IGF-1 protein.

For example, the pharmaceutical formulation may comprise one or more carriers of the present application. For example, a first vector may comprise a first recombinant nucleic acid, which may comprise a nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein, and a second vector may comprise a second recombinant nucleic acid, which may comprise a nucleotide sequence encoding the osteopontin OPN.

For example, the pharmaceutical formulation may comprise one or more carriers of the present application. For example, a first vector may comprise a first recombinant nucleic acid which may comprise a nucleotide sequence encoding insulin-like growth factor IGF-1 protein, and a second vector may comprise a second recombinant nucleic acid which may comprise a nucleotide sequence encoding osteopontin OPN.

For example, the pharmaceutical formulation may comprise one or more carriers of the present application. For example, a first vector may comprise a first recombinant nucleic acid, which may comprise a nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein, a second vector may comprise a second recombinant nucleic acid, which may comprise a nucleotide sequence encoding the insulin-like growth factor IGF-1 protein, and a third vector may comprise a third recombinant nucleic acid, which may comprise a nucleotide sequence encoding the osteopontin OPN.

For example, when the pharmaceutical formulation comprises a vector of the present application, the pharmaceutical formulation may be administered by administering the vector to a cell or subject. For example, when the pharmaceutical formulation comprises two or more vectors of the present application, the pharmaceutical formulation can be administered by administering the two or more vectors to the cell or subject simultaneously. For example, when the pharmaceutical formulation comprises two or more vectors of the present application, the pharmaceutical formulation may be administered by separately administering a portion of the two or more vectors to the cell or subject in any order. For example, when the pharmaceutical formulation comprises two or more vectors of the present application, the pharmaceutical formulation can be administered by separately administering each of the two or more vectors to the cell or subject in any order.

In the present application, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey; for example, the subject may comprise a DBA/2J mouse. In the present application, the cells may comprise bacterial cells (e.g., e.coli), yeast cells, or other eukaryotic cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells, 293T cells.

For example, the pharmaceutical formulation may be injected intraocularly. For example, the formulation may be injected in the vitreous cavity.

The present application also includes the following embodiments:

1. a recombinant nucleic acid comprising:

a) a first nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein;

b) a second nucleotide sequence encoding an insulin-like growth factor IGF-1 protein; and

c) a third nucleotide sequence encoding osteopontin OPN.

2. The recombinant nucleic acid according to embodiment 1, wherein said ciliary neurotrophic factor CNTF protein comprises an amino acid sequence selected from the group consisting of:

a) SEQ ID NO: 7;

b) and SEQ ID NO:7 an amino acid sequence having at least 98% homology;

c) and SEQ ID NO:7 an amino acid sequence having at least 95% homology;

d) and SEQ ID NO:7 an amino acid sequence having at least 90% homology; and

e) and SEQ ID NO:7 has at least 80% homology.

3. The recombinant nucleic acid of embodiment 1 or 2, wherein the first nucleotide sequence comprises a nucleotide sequence selected from the group consisting of seq id no:

a) SEQ ID NO: 1;

b) and SEQ ID NO:1 nucleotide sequence having at least 98% homology;

c) and SEQ ID NO:1 nucleotide sequence having at least 95% homology;

d) and SEQ ID NO:1 nucleotide sequence having at least 90% homology; and

e) and SEQ ID NO:1 nucleotide sequence having at least 80% homology.

4. The recombinant nucleic acid according to any one of embodiments 1-3, wherein said insulin-like growth factor IGF-1 protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

a) SEQ ID NO: 8;

b) and SEQ ID NO:8 an amino acid sequence having at least 98% homology;

c) and SEQ ID NO:8 an amino acid sequence having at least 95% homology;

d) and SEQ ID NO:8 an amino acid sequence having at least 90% homology; and

e) and SEQ ID NO:8 has at least 80% homology.

5. The recombinant nucleic acid according to any one of embodiments 1-4, wherein the second nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:

a) SEQ ID NO: 2;

b) and SEQ ID NO:2 nucleotide sequences having at least 98% homology;

c) and SEQ ID NO:2 nucleotide sequences having at least 95% homology;

d) and SEQ ID NO:2 nucleotide sequences having at least 90% homology; and

e) and SEQ ID NO:2 has at least 80% homology.

6. The recombinant nucleic acid according to any one of embodiments 1-5, wherein said osteopontin OPN comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

a) SEQ ID NO: 9;

b) and SEQ ID NO:9 an amino acid sequence having at least 98% homology;

c) and SEQ ID NO:9 an amino acid sequence having at least 95% homology;

d) and SEQ ID NO:9 an amino acid sequence having at least 90% homology; and

e) and SEQ ID NO:9 has an amino acid sequence with at least 80% homology.

7. The recombinant nucleic acid according to any one of embodiments 1-6, wherein the third nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:

a) SEQ ID NO: 3;

b) and SEQ ID NO:3 nucleotide sequences having at least 98% homology;

c) and SEQ ID NO:3 nucleotide sequences having at least 95% homology;

d) and SEQ ID NO:3 nucleotide sequences having at least 90% homology; and

e) and SEQ ID NO:3 nucleotide sequence with at least 80% homology.

8. The recombinant nucleic acid according to any one of embodiments 1-7, wherein the first, second, and third nucleotide sequences are linked directly or indirectly to each other.

9. The recombinant nucleic acid of embodiment 8, wherein said indirect linkage comprises linkage through a nucleotide sequence encoding a self-cleaving polypeptide.

10. The recombinant nucleic acid of any one of embodiments 8-9, wherein the self-cleaving polypeptide is a 2A peptide.

11. The recombinant nucleic acid of embodiment 10, wherein the 2A peptide is selected from the group consisting of: P2A peptide with an amino acid sequence shown as SEQ ID NO. 10 and T2A peptide with an amino acid sequence shown as SEQ ID NO. 11.

12. The recombinant nucleic acid according to any one of embodiments 1-11, comprising, in order from the 5 'end to the 3' end, the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence.

13. The recombinant nucleic acid of embodiment 12, comprising a nucleotide sequence selected from the group consisting of seq id no:

a) SEQ ID NO: 6;

b) and SEQ ID NO:6 nucleotide sequences having at least 98% homology;

c) and SEQ ID NO:6 nucleotide sequences having at least 95% homology;

d) and SEQ ID NO:6 nucleotide sequences having at least 90% homology; and

e) and SEQ ID NO:6 nucleotide sequences having at least 80% homology.

14. A vector, wherein the vector comprises the recombinant nucleic acid of any one of embodiments 1-13.

15. The vector of embodiment 13, wherein the recombinant nucleic acid comprises an AAV inverted terminal repeat ITR.

16. The vector of embodiment 13 or 14, wherein the vector is selected from adeno-associated viral vectors.

17. The vector of embodiment 16, wherein the adeno-associated virus comprises AAV2, AAV5, AAV7, and/or AAV 8.

18. The vector according to any one of embodiments 13-17, wherein the nucleotide sequence further comprises a promoter or enhancer.

19. The vector of embodiment 18, wherein the promoter is selected from the group consisting of: CMV, CAG and SYN.

20. The vector according to any one of embodiments 13-19, wherein said vector encodes the ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1 and osteopontin OPN independently present as a non-fusion protein upon expression.

21. Use of a vector according to any one of embodiments 13-20 for the treatment of a disease associated with optic nerve injury.

22. The use according to embodiment 21, wherein the optic nerve injury-related disease comprises acute or chronic optic nerve injury.

23. The use according to any one of embodiments 21-22, wherein the optic nerve injury-related disease is selected from the group consisting of: DOA, ischemic optic neuropathy, LHON, and glaucoma.

24. The use according to embodiment 23, wherein the optic nerve injury-related disease comprises glaucoma.

25. A pharmaceutical formulation, wherein the pharmaceutical formulation comprises the carrier according to any one of embodiments 13-20, and a pharmaceutically acceptable excipient.

26. The pharmaceutical formulation of embodiment 25, which is a liquid formulation.

27. The pharmaceutical formulation of embodiment 25 or 26 for use in treating a disease associated with optic nerve injury.

28. The use according to embodiment 27, wherein the pharmaceutical formulation results in long-term high expression of the ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1 and osteopontin OPN in retinal cells.

29. The use according to embodiment 27 or 28, wherein the pharmaceutical formulation is long acting in the treatment of a disease associated with optic nerve damage.

30. The use according to any one of embodiments 25-29, wherein said pharmaceutical preparation increases the survival rate of ganglion cells and/or their axons.

31. The use according to any one of embodiments 25-30, wherein said pharmaceutical preparation prevents or delays apoptosis of ganglion cells.

32. The use according to any one of embodiments 25-31, wherein the optic nerve injury-related disease comprises glaucoma.

33. The use according to any one of embodiments 25-32, wherein the optic nerve injury-related disease is selected from the group consisting of: acute or chronic optic nerve injury resulting in damage and/or death of the optic nerve and its ganglion cells.

34. The use according to any one of embodiments 25-33, wherein the mode of administration of the pharmaceutical formulation comprises intraocular injection.

35. The use of embodiment 34, wherein the intraocular injection comprises a vitreous cavity injection.

36. The use according to any one of embodiments 25-35, wherein intraocular injection of the pharmaceutical formulation does not cause an inflammatory response or complication.

37. A method of treating an ocular disease comprising administering to a patient two or more carriers or pharmaceutical formulations thereof, said two or more carriers comprising:

a) a first vector comprising a first recombinant nucleic acid, said first recombinant nucleic acid comprising a nucleotide sequence encoding osteopontin OPN; and

b) a second vector comprising a second recombinant nucleic acid comprising a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein, or a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein.

38. The method according to embodiment 37, wherein said first vector or said second vector further comprises a third recombinant nucleic acid, said third recombinant nucleic acid being different from said second recombinant nucleic acid and said third recombinant nucleic acid comprising a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein or a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein.

39. The method of embodiment 37, wherein said two or more vectors comprise a third vector, said third vector comprising said third recombinant nucleic acid.

40. The method according to embodiment 38 or 39, wherein said second recombinant nucleic acid comprises a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein and said third recombinant nucleic acid comprises a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein.

41. The method according to embodiment 38 or 39, wherein said second recombinant nucleic acid comprises a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein and said third recombinant nucleic acid comprises a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein.

42. A method of treating an ocular disease comprising administering to a subject in need thereof an effective amount of the vector of any one of embodiments 13-20 or the pharmaceutical formulation of any one of embodiments 25-26.

43. The method according to any one of embodiments 37-42, wherein the ocular disease comprises an optic nerve injury-related disease.

44. The method of embodiment 43, wherein the optic nerve injury-related disease comprises acute or chronic optic nerve injury.

45. The method according to embodiment 43, wherein the optic nerve injury-related disease comprises DOA, LHON, ischemic optic neuropathy and/or glaucoma.

46. The method of embodiment 43, wherein the optic nerve injury-related disease comprises glaucoma.

47. The method of any one of embodiments 42-46, comprising intraocular injection of the carrier or pharmaceutical formulation.

48. The method of embodiment 47, wherein the intraocular injection comprises a vitreous cavity injection.

49. The method according to any one of embodiments 42-48, wherein said carrier or pharmaceutical formulation results in long-lasting high expression of the ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1 and osteopontin OPN in retinal cells.

50. The method of any one of embodiments 42-49, wherein the carrier or pharmaceutical formulation is long acting to treat a disease associated with optic nerve injury.

51. The method of any one of embodiments 42-50, wherein said carrier or pharmaceutical preparation increases the survival rate of ganglion cells and/or their axons.

52. The method of any one of embodiments 42-51, wherein said carrier or pharmaceutical preparation prevents or delays apoptosis of ganglion cells.

53. The method of any one of embodiments 42-52, wherein the carrier or pharmaceutical preparation prevents or delays a reduction in the thickness of a retinal ganglion cell layer.

54. The method of any one of embodiments 42-53, wherein the carrier or pharmaceutical formulation prevents or delays a reduction in the thickness of the retinal nerve fiber layer.

55. The method according to any one of embodiments 42-54, wherein said carrier or pharmaceutical formulation prevents or delays the progression of abnormal visual field conditions.

56. The method according to any one of embodiments 42-53, which does not cause a significant inflammatory response or complication in the eye.

Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion proteins, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.

Examples

Example 1 vector design construction, expression and viral packaging, assay

As shown in FIG. 1, the vector genome comprises inverted terminal repeats (AAV ITRs) of adeno-associated virus and nucleotide sequences (SEQ ID NO: 6) of three different genes of interest (the gene encoding ciliary neurotrophic factor CNTF protein, SEQ ID NO: 1), (the gene encoding insulin-like growth factor IGF-1, SEQ ID NO: 2) and (the gene encoding osteopontin OPN, SEQ ID NO: 3) Open Reading Frame (ORF) regions encoded in tandem. Transcription was initiated using a strong CMV promoter and SV40polyA increased transcript stability. The P2A peptide self-cutting sequence (SEQ ID NO: 4) is used for connecting three target genes, so that the three genes can be simultaneously independently and efficiently expressed. Adding two restriction sites Xba I and BglII at two ends of the synthesized gene sequence, carrying out Xba I and BglII double restriction with the pAAV-MCS plasmid vector respectively, recovering restriction products, and connecting with T4DNA ligase overnight. And transforming the connecting product into competent cells, selecting a monoclonal to perform bacterial liquid sequencing, and performing sequence comparison analysis on a sequence obtained by sequencing to successfully obtain the recombinant adeno-associated virus plasmid pAAV-CNTF/IGF1/OPN with a correct sequence.

HEK293 cells were expanded and plated in 6-well plates with 3 replicate wells in parallel per group. 3 micrograms of plasmid were transfected per well at 85% confluence in HEK293 cells (transfection reagent PEI was used for transfection). And collecting cells as an infected group 48 hours after transfection, taking untransfected cells as a blank group, extracting RNA by a Rizol method, performing reverse transcription to form cDNA, and quantitatively detecting the expression level of the vector by a qPCR method.

As shown in FIG. 2, the mRNA levels of the genes encoding ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1 and osteopontin OPN in the infected group were increased by about 1500-fold compared to the blank group.

The three-plasmid system (pAAV2-CNTF/2A/IGF1/2A/OPN, pHelper, pRC) is transfected when the confluence of HEK293 cells is 85% (PEI transfection reagent is used for transfection). 10 cm culture dishes are transferred with 30 micrograms of plasmids (3 plasmid proportions 10:10:10), liquid is not changed on the day of transfection, 5% FBS DMEM is changed on the next day, cells are collected for 72 hours, the cells are put into a freezing tube, repeated freezing and thawing is carried out for 3 times at 70 ℃/37 ℃ for at least 10 minutes, after each dissolution, the cells are vigorously vortexed for 2 minutes, then the cells are centrifuged for 5 minutes at 14000 rpm, the supernatant is taken, the viruses are purified by gradient centrifugation of cesium chloride solution, and the fluorescence quantitative PCR method is used for detecting the physical titer of the recombinant nucleic acids rAAV-CNTF/IGF1/OPN, namely 1.5 × 10¹²vg/mL。

The rAAV-CNTF vector and nucleic acid, and AAV-mCherry empty-shell virus vector and nucleic acid preparation method refer to the above example 1.

Example 2 mouse model of glaucoma

Experimental SPF-grade DBA/2J mice (purchased in Jackson lab, USA) were bred under standard photoperiod conditions, with C57/BL6 mice as controls. The intraocular pressure of the mice was measured once a week, and the measurement time was fixed at 9-11 am.

As shown in FIG. 3, the DBA/2J mice exhibited elevated ocular pressure from month 7 and persisted.

DBA/2J mice 7 months old and with an intraocular pressure of >18mmHg were selected and randomized into 3 groups, blank (PBS), experimental group A (control nucleic acid rAAV-CNTF) and experimental group B (recombinant nucleic acid rAAV-CNTF/IGF1/OPN as described herein). After anesthetizing the mice by intraperitoneal injection of 5% chloral hydrate, the outside of the eyes and eyeballs of the mice are cleaned and disinfected. The insulin needle is used for opening a hole at the edge of a mouse horn, and the micro-syringe injects 1-2 microliters of the recombinant nucleic acid preparation in the application in the vitreous cavity, and the recombinant nucleic acid preparation consists of the recombinant nucleic acid in the application and a pharmaceutically acceptable carrier or excipient. Mice were raised in standard environment after surgery and had free diet.

After the operation, the eyes of all mice have no obvious abnormality, no conjunctival congestion, no secretion and no endophthalmitis by using an ophthalmoscope every 2 days, which shows that the intravitreal injection 1 × 10¹⁰The vp (total number of viral particles) dose of the recombinant nucleic acid described herein is safe.

Experimental mice were sacrificed 2 months after intravitreal injection, in each case blank (PBS), experimental group a (control nucleic acid rAAV-CNTF) and experimental group B (recombinant nucleic acid rAAV-CNTF/IGF1/OPN described herein), eyeballs were removed, retinas were rapidly detached on ice, and stored at-80 ℃ after rapid freezing with liquid nitrogen. Following lysis of retinal tissue by RIPA lysate, immunoblotting (WB) was performed.

As shown in FIG. 4, the mouse retinas of both experimental groups A and B detected overexpression of the ciliary neurotrophic factor CNTF protein (FIG. 4a), and the expression level was almost the same. Overexpression of insulin-like growth factor IGF-1 and osteopontin OPN was detected only in the retinas of mice of experimental group B (fig. 4B and 4 c). The AAV-CNTF/IGF1/OPN polygene vector successfully expresses three target proteins in mouse retina, and compared with the AAV-CNTF monogene vector, the protein expression level is not influenced.

The mice killed by the broken ridges are respectively a normal group (DBA/2J mice group at month 6), a blank group (PBS group), an experimental group A (control nucleic acid rAAV-CNTF group) and an experimental group B (recombinant nucleic acid rAAV-CNTF/IGF1/OPN group described in the application), wherein the blank group, the experimental group A and the experimental group B are operated at month 7, and are killed and the materials are obtained and detected at month 9. The eyeball is taken out and fixed in the fixing solution for 20 minutes, then the retina is stripped out and cut into 4-petal shape, and 4% paraformaldehyde is fixed at 4 ℃ overnight. Punching 1% TritonX-100 for 2 hours, and sealing with sealing liquid for 1 hour. The TUJ1 antibody (knob-specific antibody) was incubated overnight at 4 ℃ for the primary antibody and 2 hours at room temperature for the secondary antibody. Slides were mounted 15 min after DAPI staining of nuclei. And (3) respectively selecting 2 peripheral visual fields (3 mm from the optic disc) and 1 central visual field (1 mm from the optic disc) of the temporal quadrant, the upper quadrant, the nasal quadrant and the lower quadrant of the retina under a fluorescence microscope for photographing and counting. And (3) collecting 12 visual field images of each retina, counting the number of all node cells in the visual field, and calculating the number of retinal ganglion cells in unit area.

As shown in FIG. 5, the Retinal Ganglion Cell (RGC) density at month 9 was significantly lower than that at month 6 in DBA/2J mice, indicating that ocular hypertension caused damage to the optic nerve and death of ganglion cells in the mice. The survival rate of the retinal ganglion cells of the mice in the experimental group B is obviously higher than that of the blank group (improved by 28 percent), which shows that the expression of the ciliary neurotrophic factor CNTF protein in the retina has certain protective effect on the ganglion cells, and the protective effect is not related to the level of intraocular pressure. Wherein, the protective effect of three target proteins of the expression of the ciliary neurotrophic factor CNTF protein, the insulin-like growth factor IGF-1 and the osteopontin OPN on the ganglion cells is stronger than that of the expression of the ciliary neurotrophic factor CNTF protein alone.

After the mice were sacrificed by cutting the spine, the normal group (DBA/2J mice group at month 6), the blank group (PBS group), the experimental group A (control nucleic acid rAAV-CNTF group) and the experimental group B (recombinant nucleic acid rAAV-CNTF/IGF1/OPN group) were obtained, and the optic nerve was immediately cut from the retrobulbar position, the skull was carefully opened to remove the brain tissue, and the entire optic nerve length was dissected. The optic nerve was then washed once in PBS, fixed by immersion in 5% glutaraldehyde, and then fixed with 1% osmium tetroxide for 180 minutes. After fixation, the optic nerve was washed, dehydrated in graded alcohol (70% for 15 minutes; 95% for 15 minutes; 100% for 3X 15 minutes) and immersed in graded propylene oxide for 2 days. Upon the last infusion of the gradient propylene oxide, the diluted resin was replaced with 100% resin and the optic nerve was placed in a mold and heated to 65 ℃ for 48 hours. Sections of the optic nerve 1 micron thick were transected using a Leica EMUC6 microtome (Leica Microsystems) and collected fixed on polylysine coated slides. After drying and adhering to the slide, the half thin sections were etched to remove the resin surrounding the nerves (soaked for 3 seconds in a mixed solution of 10 ml of propylene oxide, 10 ml of 100% ethanol, 10 g of NaOH), and stained with toluidine blue. Finally, washing the film by distilled water and sealing the film. The entire cross section of the optic nerve was imaged using a bright field microscope with Lucia G software and a 10-fold objective. Three high resolution representative images of each optic nerve were then captured using a 100-fold oil immersion objective. Axons in these images were blind counted using Image-J to estimate the total number of axons remaining.

As shown in FIG. 6, the survival rate of optic nerve of mice in experimental group B is significantly higher than that of blank group (increased by 25%), and the optic nerve protection effect of the mice expressing the ciliary neurotrophic factor CNTF protein, the insulin-like growth factors IGF-1 and the osteopontin OPN is better than that of the mice expressing the ciliary neurotrophic factor CNTF protein alone.

Example 3 Rabbit eyeball model

10 New Zealand white rabbits were divided into 2 groups, a blank group (PBS group) and an infected group (the recombinant nucleic acid rAAV-CNTF/IGF1/OPN group described in this application), 50 microliters of PBS and rAAV-CNTF/IGF1/OPN were respectively aspirated to penetrate the pars plana of the ciliary body 3 mm from the limbus of the corneal tissue and enter the vitreous cavity, and intravitreal injection was performed. The eye state of the mice was observed daily in standard environmental culture, free diet.

The two groups of rabbits were subjected to slit lamp and intraocular pressure examination at 1, 3, 7 and 30 days after operation. All rabbits had no obvious abnormality, no conjunctival congestion, no secretion, no endophthalmitis, and no increase in intraocular pressure.

As shown in fig. 7, fundus photographic results for one month after the operation. There were no significant complications or damage to retinal vessels and optic nerves in all rabbits, indicating that the normal standard intravitreal injection did not produce significant inflammatory responses or other complications.

As shown in FIG. 8, H & E staining of tissue sections revealed no abnormalities in retinal hierarchy in all rabbits.

The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.

Sequence listing

<110> Wuhan Newcastle Biotechnology Ltd

<120> multi-neurotrophic factor combined expression vector and application thereof

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Asn Leu Asp Ser Ala Asp Gly Met Pro Val Ala Ser Thr Asp Gln Trp

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Ser Glu Leu Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr

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Arg Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val

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His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu

100 105 110

Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met Ile

115 120 125

Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gly Met Pro Ile

130 135 140

Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys

145 150 155 160

Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu

165 170 175

Arg Phe Ile Ser Ser His Gln Thr Gly Ile Pro Ala Arg Gly Ser His

180 185 190

Tyr Ile Ala Asn Asn Lys Lys Met

195 200

<210>8

<211>158

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>IGF1

<400>8

Met Gly Lys Ile Ser Ser Leu Pro Thr Gln Leu Phe Lys Cys Cys Phe

1 5 10 15

Cys Asp Phe Leu Lys Val Lys Met His Thr Met Ser Ser Ser His Leu

20 25 30

Phe Tyr Leu Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala

35 40 45

Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe

50 55 60

Val CysGly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly

65 70 75 80

Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys

85 90 95

Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu

100 105 110

Lys Pro Ala Lys Ser Ala Arg Ser Val Arg Ala Gln Arg His Thr Asp

115 120 125

Met Pro Lys Thr Gln Lys Tyr Gln Pro Pro Ser Thr Asn Lys Asn Thr

130 135 140

Lys Ser Gln Arg Arg Lys Gly Ser Thr Phe Glu Glu Arg Lys

145 150 155

<210>9

<211>327

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>OPN

<400>9

Met Gly Ile Val Pro Arg Ser Leu Asp Lys Lys Ala His Arg Val Gln

1 5 10 15

Phe Gln Leu Asn Arg Ile Lys Ala Lys Ile Glu Leu Pro Trp Gly Ser

20 25 30

Leu Gln LeuAsp Cys Leu Met Lys Thr Leu Lys Glu Leu Tyr Asn Lys

35 40 45

Tyr Pro Asp Ala Val Ala Thr Trp Leu Asn Pro Asp Pro Ser Gln Lys

50 55 60

Gln Asn Leu Leu Ala Pro Gln Asn Ala Val Ser Ser Glu Glu Thr Asn

65 70 75 80

Asp Phe Lys Gln Glu Thr Leu Pro Ser Lys Ser Asn Glu Ser His Asp

85 90 95

His Met Asp Asp Met Asp Asp Glu Asp Asp Asp Asp His Val Asp Ser

100 105 110

Gln Asp Ser Ile Asp Ser Asn Asp Ser Asp Asp Val Asp Asp Thr Asp

115 120 125

Asp Ser His Gln Ser Asp Glu Ser His His Ser Asp Glu Ser Asp Glu

130 135 140

Leu Val Thr Asp Phe Pro Thr Asp Leu Pro Ala Thr Glu Val Phe Thr

145 150 155 160

Pro Val Val Pro Thr Val Asp Thr Tyr Asp Gly Arg Gly Asp Ser Val

165 170 175

Val Tyr Gly Leu Arg Ser Lys Ser Lys Lys Phe Arg Arg Pro Asp Ile

180 185 190

Gln Tyr Pro Asp Ala ThrAsp Glu Asp Ile Thr Ser His Met Glu Ser

195 200 205

Glu Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro Val Ala Gln Asp Leu

210 215 220

Asn Ala Pro Ser Asp Trp Asp Ser Arg Gly Lys Asp Ser Tyr Glu Thr

225 230 235 240

Ser Gln Leu Asp Asp Gln Ser Ala Glu Thr His Ser His Lys Gln Ser

245 250 255

Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser Asn Glu His Ser Asp

260 265 270

Val Ile Asp Ser Gln Glu Leu Ser Lys Val Ser Arg Glu Phe His Ser

275 280 285

His Glu Phe His Ser His Glu Asp Met Leu Val Val Asp Pro Lys Ser

290 295 300

Lys Glu Glu Asp Lys His Leu Lys Phe Arg Ile Ser His Glu Leu Asp

305 310 315 320

Ser Ala Ser Ser Glu Val Asn

325

<210>10

<211>22

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>P2A

<400>10

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210>11

<211>21

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>T2A

<400>11

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

Claims

1. A recombinant nucleic acid, wherein the recombinant nucleic acid comprises:

a first nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein;

a second nucleotide sequence encoding an insulin-like growth factor IGF-1 protein; and

a third nucleotide sequence encoding osteopontin OPN.

2. The recombinant nucleic acid of claim 1, wherein:

the sequence of the ciliary neurotrophic factor CNTF protein comprises a protein sequence selected from the following group:

a) the protein sequence is similar to SEQ ID NO:7 are the same;

3. The recombinant nucleic acid of claim 1 or 2, wherein:

said first nucleotide sequence comprises a nucleotide sequence selected from the group consisting of:

a) the first nucleotide sequence is identical to SEQ ID NO:1 are the same;

4. The recombinant nucleic acid of any one of claims 1-3, wherein:

the sequence of the insulin-like growth factor IGF-1 protein comprises a protein sequence selected from the group consisting of:

a) the protein sequence is similar to SEQ ID NO:8 are the same;

5. The recombinant nucleic acid of any one of claims 1-4, wherein:

said second nucleotide sequence comprises a nucleotide sequence selected from the group consisting of:

a) and the second nucleotide sequence is identical to SEQ ID NO:2 are the same;

6. The recombinant nucleic acid of any one of claims 1-5, wherein:

the sequence of osteopontin OPN comprises a protein sequence selected from the group consisting of:

a) the protein sequence is similar to SEQ ID NO:9 are the same;

7. The recombinant nucleic acid of any one of claims 1-6, wherein:

the third nucleotide sequence comprises a nucleotide sequence selected from the group consisting of:

a) and the third nucleotide sequence is similar to SEQ ID NO:3 are the same;

8. The recombinant nucleic acid of any one of claims 1-7, comprising a self-cleaving polypeptide-encoding nucleotide sequence of a different origin between two adjacent of said nucleotide sequences to reduce the probability of homologous recombination.

9. The recombinant nucleic acid of claim 8, wherein the self-cleaving polypeptide is a 2A peptide, preferably the self-cleaving polypeptide is a P2A peptide (SEQ ID NO:10) and a T2A peptide (SEQ ID NO:11), respectively.

10. The recombinant nucleic acid of claim 8 or 9, wherein the nucleotide sequence encoding the self-cleaving polypeptide and the nucleotide sequence are directly linked without an additional nucleotide sequence therebetween.

11. The recombinant nucleic acid of any one of claims 1-10, wherein the three nucleotide sequences are in the order of, in the 5 'to 3' direction, the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence, in tandem.

12. The recombinant nucleic acid of claim 11, wherein the recombinant nucleic acid is selected from the group consisting of nucleotide sequences of seq id no:

a) the nucleotide sequence is similar to SEQ ID NO:6 are the same;

13. A vector comprising the recombinant nucleic acid of any one of claims 1-11.

14. The vector of claim 13, wherein the recombinant nucleic acid comprises an AAV inverted terminal repeat ITR.

15. The vector of claim 13 or 14, wherein the vector is selected from adeno-associated viral vectors.

16. The vector of claim 15, wherein the adeno-associated virus is selected from AAV2, AAV5, AAV7 or AAV8 or a combination thereof.

17. The vector according to any one of claims 13 to 16, wherein the nucleotide sequence further comprises a promoter or enhancer, preferably wherein the promoter is selected from CMV, CAG or SYN.

18. The vector according to any one of claims 13 to 17, wherein said vector encodes the ciliary neurotrophic factor CNTF protein, the insulin-like growth factor IGF-1 and the osteopontin OPN independently in the form of a non-fusion protein upon expression.

19. Use of a vector according to any one of claims 13 to 18 for the treatment of a disease associated with optic nerve injury.

20. The use of claim 19, wherein the disease associated with optic nerve injury is acute or chronic optic nerve injury.

21. The use according to claim 19, wherein the disease associated with optic nerve damage is DOA, ischemic optic neuropathy, LHON, or glaucoma.

22. The use of claim 19, wherein the disease associated with optic nerve damage is glaucoma.

23. A pharmaceutical formulation comprising the vector of any one of claims 13-18, and a pharmaceutically acceptable excipient.

24. The pharmaceutical formulation of claim 13, wherein the pharmaceutical formulation is a liquid formulation.

25. Use of a pharmaceutical formulation according to claim 23 or 24 for the treatment of a disease associated with optic nerve damage.

26. The use according to claim 25, wherein said pharmaceutical formulation is capable of causing long-lasting high expression of the ciliary neurotrophic factor CNTF protein, the insulin-like growth factor IGF-1 and osteopontin OPN in retinal cells.

27. The use of claim 25 or 26, wherein the pharmaceutical formulation is capable of long-acting treatment of a disease associated with optic nerve damage.

28. The use of any one of claims 25 to 27, wherein the pharmaceutical preparation is capable of increasing the survival rate of ganglion cells and/or their axons.

29. The use of any one of claims 25 to 28, wherein the pharmaceutical formulation prevents or delays apoptosis of ganglion cells.

30. The use according to any one of claims 25 to 29, wherein the disease associated with optic nerve damage is glaucoma.

31. The use according to any one of claims 25 to 30, wherein the disease associated with optic nerve injury is acute or chronic optic nerve injury resulting in injury and/or death of the optic nerve and its ganglion cells.

32. The use of any one of claims 25 to 31, wherein the pharmaceutical formulation is injected intraocularly.

33. The use of claim 32, wherein the intraocular injection is a vitreous cavity injection.

34. The use of any one of claims 25 to 33, wherein intraocular injection of the pharmaceutical formulation does not induce a significant inflammatory response or other complications.

35. A method of treating an ocular disease comprising administering to a patient two or more carriers or pharmaceutical formulations thereof, said two or more carriers comprising:

36. The method of claim 35, wherein said first vector or said second vector further comprises a third recombinant nucleic acid comprising a nucleotide sequence encoding a ciliary neurotrophic factor CNTF protein or a nucleotide sequence encoding an insulin-like growth factor IGF-1 protein.

37. The method of claim 35, wherein said two or more vectors comprise a third vector, said third vector comprising a third recombinant nucleic acid.

38. The method according to claim 36 or 37, wherein said second recombinant nucleic acid comprises a nucleotide sequence encoding the ciliary neurotrophic factor CNTF protein and said third recombinant nucleic acid comprises a nucleotide sequence encoding the insulin-like growth factor IGF-1 protein.

39. The method of claim 36 or 37, wherein said second recombinant nucleic acid comprises a nucleotide sequence encoding insulin-like growth factor IGF-1 protein and said third recombinant nucleic acid comprises a nucleotide sequence encoding ciliary neurotrophic factor CNTF protein.

40. A method of treating an ocular disorder comprising administering to a patient the vector of any one of claims 13-18 or the pharmaceutical formulation of any one of claims 23-24.

41. The method of any one of claims 35-40, wherein the ocular disease is a disease associated with optic nerve damage.

42. The method of claim 41, wherein the disease associated with optic nerve injury is acute or chronic optic nerve injury.

43. The method of claim 41, wherein the optic nerve injury-related disorder is DOA, LHON, ischemic optic neuropathy or glaucoma; preferably, the disease associated with optic nerve damage is glaucoma.

44. The method of any one of claims 35-43, wherein the carrier or pharmaceutical formulation is injected intraocularly.

45. The method of claim 44, wherein the intraocular injection is a vitreous cavity injection.

46. The method of any one of claims 35 to 45, wherein said carrier or pharmaceutical formulation is capable of causing long-term high expression of the ciliary neurotrophic factor CNTF protein, insulin-like growth factor IGF-1 and osteopontin OPN in retinal cells.

47. The method of any one of claims 35-46, wherein the carrier or pharmaceutical formulation is capable of long-acting treatment of a disorder associated with optic nerve injury.

48. The method of any one of claims 35 to 47, wherein the carrier or pharmaceutical agent is capable of increasing the survival rate of ganglion cells and/or their axons.

49. A method as claimed in any one of claims 35 to 48 wherein the carrier or pharmaceutical agent is capable of preventing or delaying the apoptosis of ganglion cells.

50. A method as claimed in any one of claims 35 to 49 wherein the carrier or pharmaceutical agent is capable of preventing or delaying the reduction in the thickness of the retinal ganglion cell layer.

51. The method of any one of claims 35 to 50, wherein the carrier or pharmaceutical agent prevents or delays a reduction in the thickness of the retinal nerve fiber layer.

52. The method of any one of claims 35 to 51, wherein the carrier or pharmaceutical agent prevents or delays the progression of the abnormal visual field condition.

53. The method of any one of claims 35-52, wherein no significant inflammatory response or other complications are induced in the eye.