WO2020082417A1 - 基因治疗Leber遗传性视神经病变 - Google Patents

基因治疗Leber遗传性视神经病变 Download PDF

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WO2020082417A1
WO2020082417A1 PCT/CN2018/113799 CN2018113799W WO2020082417A1 WO 2020082417 A1 WO2020082417 A1 WO 2020082417A1 CN 2018113799 W CN2018113799 W CN 2018113799W WO 2020082417 A1 WO2020082417 A1 WO 2020082417A1
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associated virus
polypeptide
recombinant adeno
drug
another preferred
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PCT/CN2018/113799
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English (en)
French (fr)
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李斌
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武汉纽福斯生物科技有限公司
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Priority to JP2021521870A priority Critical patent/JP2021529001A/ja
Priority to CN202110786772.2A priority patent/CN113528510A/zh
Priority to CN201980003485.0A priority patent/CN110876269B/zh
Priority to MX2020013772A priority patent/MX2020013772A/es
Priority to CN202110786630.6A priority patent/CN113476484A/zh
Priority to EP19826653.8A priority patent/EP3814492A4/en
Priority to SG11202012044QA priority patent/SG11202012044QA/en
Priority to KR1020217001385A priority patent/KR102627561B1/ko
Priority to PCT/CN2019/094136 priority patent/WO2020001657A1/en
Priority to KR1020247001775A priority patent/KR20240014102A/ko
Priority to AU2019296451A priority patent/AU2019296451B2/en
Priority to CA3103740A priority patent/CA3103740A1/en
Priority to BR112020026361-3A priority patent/BR112020026361A2/pt
Priority to SG11202101032VA priority patent/SG11202101032VA/en
Priority to AU2019323434A priority patent/AU2019323434A1/en
Priority to PCT/CN2019/101538 priority patent/WO2020038352A1/en
Priority to CN201980054770.5A priority patent/CN112584874A/zh
Priority to JP2021509893A priority patent/JP7403852B2/ja
Priority to CA3109432A priority patent/CA3109432A1/en
Priority to EP19853225.1A priority patent/EP3840785A4/en
Priority to KR1020217007727A priority patent/KR20210068014A/ko
Priority to US16/836,644 priority patent/US11034954B2/en
Publication of WO2020082417A1 publication Critical patent/WO2020082417A1/zh
Priority to US17/181,849 priority patent/US11352645B2/en
Priority to US17/317,295 priority patent/US20220340895A1/en
Priority to US17/320,388 priority patent/US11332741B1/en
Priority to AU2021204690A priority patent/AU2021204690A1/en
Priority to US17/726,833 priority patent/US20220259619A1/en
Priority to JP2023029170A priority patent/JP2023078173A/ja
Priority to JP2023205807A priority patent/JP2024028861A/ja
Priority to AU2023285773A priority patent/AU2023285773A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the invention relates to the field of gene therapy, in particular to gene therapy Leber hereditary optic neuropathy.
  • Leber hereditary optic neuropathy is a genetic disease that mainly affects the macular papillary tract fibers and causes degeneration of the optic nerve. It is a maternal genetic disease whose etiology and mechanism have not been elucidated. It was first reported in 1858 and in 1871 He was named after Leber, the first German scholar to describe its clinical features. LHON is relatively rare, and the clinical manifestations are simultaneous or successive acute or subacute painless vision loss in both eyes, which may be accompanied by loss of central visual field and color vision impairment. At present, neurotrophic drugs or traditional Chinese medicine are often used for treatment, but the efficacy is not good enough. Because there is no effective treatment, only a few can get better once the onset occurs, and most patients end up with blindness. Therefore, since the discovery of the disease, it has attracted the attention of some doctors and has been exploring the etiology, mechanism and treatment.
  • HGP Human Genome Project
  • LHON is characterized by multiple young adults, incomplete penetrance, and gender bias; the incidence rate in Asia is about 1.18 / 10000, the primary mutation site m.11778G> A mutation rate accounts for about 87% -92.9%, and China One mutation rate is about 90%. In other words, there are about 150,000 cases of LHON with m.11778G> A mutation in the primary mutation site in China.
  • the object of the present invention is to provide a safer method and medicine for gene therapy eye.
  • a pharmaceutical combination comprising:
  • a first drug containing (a) a first active ingredient, the first active ingredient is a recombinant adeno-associated virus, the recombinant adeno-associated virus comprises an exogenous nucleic acid encoding a polypeptide, the polypeptide Polypeptides for restoring or enhancing the function of optic nerve or retinal cells; and (b) pharmaceutically acceptable carriers; and
  • the restoration or enhancement of optic nerve or retinal cell function includes treatment or improvement of optic nerve or retinal (cell) disease, and / or restoration or enhancement of the subject's vision.
  • the retinal cells are photoreceptors (including cone cells and / or rod cells), retinal ganglion cells, Mueller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment Epithelial Cells.
  • the polypeptide is a polypeptide that restores or enhances the function of the optic nerve, preferably a polypeptide for treating or improving optic neuropathy, and more preferably a polypeptide for treating or improving Leber's hereditary optic neuropathy.
  • polypeptide is selected from the group consisting of ND1, ND4, ND6, or a combination thereof.
  • polypeptide is ND4.
  • the ND4 coding sequence is shown in SEQ ID NO.:7.
  • the content of the recombinant adeno-associated virus is 1 ⁇ 10 7 vg / 0.05ml-1 ⁇ 10 14 vg / 0.05ml, preferably 1 ⁇ 10 9 vg / 0.05ml-1 ⁇ 10 12 vg / 0.05ml, more preferably 1 ⁇ 10 10 vg / 0.05ml-1 ⁇ 10 11 vg / 0.05ml.
  • the recombinant adeno-associated virus further contains the coding sequence of the mitochondrial targeting peptide.
  • the coding sequence of the mitochondrial targeting peptide includes: COX8 coding sequence (including optimized or unoptimized), COX10 coding sequence, OPA1 coding sequence, or a combination thereof.
  • the COX8 coding sequence has the sequence shown in SEQ ID NO.:1.
  • the optimized COX10 coding sequence has the sequence shown in SEQ ID NO .: 2.
  • the unoptimized COX10 coding sequence has the sequence shown in SEQ ID NO.:3.
  • the coding sequence of OPA1 has the sequence shown in SEQ ID NO.:4.
  • the recombinant adeno-associated virus contains a fusion nucleic acid, and the fusion nucleic acid has a structure of formula I from the 5 'end to the 3' end:
  • Each "-" is independently a bond or nucleotide linking sequence
  • Z0 is none, or 5'-UTR sequence
  • Z1 is the coding sequence of mitochondrial targeting peptide
  • Z2 is the coding sequence of the polypeptide (ie, the exogenous nucleic acid encoding the polypeptide).
  • Z3 is a 3'-UTR sequence.
  • each nucleotide linking sequence is 0-21 nt, preferably 3-12 nt.
  • the coding sequence of the mitochondrial targeting peptide is an optimized COX10 coding sequence.
  • the structure of the fusion nucleic acid from the 5'-3 'end is COX10-ND4-UTR.
  • the fusion nucleic acid sequence is shown in SEQ ID NO .: 5 or 6.
  • sequence is as shown in SEQ ID NO .: 5 or 6 in the fusion nucleic acid
  • Bits 1-84 are the COX10 coding sequence
  • Position 85-1464 is the nucleotide sequence encoding human NADH dehydrogenase subunit 4 protein
  • Positions 1465-2889 are 3'-UTR sequences.
  • the serotype of the recombinant adeno-associated virus is selected from: AAV2, AAV5, AAV7, AAV8, or a combination thereof; preferably AAV2.
  • the dosage form of the first drug is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.
  • the dosage form of the first drug is an injection dosage form.
  • the second drug is administered intravenously, subcutaneously, orally, or a combination thereof.
  • the dosage form of the second drug is preferably an oral preparation.
  • the drug combination further includes: (C) a third drug, and the third drug includes: sodium creatine phosphate, methylprednisolone, or a combination thereof.
  • the pharmaceutical combination contains:
  • the first drug which contains (a) a therapeutically effective amount of the first active ingredient, and (b) a pharmaceutically acceptable carrier;
  • the drug combination is used to restore the subject's vision or treat eye diseases.
  • the first drug and the second drug in the drug combination are independent of each other.
  • a kit includes:
  • the first preparation is a recombinant adeno-associated virus
  • the recombinant adeno-associated virus contains an exogenous nucleic acid encoding a polypeptide
  • the polypeptide is a polypeptide that restores or enhances the function of the optic nerve or retinal cells
  • the kit further includes a label or instructions, the label or instructions indicate the following: before and / or after the first formulation is administered, the second formulation is administered to the desired subject.
  • first preparation and the second preparation are independent of each other.
  • the dosage form of the first formulation is selected from the group consisting of lyophilized formulations, liquid formulations, or a combination thereof.
  • the dosage form of the pharmaceutical preparation is an injection dosage form.
  • the administration method of the second formulation includes intravenous administration, subcutaneous administration, oral administration, or a combination thereof.
  • the dosage form of the second preparation is preferably an oral preparation.
  • a third aspect of the present invention there is provided the use of the pharmaceutical combination according to the first aspect of the present invention, which is used to prepare a medicament or preparation for restoring or improving the vision of a subject and / or treating eye diseases .
  • the restoration or enhancement of the subject's vision includes restoration or enhancement of the optic nerve or retinal cell function.
  • the ocular disease is optic neuropathy, glaucoma, retinitis pigmentosa, macular degeneration, retinal schizophrenia, Leber's congenital cataract, diabetic retinopathy, total color blindness or color blindness, Degenerative retinal diseases, or a combination thereof.
  • the eye disease is optic neuropathy.
  • the formulation or composition is used to treat optic neuropathy, preferably hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).
  • optic neuropathy preferably hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).
  • LHON Leber's hereditary optic neuropathy
  • the formulation or composition is used to expand or restore the photoreceptor function of the retina, restore the subject's vision (or photosensitive ability), and / or treat degenerative diseases of the retina.
  • the degenerative retinal disease is selected from the group consisting of retinal dystrophy (eg rod dystrophy, rod cone dystrophy, cone rod dystrophy, cone dystrophy or macular dystrophy) ), Retinal or macular degeneration, retinitis pigmentosa, other diseases, or a combination thereof.
  • retinal dystrophy eg rod dystrophy, rod cone dystrophy, cone rod dystrophy, cone dystrophy or macular dystrophy
  • Retinal or macular degeneration retinitis pigmentosa
  • other diseases or a combination thereof.
  • a method for treating ocular diseases comprising the steps of: administering to a subject in need thereof, the drug combination according to the first aspect of the present invention or the drug according to the second aspect of the present invention box.
  • other active substances for treating eye diseases are used in combination before, simultaneously and / or after using the drug combination or the kit.
  • a treatment method includes the steps of:
  • Metsola is administered to the subject before, simultaneously and / or after administration of the recombinant adeno-associated virus into the eye of the subject.
  • the recombinant adeno-associated virus is a recombinant adeno-associated virus that has the function of restoring or improving the vision of the subject and / or treating eye diseases.
  • the recombinant adeno-associated virus contains an exogenous nucleic acid encoding a polypeptide, and the polypeptide is a polypeptide that restores or enhances the function of the optic nerve or retinal cells.
  • the administration method of the Metsola includes: intravenous administration, subcutaneous administration, oral administration, or a combination thereof.
  • the administration method of the Metso is oral administration.
  • the treatment method includes the steps of:
  • Pre-operative pre-treatment which includes administering Metsola to the desired subject;
  • the recombinant adeno-associated virus comprising an exogenous nucleic acid encoding a polypeptide, the polypeptide being a polypeptide that restores or enhances the function of the optic nerve or retinal cells;
  • the subject is a human or non-human mammal.
  • the method of administration of Metsola in the preoperative pretreatment is oral administration.
  • the amount of Metso administered is 28-36 mg / 60 kg body weight each time, preferably 30-34 mg / 60 kg body weight each time.
  • the administration interval of the Metso is at least once every three days, preferably at least once every two days, and more preferably at least once a day.
  • the application frequency of the Metso is at least 1, preferably not less than 3 times, preferably not less than 5 times, more preferably not less than 7 times.
  • the administration time of the Metso is at least 3 days, preferably not less than 5 days, preferably not less than one week.
  • the preoperative pretreatment includes:
  • Metso is administered to the desired subject at an interval of once a day, at an amount of 30-34 mg / 60 kg body weight each time, for at least one week.
  • the pre-operative pre-treatment further includes topical (eye) administration of antibiotic eye drops and / or ointment one day before the operation.
  • step (2) the recombinant adeno-associated virus is administered intraocularly, preferably by subretinal, direct retina, suprachoroidal, or intravitreal injection.
  • the recombinant adeno-associated virus is administered by intravitreal injection.
  • the administration of the recombinant adeno-associated virus is one-eye injection or two-eye injection.
  • the application amount of the recombinant adeno-associated virus is 1 ⁇ 10 7 vg / 0.05ml-1 ⁇ 10 14 vg / 0.05ml, preferably 1 ⁇ 10 9 vg / 0.05ml-1 ⁇ 10 12 vg / 0.05ml, more preferably 1 ⁇ 10 10 vg / 0.05ml-1 ⁇ 10 11 vg / 0.05ml.
  • the post-operative treatment further includes: administering sodium creatine phosphate and / or methylprednisolone to the subject before administering Metsola.
  • the administration method of the sodium creatine phosphate and / or methylprednisolone includes intravenous administration, subcutaneous administration, oral administration, or a combination thereof.
  • the sodium creatine phosphate and / or methylprednisolone are administered intravenously.
  • the recombinant adeno-associated virus is administered first, followed by sodium creatine phosphate; the (first) application interval between the recombinant adeno-associated virus and the sodium creatine phosphate is at most 24 hours, preferably 4-20h, better 6-18h, better 10-14h.
  • the application amount of the sodium creatine phosphate is 1.5-2.5 g / 60 kg body weight each time, preferably 1.8-2.2 g / 60 kg body weight each time.
  • the application interval of the sodium creatine phosphate is at least once every two days, preferably at least once a day.
  • the application frequency of the sodium creatine phosphate is at least 1 time, preferably not less than 2 times.
  • the application time of the sodium creatine phosphate is at least 1 day, preferably 2-4 days.
  • the recombinant adeno-associated virus is administered first, followed by methylprednisolone; the (first) application interval between the recombinant adeno-associated virus and the methylprednisolone is at most 24h, preferably 4 -20h, better 6-18h, better 10-14h.
  • the administration amount of methylprednisolone is 70-90 mg / 60 kg body weight each time, preferably 75-85 mg / 60 kg body weight each time, preferably 78-82 mg / 60 kg body weight each time.
  • the administration interval of the methylprednisolone is at least once every two days, preferably at least once a day.
  • the application frequency of the methylprednisolone is at least 1 time, preferably not less than 2 times.
  • the administration time of the methylprednisolone is at least 1 day, preferably 2-4 days.
  • step (3) within one week of administration of recombinant adeno-associated virus, the amount of Metso administered is 28-36 mg / 60 kg body weight each time, preferably 30-34 mg / time 60kg body weight.
  • step (3) the application amount of Metso decreases weekly, and the decreasing amount is 1-10 mg / 60 kg body weight, preferably 2-8 mg / 60 kg body weight.
  • the administration interval of the Metso is at least once every three days, preferably at least once every two days, and more preferably at least once a day.
  • the administration time of the Metso is at least 2 weeks, preferably 3 weeks to 10 weeks, more preferably 4 weeks to 7 weeks.
  • step (3) the administration method of the Metso is oral administration.
  • postoperative treatment includes:
  • sodium creatine phosphate and methylprednisolone are administered to the desired subjects at an interval of once a day for 3 days, wherein
  • the application amount of sodium is 1.8-2.2g / 60kg body weight each time, and the application amount of methylprednisolone is 78-82mg / 60kg body weight each time;
  • Metsola is administered at a dose of 30-34mg / 60kg body weight each time, and the application amount is reduced weekly, the decrement is 2-8mg / 60kg body weight, once a day, preferably For at least 4 weeks.
  • a use of Metso for preparing a formulation or composition for reducing side effects or complications of intraocular administration of recombinant adeno-associated virus is provided.
  • the side effects or complications include: cataract extraction, uveitis, vitreitis, keratitis, anterior chamber inflammation, vitreous hemorrhage, eye pain, allergic conjunctivitis, ocular hypertension, or combination.
  • the reduction is a preventive reduction.
  • the formulation or composition is used to reduce the incidence of side effects or complications of intraocular administration of recombinant adeno-associated virus.
  • the formulation or composition is also used to increase the therapeutic effect of the recombinant adeno-associated virus.
  • the therapeutic effect is to improve or restore the subject's vision and / or to treat eye diseases.
  • the therapeutic effect is the effect of treating eye diseases selected from the group consisting of:
  • Optic neuropathy Optic neuropathy, glaucoma, retinitis pigmentosa, macular degeneration, retinal schizophrenia, Leber's congenital cataract, diabetic retinopathy, panchromatic or color blindness, retinal degenerative diseases, or a combination thereof.
  • Figure 1 shows a schematic diagram of the function of ND4 fusion protein with MTS sequence entering into mitochondria.
  • the inventor unexpectedly discovered that the administration of Metsola before and / or after gene therapy (ie, administration of the recombinant adeno-associated virus described in the present invention) can not only significantly improve the therapeutic effect, but also Effectively prevent and / or treat complications of gene therapy, such as selective cataract extraction, uveitis, anterior chamber inflammation, and vitreitis.
  • the present invention has been completed.
  • the inventor carried out randomized multi-center clinical research in accordance with relevant national laws and regulations, clinical trials were approved by the ethics committee, and informed consent was signed with patients, gene therapy for LHON patients, and regular safety inspections, except for a small amount of high intraocular pressure It can recover by itself without any serious complications and has high safety. This study confirmed the safety and effectiveness of the gene therapy of this research team.
  • Gene therapy uses ectopic expression technology, as shown in Figure 1, that is, adeno-associated virus will bring foreign nucleic acid (such as ND4 gene) to the cell to express normal protein, and the small peptide chain anchored in front can guide the target protein It enters the mitochondria and replaces defective proteins caused by genetic mutations to perform normal physiological functions.
  • adeno-associated virus will bring foreign nucleic acid (such as ND4 gene) to the cell to express normal protein, and the small peptide chain anchored in front can guide the target protein It enters the mitochondria and replaces defective proteins caused by genetic mutations to perform normal physiological functions.
  • the terms "containing” or “including (including)” may be open, semi-closed, and closed. In other words, the term also includes “consisting essentially of” or “consisting of”.
  • administering refers to physically introducing the product of the present invention into a subject using any of various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, and peritoneal Internal, spinal cord or other parenteral routes of administration, for example by injection or infusion.
  • Before surgery means before administration of the recombinant adeno-associated virus of the present invention.
  • Postoperative refers to after administration of the recombinant adeno-associated virus of the present invention.
  • Metso that is, methylprednisolone tablets, the main component is: methylprednisolone, and its chemical name is: 11 ⁇ , 17,21-trihydroxy-6 ⁇ -methylpregnant-1,4-diene-3,20- Dione.
  • Molecular formula C 22 H 30 O 5 , molecular weight: 374.48. Its structural formula is shown as formula II:
  • the present invention has found through extensive research and clinical experiments that Metsol not only can improve the therapeutic effect of recombinant adeno-associated virus, but also can effectively prevent and / or treat complications of gene therapy, such as cataract extraction, uveitis, anterior chamber Inflammation, vitreitis, keratitis, vitreous hemorrhage, eye pain, allergic conjunctivitis, high intraocular pressure, etc.
  • the recombinant adeno-associated virus can restore or improve the vision of the subject and / or treat the eye diseases.
  • the recombinant adeno-associated virus is as described in the first aspect of the present invention, the recombinant adeno-associated virus comprises an exogenous nucleic acid encoding a polypeptide, the polypeptide is to restore or enhance the function of the optic nerve or retinal cells Of peptides.
  • the restoration or enhancement of optic nerve or retinal cell function includes treatment or improvement of optic nerve or retinal (cell) disease, restoration or improvement of the subject's vision.
  • the retinal cells are photoreceptors (including cone cells and / or rod cells), retinal ganglion cells, Mueller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment Epithelial Cells.
  • the polypeptide is a polypeptide that restores or enhances the function of the optic nerve, preferably a polypeptide for treating or improving optic neuropathy, and more preferably a polypeptide for treating or improving Leber's hereditary optic neuropathy.
  • polypeptide is selected from the group consisting of ND1, ND4, ND6, or a combination thereof.
  • polypeptide is ND4.
  • the ND4 coding sequence is shown in SEQ ID NO.:7.
  • the polypeptide may also be a neuroprotective factor, an anti-angiogenic polypeptide, an anti-apoptotic factor, or a polypeptide that enhances retinal cell function.
  • glial-derived neurotrophic factor fibroblast growth factor 2, neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, brain-derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked apoptosis Inhibitor protein, retinal cleavage protein, RPE65, retinitis pigmentosa GTPase interacting protein-1, peripheral protein, peripheral protein-2, rhodopsin or sonic hedgehog factor.
  • the recombinant adeno-associated virus further contains the coding sequence of the mitochondrial targeting peptide.
  • the coding sequence of the mitochondrial targeting peptide includes: COX8 coding sequence (including optimized or unoptimized), COX10 coding sequence, OPA1 coding sequence, or a combination thereof.
  • COX8 coding sequence (87 bp) is shown in SEQ ID NO.:1.
  • the coding sequence (84 bp) of the unoptimized COX10 is shown in SEQ ID NO.:3.
  • the coding sequence (266 bp) of OPA1 is shown in SEQ ID NO.:4.
  • the optimized COX10 coding sequence (84 bp) is shown in SEQ ID NO .: 2.
  • the optimized COX10 coding sequence is not only shorter, but also more accurate in positioning, and more target proteins are transported into the mitochondria.
  • the recombinant adeno-associated virus contains a fusion nucleic acid, and the fusion nucleic acid has a structure represented by Formula I from the 5 'end to the 3' end:
  • each "-" is independently a bond or nucleotide linking sequence
  • Z0 is none, or 5'-UTR sequence
  • Z1 is the coding sequence of mitochondrial targeting peptide
  • Z2 is the coding sequence of the polypeptide (ie, the exogenous nucleic acid encoding the polypeptide).
  • Z3 is a 3'-UTR sequence.
  • each nucleotide linking sequence is 0-21 nt, preferably 3-12 nt.
  • the coding sequence of the mitochondrial targeting peptide is an optimized COX10 coding sequence.
  • the structure of the fusion nucleic acid from the 5'-3 'end is COX10-ND4-UTR.
  • the fusion nucleic acid sequence is shown in SEQ ID NO .: 5 or 6.
  • the sequence is as shown in SEQ ID NO .: 5 in the fusion nucleic acid, from 1bp to 84bp position is the optimized COX10 sequence (total 84bp); 85bp to 1464bp position is the optimized ND4 gene, namely The nucleic acid encoding human NADH dehydrogenase subunit 4 protein (total 1380bp), the position from 1465bp to 2889bp is UTR sequence (total 1425bp, also known as 3'UTR).
  • the COX10 sequence guides the ND4 protein into the mitochondria and exerts its physiological function; 3'UTR is a non-coding sequence, designed behind the ND4 protein, and its role is to stabilize the expression of the mitochondrial targeting sequence and ND4.
  • the sequence is as shown in SEQ ID NO .: 6 in the fusion nucleic acid, from 1bp to 84bp position is unoptimized COX10 sequence (total 84bp); 85bp to 1464bp position is unoptimized ND4 gene, That is to say, the nucleic acid encoding the human NADH dehydrogenase subunit 4 protein (total 1380bp), the position from 1465bp to 2889bp is the UTR sequence (total 1425bp, also known as 3'UTR).
  • the COX10 sequence guides the ND4 protein into the mitochondria and exerts its physiological function; 3'UTR is a non-coding sequence, designed behind the ND4 protein, and its role is to stabilize the expression of the mitochondrial targeting sequence and ND4.
  • the pharmaceutical combination of the present invention includes:
  • a first drug containing (a) a first active ingredient, the first active ingredient is a recombinant adeno-associated virus, the recombinant adeno-associated virus comprises an exogenous nucleic acid encoding a polypeptide, the polypeptide Polypeptides for restoring or enhancing the function of optic nerve or retinal cells; and (b) pharmaceutically acceptable carriers; and
  • the kit of the present invention includes:
  • the first preparation is a recombinant adeno-associated virus
  • the recombinant adeno-associated virus contains an exogenous nucleic acid encoding a polypeptide
  • the polypeptide is a polypeptide that restores or enhances the function of the optic nerve or retinal cells
  • composition may further include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable ingredients refer to substances that are suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergies), that is, substances with reasonable benefit / risk ratios .
  • the term "effective amount” refers to an amount that is functional or active against humans and / or animals and acceptable to humans and / or animals.
  • preventive and / or therapeutically effective amount refers to an amount that can produce a prophylactic effect and / or therapeutic function or activity on humans and / or animals and is acceptable to humans and / or animals.
  • the term "pharmaceutically acceptable carrier” refers to a carrier for administration of therapeutic agents, including various excipients and diluents.
  • the term refers to pharmaceutical carriers that are not themselves essential active ingredients and that are not excessively toxic after application. Suitable carriers are well known to those of ordinary skill in the art.
  • the pharmaceutical combination or kit of the present invention When the pharmaceutical combination or kit of the present invention is administered, a safe and effective amount of the composition, active ingredient combination or pharmaceutical composition of the present invention is administered to a mammal.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skills of a skilled physician.
  • the first medicine (or first preparation) and the first medicine (or first preparation) in the pharmaceutical combination and kit of the present invention are administered simultaneously or sequentially by different routes, including but not limited to: oral administration , Injection administration, intratumoral administration, implantation administration, intracavity administration, anal administration, transdermal administration, internal and external application;
  • preferred dosage forms include: various dosage forms for oral administration, implants, and injections.
  • the preferred injection administration method is intraocular or intravitreal injection
  • the preferred mode of administration is oral.
  • restoring or enhancing the function of the optic nerve or retinal cells means recovering or enhancing the ability of the optic nerve or retinal cells to receive and transmit light signals through treatment or improvement of optic nerve or retinal (cell) disease, thereby restoring or enhancing Subject's eyesight. Therefore, the optic nerve or retina will have an increased ability to receive optical signals and transmit such signals compared to optic nerves or retinas that have not been treated as described herein. The increase can be any amount.
  • restoring vision in a subject means that the subject shows improved vision compared to before treatment, for example using a vision test as described herein.
  • Recovery includes any degree of improvement, including complete restoration of vision to perfect or near-perfect vision.
  • the treatment of ocular diseases means the administration of recombinant adeno-associated virus as described herein to improve or alleviate one or more symptoms of the disease selected from the group consisting of optic neuropathy, glaucoma, pigmented retina Inflammation, macular degeneration, retinal splitting disease, Liebherr's congenital cataract, diabetic retinopathy, panchromatic or color blindness, retinal degenerative diseases, or a combination thereof. Improvements or reductions can lead to peripheral or central vision, and / or improvement of vision during the day or night.
  • the present invention is a combination medicine before and after gene therapy. Compared with the existing technology, it greatly improves the effectiveness and safety of gene therapy for eye diseases (such as Leber disease), especially the safety, and effectively reduces the gene therapy eye Complications of some diseases (such as uveitis, etc.).
  • the method of the present invention improves the safety of gene therapy Leber disease is a great breakthrough in the field of gene therapy.
  • the present invention only takes recombinant adeno-associated virus carrying the ND4 coding sequence as an example, but is not limited thereto.
  • Recombinant adeno-associated virus rAAV2 / 2-ND4 was prepared by referring to the method in Example 1 of CN 102634527 B.
  • Plasmid preparation The fusion nucleic acid shown in SEQ ID NO .: 6 was synthesized by Chengdu Qingke Zixi Biotechnology Co., Ltd. Amplify the full-length gene by PCR, cut the fusion gene by EcoRI / SalI to form a sticky end, and insert the fusion gene into the adeno-associated virus vector pSNaV with EcoRI / SalI cleavage site, namely pSNaV / rAAV2 / 2-ND4 (Hereinafter referred to as pAAV2-ND4).
  • the screening and identification procedures of recombinants are the same as CN102634527B, and are briefly described as follows: Take LB plates after culturing at 37 °C, blue spots and white spots appear, white is recombinant clones. Pick white colonies and add them to LB liquid medium containing Amp 100mg / L, and incubate at 37 ° C and 200rpm for 8h. After culturing, the bacterial solution was taken to extract the plasmid. The plasmid extraction procedure was based on the Biomiga instruction manual and identified by EcoRI / SalI digestion.
  • HEK293 cells were seeded in a 225cm 2 cell culture flask with a density of 3.0 ⁇ 10 7 cells / mL, the medium was DMEM + 10% bovine serum, and the solution contained 37% 5% CO 2 Incubate in the incubator overnight. The medium was changed on the day of transfection, and culture was continued with fresh DMEM medium containing 10% bovine serum. When the cells grow to 80-90%, the medium is discarded, and pAAV2-ND4 is transfected with the PlasmidTrans II (VGTC) transfection kit (for specific transfection steps, see CN 102634527 B Example 1). 48 hours after transfection, cells were collected.
  • VGTC PlasmidTrans II
  • Virus collection 1) Prepare dry ice ethanol bath (or liquid nitrogen) and 37 ° C water bath; 2) Collect the toxin-producing cells together with the culture medium into a 15ml centrifuge tube; 3) 1000rpm / min, centrifuge Separate the cells and supernatant for 3 minutes, store the supernatant separately, and resuspend the cells in 1ml of PBS; 4) Repeat the transfer of the cell suspension in a dry ice ethanol bath and a 37 ° C water bath, freeze-thaw four times, freeze and thaw each 10 Minutes, a little shock after each melt.
  • Humoral immune response AAV2 humoral immune examination
  • the mutation site of gene detection is mtDNA 11778 mutation.
  • Humoral immune response AAV2 humoral immune examination to confirm that the rAAV2 immunity of the patient is at a low level before medication;
  • Laminar flow operating room should be prepared for disinfection measures and surgical consumables
  • the gene drug is kept by a special person. Before the medicine is taken, check the patient information and make sure that the medicine package is complete and free of pollution. Strictly do a good job of disinfecting the outer packaging of the medicine; when the medicine is drawn, slowly and smoothly operate, complete the medicine suction at one time, do not repeatedly pump and touch the bottle wall Drug contamination; after aspiration of the drug, the injection needs to be completed within half an hour. After the injection is completed, keep the drug packaging and the remaining drugs at -20 °C for future reference. (Note: During the process of pumping drugs, absolutely sterile operation is required. If the drug falls on the ground or the suction needle touches the contaminated area, the drugs must be discarded. Any drugs that are at risk of contamination must not be injected into the patient's vitreous cavity.)
  • the assistant also prepares for injection eye anesthesia, disinfection, eyelid opening, etc., rinses the conjunctival sac 3 times with 0.5% povidone iodine, and performs intravitreal injection according to the intravitreal injection specifications. Immediately after pulling out the needle, press the injection point with a cotton swab and massage for 10 seconds to prevent drug leakage.
  • antibiotic eye ointment is applied to the eye, and sterile gauze is used to cover the eye.
  • the patient lay flat for 20 minutes and returns to the ward;
  • Metso reduced the amount, po, 24mg / 60kg; Qd * 7d;
  • Metso reduced the amount, po, 8mg / 60kg; Qd * 7d;
  • Metso reduced the amount, po, 6mg / 60kg; Qd * 7d;
  • Metso reduced the amount, po, 4mg / 60kg; Qd * 7d.
  • the second day after surgery vision, intraocular pressure, slit lamp, fundus;
  • the third day after surgery vision, intraocular pressure, slit lamp, fundus.
  • the clinical-grade rAAV2-ND4 dosage form used in this study is an injection; the recombinant adeno-associated virus rAAV2-ND4 is a recombinant adeno-associated virus prepared in Example 1 and containing the fusion nucleic acid shown in SEQ ID NO .: 6.
  • Table 1 145 patients within 3 days of treatment efficacy analysis
  • Uveitis has a variety of causes, and each type of clinical manifestation, progression, and treatment drugs and time are different. Therefore, doctors need to develop an individualized treatment plan for the patient according to the specific situation of the patient.
  • High intraocular pressure is a mild complication. It is the most common complication in ophthalmic surgery. Under normal circumstances, patients can return to normal levels on their own. They can also reduce intraocular pressure to normal levels after using intraocular pressure drops.
  • the treatment method of the present invention can effectively treat eye diseases (such as Leber's hereditary optic neuropathy), and there are no related complications, such as uveitis, and the side effects are very small and very safe.
  • eye diseases such as Leber's hereditary optic neuropathy
  • Example 2 Replace the recombinant adeno-associated virus rAAV2-ND4 containing the fusion nucleic acid shown in SEQ ID NO .: 6 in Example 2 with the recombinant adeno-associated virus containing the fusion nucleic acid shown in SEQ ID NO .: 5, in other experimental methods and experiments
  • the reagents used are exactly the same as in Example 2. The results show that this treatment method can effectively treat eye diseases, and there are no related complications, such as uveitis, and the side effects are very small and very safe.

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Abstract

本发明公开了基因治疗Leber遗传性视神经病变。施用本发明所述的重组腺相关病毒之前和/或之后施用美卓乐，可以提高治疗效果，并有效预防和/或治疗基因治疗引起的并发症。

Description

基因治疗Leber遗传性视神经病变 技术领域
本发明涉及基因治疗领域，具体涉及基因治疗Leber遗传性视神经病变。
背景技术
Leber遗传性视神经病变(LHON)是一种主要累及黄斑乳头束纤维，导致视神经退行性变的遗传性疾病，是一种病因及机制尚未阐明的母系遗传病，最早报道于1858年，并于1871年以第一个描述其临床特征的德国学者Leber名字命名。LHON较为罕见，临床表现为双眼同时或先后急性或亚急性无痛性视力减退，同时可伴有中心视野缺失及色觉障碍。目前常使用神经营养药物治疗或使用中医中药治疗，但均疗效欠佳。因无有效治疗方法，一旦发病，仅少数可以好转，多数患者最终走向失明的结局。故自发现该病以来，就受到部分医者关注，也一直在探寻病因、机制及治疗方法。
人类基因组计划(human genome project,HGP)实施是医学的一大进步，医者终究对多数疾病可以追根索源。部分研究者借助HGP衍生的生物工程技术对LHON进行了分子水平的研究。1988年Wallace [2]发现第一个LHON相关突变位点m.11778G>A，后来研究者又陆续发现60多个线粒体DNA(mitochondrion DNA，mtDNA)突变位点。目前国际公认，包括m.11778G>A，m.3460G>A和m.14484T>c这三个常见原发突变位点在内的mtDNA突变是LHON发病的分子基础。
随着分子生物学水平的提高，以基因测序、基因诊断、基因治疗为代表的基因工程技术日趋成熟，人类对疾病本质的认识进一步深化、治疗手段更为精准。
人眼因其特殊生理及结构特点使其成为研究者心目中的基因治疗的首选器官。2007年是眼部疾病基因治疗的元年，美国和英国的三个研究小组针对Leber先天黑矇症(LCA)的基因治疗成功地开展了临床试验，在安全性和有效性方面均取得满意结果，开启了眼部疾病基因治疗的先河。随后，包括视网膜色素变性(RP)在内的多达31项眼部疾病基因治疗临床试验被批准。
LHON呈青壮年多发、不完全外显及性别偏向的特征；在亚洲区约1.18/10000的发病率，原发突变位点m.11778G>A突变率约占87％-92.9％，而中国这一突变率约90％。换言之，中国大约有15万例原发突变位点m.11778G>A突变而发病的LHON患者。2011年完成全球首例LHON患者的基因治疗临床研究，参与临床研究的9例LHON基因治疗的患者至今已经观察7年，其疗效和安全性得到了充分的验证，注射药物后患者的视网膜外观和形态没有异常。2015年，美国Guy教授等也做了类似的研究，他们治疗14例患者，有6例视力提高，但有两例发生葡萄膜炎。法国GenSight Biologics进行LHON基因治疗的临床试验15例，出现了2例选择性白内障摘除、2个严重的前房炎症和玻璃体炎症事件。
因此，本领域急需开发新的更安全的基因治疗方法和药物。
发明内容
本发明的目的是提供一种更安全的基因治疗眼部方法和药物。
本发明的第一方面，提供了一种药物组合，所述药物组合包括：
(A)第一药物，所述第一药物含有(a)第一活性成分，所述第一活性成分为重组腺相关病毒，所述重组腺相关病毒包含编码多肽的外源核酸，所述多肽为恢复或增强视神经或视网膜细胞功能的多肽；和(b)药学上可接受的载体；和
(B)第二药物，所述第二药物为美卓乐。
在另一优选例中，所述恢复或增强视神经或视网膜细胞功能包括治疗或改善视神经或视网膜(细胞)病变，和/或恢复或提高受试者视力。
在另一优选例中，所述视网膜细胞为光感受器(包括视锥细胞和/或视杆细胞)、视网膜神经节细胞、缪勒细胞、双极细胞、无长突细胞、水平细胞或视网膜色素上皮细胞。
在另一优选例中，所述多肽为恢复或增强视神经功能的多肽，较佳地为治疗或改善视神经病变的多肽，更佳地为治疗或改善Leber遗传性视神经病变的多肽。
在另一优选例中，所述多肽选自下组：ND1、ND4、ND6、或其组合。较佳地，所述多肽为ND4。
在另一优选例中，所述ND4的编码序列如SEQ ID NO.:7所示。
在另一优选例中，所述重组腺相关病毒的含量为1×10 7vg/0.05ml-1×10 14vg/0.05ml，较佳地1×10 9vg/0.05ml-1×10 12vg/0.05ml，更佳地1×10 10vg/0.05ml-1×10 11vg/0.05ml。
在另一优选例中，所述重组腺相关病毒还含有线粒体靶向肽的编码序列。
在另一优选例中，所述线粒体靶向肽的编码序列包括：COX8的编码序列(包括优化或未优化的)、COX10的编码序列、OPA1的编码序列、或其组合。
在另一优选例中，所述COX8的编码序列具有如SEQ ID NO.:1所示的序列。
在另一优选例中，所述优化的COX10的编码序列具有如SEQ ID NO.:2所示的序列。
在另一优选例中，所述未优化的COX10编码序列具有如SEQ ID NO.:3所示的序列。
在另一优选例中，所述OPA1的编码序列具有如SEQ ID NO.:4所示的序列。
在另一优选例中，所述重组腺相关病毒含有融合核酸，所述融合核酸从5’端-3’端具有式I结构：
Z0-Z1-Z2-Z3  (I)
式中，
各“-”独立地为键或核苷酸连接序列；
Z0为无、或5’-UTR序列；
Z1为线粒体靶向肽的编码序列；
Z2为所述多肽的编码序列(即编码所述多肽的外源核酸)；和
Z3为3’-UTR序列。
在另一优选例中，各个核苷酸连接序列的长度为0-21nt，较佳地3-12nt。
在另一优选例中，所述线粒体靶向肽的编码序列为优化的COX10的编码序列。
在另一优选例中，所述融合核酸从5’-3’端的结构为COX10-ND4-UTR。
在另一优选例中，所述融合核酸序列如SEQ ID NO.:5或6所示。
在另一优选例中，所述序列如SEQ ID NO.:5或6所示的融合核酸中，
第1-84位为COX10的编码序列；
第85-1464位为编码人NADH脱氢酶亚单位4蛋白的核苷酸序列；
第1465-2889位为3’-UTR序列。
在另一优选例中，所述重组腺相关病毒的血清型选自：AAV2、AAV5、AAV7、AAV8、或其组合；较佳地为AAV2。
在另一优选例中，所述第一药物的剂型选自下组：冻干制剂、液体制剂、或其组合。较佳地，所述第一药物的剂型为注射剂型。
在另一优选例中，所述第二药物的施用方式包括：经静脉施用、经皮下施用、口服、或其组合。较佳地，所述第二药物的剂型优选为口服制剂。
在另一优选例中，所述药物组合还包括：(C)第三药物，所述第三药物包括：磷酸肌酸钠、甲强龙、或其组合。
在另一优选例中，所述药物组合含有：
(A)第一药物，所述第一药物含有(a)治疗有效量的第一活性成分，以及(b)药学上可接受的载体；
(B)预防和/或治疗有效量的第二药物。
在另一优选例中，所述药物组合用于恢复受试者视力或治疗眼部疾病。
在另一优选例中，所述药物组合中的第一药物和第二药物为各自独立的。
本发明的第二方面，提供了一种药盒，所述药盒包括：
(A)第一制剂，所述第一制剂为重组腺相关病毒，所述重组腺相关病毒包含编码多肽的外源核酸，所述多肽为恢复或增强视神经或视网膜细胞功能的多肽；和
(B)第二制剂，所述第二制剂为美卓乐。
在另一优选例中，所述药盒还包括标签或说明书，所述标签或说明书中注明以下内容：在第一制剂施用前和/或施用后，向所需要的对象施用第二制剂。
在另一优选例中，所述第一制剂和第二制剂为各自独立的。
在另一优选例中，所述第一制剂的剂型选自下组：冻干制剂、液体制剂、或其组合。较佳地，所述药物制剂的剂型为注射剂型。
在另一优选例中，所述第二制剂的施用方式包括：经静脉施用、经皮下施用、口服、或其组合。较佳地，所述第二制剂的剂型优选为口服制剂。
本发明的第三方面，提供了如本发明第一方面所述的药物组合的用途，所述组合物用于制备用于恢复或提高受试者视力和/或治疗眼部疾病的药物或制剂。
在另一优选例中，所述恢复或提高受试者视力包括恢复或增强视神经或视网膜细胞功能。
在另一优选例中，所述眼部疾病为视神经病变、青光眼、色素性视网膜炎、黄斑变性、视网膜劈裂症、利伯氏先天性黑内障、糖尿病性视网膜病、全色盲或色盲、视网膜退化性疾病、或其组合。
在另一优选例中，所述眼部疾病为视神经病变。
在另一优选例中，所述制剂或组合物用于治疗视神经病变，较佳地为遗传性视神经病变，较佳地为Leber遗传性视神经病变(LHON)。
在另一优选例中，所述制剂或组合物用于扩大或恢复视网膜的光感受器功能、恢复受试者视力(或感光能力)、和/或治疗视网膜退化性疾病。
在另一优选例中，所述视网膜退化性疾病选自下组：视网膜营养不良(如视杆营养不良、视杆视锥营养不良、视锥视杆营养不良、视锥营养不良或黄斑营养不良)、视网膜或黄斑退化、视网膜色素变性、其他疾病、或其组合。
本发明的第四方面，提供了一种治疗眼部疾病的方法，所述方法包括步骤：给需要的对象，施用本发明第一方面所述的药物组合或本发明第二方面所述的药盒。
在另一优选例中，在使用所述药物组合或所述药盒之前、同时和/或之后，配合使用其他治疗眼部疾病的活性物质。
本发明的第五方面，提供了一种治疗方法，所述治疗方法包括步骤：
在向所需要的对象眼内施用重组腺相关病毒之前、同时和/或之后，向所述对象施用美卓乐。
在另一优选例中，所述重组腺相关病毒为具有恢复或提高受试者视力和/或治疗眼部疾病功能的重组腺相关病毒。
在另一优选例中，所述重组腺相关病毒包含编码多肽的外源核酸，所述多肽为恢复或增强视神经或视网膜细胞功能的多肽。
在另一优选例中，所述美卓乐的施用方式包括：经静脉施用、经皮下施用、口服、或其组合。较佳地，所述美卓乐的施用方式为口服施用。
在另一优选例中，所述治疗方法包括步骤：
(1)术前预处理，所述术前预处理包括向所需要的对象施用美卓乐；
(2)向所述对象眼内施用重组腺相关病毒，所述重组腺相关病毒包含编码多肽的外源核酸，所述多肽为恢复或增强视神经或视网膜细胞功能的多肽；和
(3)术后处理，所述术后处理包括向所述对象施用美卓乐。
在另一优选例中，所述的对象为人或非人哺乳动物。
在另一优选例中，所述术前预处理中美卓乐的施用方式为口服施用。
在另一优选例中，步骤(1)中，所述美卓乐的施用量为每次28-36mg/60kg体重，较佳地为每次30-34mg/60kg体重。
在另一优选例中，步骤(1)中，所述美卓乐的施用间隔为至少每三天一次，较佳地至少两天一次，更佳地至少每天一次。
在另一优选例中，步骤(1)中，所述美卓乐的施用次数为至少1次，较佳地不低于3次，较佳地不低于5次，更佳地不低于7次。
在另一优选例中，步骤(1)中，所述美卓乐的施用时间为至少3天，较佳地不低于5天，较佳地不低于一周。
在另一优选例中，所述术前预处理包括：
向所需要的对象施用美卓乐，施用间隔为每天一次，施用量为每次30-34mg/60kg体重，施用至少一周。
在另一优选例中，步骤(1)中，所述术前预处理还包括术前一天局部(眼部)施用抗生素眼药水和/或眼膏。
在另一优选例中，步骤(2)中，所述重组腺相关病毒的施用是通过眼内施用，较佳地通过视网膜下、直接视网膜、脉络膜上或玻璃体内注射。
在另一优选例中，所述重组腺相关病毒的施用是通过玻璃体内注射。
在另一优选例中，所述重组腺相关病毒的施用是单眼注射或双眼注射。
在另一优选例中，所述重组腺相关病毒的施用量为1×10 7vg/0.05ml-1×10 14vg/0.05ml，较佳地1×10 9vg/0.05ml-1×10 12vg/0.05ml，更佳地1×10 10vg/0.05ml-1×10 11vg/0.05ml。
在另一优选例中，步骤(3)中，所述术后处理还包括：在施用美卓乐前，向所述对象施用磷酸肌酸钠和/或甲强龙。
在另一优选例中，所述磷酸肌酸钠和/或甲强龙的施用方式包括：经静脉施用、经皮下施用、口服、或其组合。较佳地，所述磷酸肌酸钠和/或甲强龙经静脉施用。
在另一优选例中，先施用所述重组腺相关病毒，再施用磷酸肌酸钠；所述重组腺相关病毒与所述磷酸肌酸钠的(第一次)施用间隔为至多24h，较佳地4-20h，更佳地6-18h，更佳地10-14h。
在另一优选例中，所述磷酸肌酸钠的施用量为每次1.5-2.5g/60kg体重，较佳地每次1.8-2.2g/60kg体重。
在另一优选例中，所述磷酸肌酸钠的施用间隔为至少每两天一次，较佳地至少每天一次。
在另一优选例中，所述磷酸肌酸钠的施用次数为至少1次，较佳地不低于2次。
在另一优选例中，所述磷酸肌酸钠的施用时间为至少1天，较佳地2-4天。
在另一优选例中，先施用所述重组腺相关病毒，再施用甲强龙；所述重组腺相关病毒与所述甲强龙的(第一次)施用间隔为至多24h，较佳地4-20h，更佳地6-18h，更佳地10-14h。
在另一优选例中，所述甲强龙的施用量为每次70-90mg/60kg体重，较佳地每次75-85mg/60kg体重，较佳地每次78-82mg/60kg体重。
在另一优选例中，所述甲强龙的施用间隔为至少每两天一次，较佳地至少每天一次。
在另一优选例中，所述甲强龙的施用次数为至少1次，较佳地不低于2次。
在另一优选例中，所述甲强龙的施用时间为至少1天，较佳地2-4天。
在另一优选例中，步骤(3)中，在施用重组腺相关病毒的一周内，所述美卓乐的施用量为每次28-36mg/60kg体重，较佳地每次30-34mg/60kg体重。
在另一优选例中，步骤(3)中，所述美卓乐的施用量每周递减，递减量为1-10mg/60kg体重，较佳地2-8mg/60kg体重。
在另一优选例中，步骤(3)中，所述美卓乐的施用间隔为至少每三天一次，较佳地至少两天一次，更佳地至少每天一次。
在另一优选例中，步骤(3)中，所述美卓乐的施用时间为至少2周，较佳地3周-10周，更佳地4周-7周。
在另一优选例中，步骤(3)中，所述美卓乐的施用方式为口服施用。
在另一优选例中，步骤(3)中，术后处理包括：
在施用重组腺相关病毒的第0天(当天)至第2天，向所需要的对象施用磷酸肌酸钠和甲强龙，施用间隔均为每天一次，均施用3天，其中所述磷酸肌酸钠的施用量为每次1.8-2.2g/60kg体重，所述甲强龙的施用量为每次78-82mg/60kg体重；
在施用重组腺相关病毒的第3天开始施用美卓乐，施用量为每次30-34mg/60kg体重，施用量每周递减，递减量为2-8mg/60kg体重，每天一次，较佳地，至少施用4周。
本发明的第六方面，提供了一种美卓乐的用途，用于制备一制剂或组合物，所述制剂或组合物用于降低眼内施用重组腺相关病毒的副作用或并发症。
在另一优选例中，所述副作用或并发症包括：白内障摘除、葡萄膜炎、玻璃体炎、角膜炎、前房炎症、玻璃体积血、眼痛、过敏性结膜炎、高眼压、或其组合。
在另一优选例中，所述降低为预防性地降低。
在另一优选例中，所述制剂或组合物用于降低眼内施用重组腺相关病毒的副作用或并发症的发生率。
在另一优选例中，所述制剂或组合物还用于提高所述重组腺相关病毒的治疗效果。
在另一优选例中，所述治疗效果为提高或恢复受试者视力和/或治疗眼部疾病的效果。
在另一优选例中，所述治疗效果为治疗选自下组眼部疾病的效果：
视神经病变、青光眼、色素性视网膜炎、黄斑变性、视网膜劈裂症、利伯氏先天性黑 内障、糖尿病性视网膜病、全色盲或色盲、视网膜退化性疾病、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了带有MTS序列的ND4融合蛋白进入到线粒体里发挥作用的示意图。
具体实施方式
本发明人经过广泛而深入地研究，意外地发现了在基因治疗(即施用本发明所述的重组腺相关病毒)之前和/或之后施用美卓乐，不仅可以显著提高治疗的效果，还可以有效预防和/或治疗基因治疗的并发症，如选择性白内障摘除、葡萄膜炎、前房炎症、玻璃体炎。在此基础上，完成了本发明。
在前期做了充足的理论准备后，自2008年起，本发明人在基础和临床做了大量工作，从基因治疗载体的构建到安全性检测和评价的探究，从大量的动物实验到临床试验，本发明从多个方面严谨的证实了基因治疗LHON的安全性。
本发明人根据国家相关法规开展随机多中心临床研究，临床试验均通过了伦理委员会审批，以及和病人签署了知情同意书，基因治疗LHON患者，并定期进行安全性检查，除有少量高眼压，可自行恢复，无任何严重并发症，安全性高。此研究证实本研究团队的基因治疗的安全性和有效性好。
基因治疗采用的是异位表达技术，如图1所示，即通过腺相关病毒将外源核酸(如ND4基因)带到细胞表达正常的蛋白，前面锚定的小肽链，能够引导目的蛋白进入到线粒体内，替代因为基因突变导致的缺陷蛋白，发挥正常的生理功能。这一研究处于全球眼科基因治疗领域最前沿，是全球领先的技术。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
术语“给予”或“施用”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内，肌内，皮下，腹膜内，脊髓或其它肠胃外给药途径，例如通过注射或输注。
“术前”是指施用本发明所述重组腺相关病毒之前。
“术后”是指施用本发明所述重组腺相关病毒之后。
美卓乐(甲泼尼龙片)
美卓乐，即甲泼尼龙片，主要成份为：甲泼尼龙，其化学名称为：11β,17,21-三羟基-6α-甲基孕甾-1,4-二烯-3,20-二酮。分子式：C 22H 30O 5，分子量：374.48。其结构式如式II所示：
Figure PCTCN2018113799-appb-000001
本发明经过大量的研究和临床实验发现，美卓乐不仅可以提高重组腺相关病毒的治疗效果，还可以有效地预防和/或治疗基因治疗的并发症，如白内障摘除、葡萄膜炎、前房炎症、玻璃体炎、角膜炎、玻璃体积血、眼痛、过敏性结膜炎、高眼压等。
重组腺相关病毒
在本发明中，所述重组腺相关病毒可以恢复或提高受试者视力和/或治疗眼部疾病的重组腺相关病毒。在本发明的一个实施方式中，所述重组腺相关病毒如本发明第一方面所述，所述重组腺相关病毒包含编码多肽的外源核酸，所述多肽为恢复或增强视神经或视网膜细胞功能的多肽。
在另一优选例中，所述恢复或增强视神经或视网膜细胞功能包括治疗或改善视神经或视网膜(细胞)病变，恢复或提高受试者视力。
在另一优选例中，所述视网膜细胞为光感受器(包括视锥细胞和/或视杆细胞)、视网膜神经节细胞、缪勒细胞、双极细胞、无长突细胞、水平细胞或视网膜色素上皮细胞。
在另一优选例中，所述多肽为恢复或增强视神经功能的多肽，较佳地为治疗或改善视神经病变的多肽，更佳地为治疗或改善Leber遗传性视神经病变的多肽。
在另一优选例中，所述多肽选自下组：ND1、ND4、ND6、或其组合。较佳地，所述多肽为ND4。
在另一优选例中，所述ND4的编码序列如SEQ ID NO.:7所示。
在另一优选例中，所述多肽还可以是神经保护因子、抗血管生成多肽、抗凋亡因子或 增强视网膜细胞功能的多肽。例如，胶质源性神经营养因子、成纤维细胞生长因子2、神经营养因子、睫状神经营养因子、神经生长因子、脑源性神经营养因子、表皮生长因子、视紫红质、X连锁凋亡抑制蛋白、视网膜劈裂蛋白、RPE65、色素性视网膜炎GTP酶相互作用蛋白-1、外周蛋白、外周蛋白-2、视紫红质或音猬因子。
在另一优选例中，所述重组腺相关病毒还含有线粒体靶向肽的编码序列。
在另一优选例中，所述线粒体靶向肽的编码序列包括：COX8的编码序列(包括优化或未优化的)、COX10的编码序列、OPA1的编码序列、或其组合。
在另一优选例中，所述COX8的编码序列(87bp)如SEQ ID NO.:1所示。
Figure PCTCN2018113799-appb-000002
在另一优选例中，所述未优化的COX10的编码序列(84bp)如SEQ ID NO.:3所示。
Figure PCTCN2018113799-appb-000003
在另一优选例中，所述OPA1的编码序列(266bp)如SEQ ID NO.:4所示。
Figure PCTCN2018113799-appb-000004
在另一优选例中，所述优化的COX10的编码序列(84bp)如SEQ ID NO.:2所示。相比未优化的COX10的编码序列和其它的线粒体靶向肽编码序列，优化的COX10的编码序列不仅更短，而且定位更准确，更多的目标蛋白转运到线粒体中。
Figure PCTCN2018113799-appb-000005
在另一优选例中，所述重组腺相关病毒含有融合核酸，所述融合核酸从5’端-3’端具有式I所示的结构：
Z0-Z1-Z2-Z3  (I)
式中，各“-”独立地为键或核苷酸连接序列；
Z0为无、或5’-UTR序列；
Z1为线粒体靶向肽的编码序列；
Z2为所述多肽的编码序列(即编码所述多肽的外源核酸)；和
Z3为3’-UTR序列。
在另一优选例中，各个核苷酸连接序列的长度为0-21nt，较佳地3-12nt。
在另一优选例中，所述线粒体靶向肽的编码序列为优化的COX10的编码序列。
在另一优选例中，所述融合核酸从5’-3’端的结构为COX10-ND4-UTR。
在另一优选例中，所述融合核酸序列如SEQ ID NO.:5或6所示。
在另一优选例中，所述序列如SEQ ID NO.:5所示的融合核酸中，自1bp至84bp位置为优化的COX10序列(共84bp)；85bp至1464bp位置为优化ND4基因，即所述的编码人NADH脱氢酶亚单位4蛋白的核酸(共1380bp)，1465bp至2889bp位置为UTR序列(共1425bp，又称3’UTR)。COX10序列引导ND4蛋白进入到线粒体中，发挥其生理功能；3’UTR是非编码序列，设计在ND4蛋白的后面，其作用是稳定线粒体靶向序列和ND4的表达。
Figure PCTCN2018113799-appb-000006
Figure PCTCN2018113799-appb-000007
在另一优选例中，所述序列如SEQ ID NO.:6所示的融合核酸中，自1bp至84bp位置为未优化的COX10序列(共84bp)；85bp至1464bp位置为未优化ND4基因，即所述的编码人NADH脱氢酶亚单位4蛋白的核酸(共1380bp)，1465bp至2889bp位置为UTR序列(共1425bp，又称3’UTR)。COX10序列引导ND4蛋白进入到线粒体中，发挥其生理功能；3’UTR是非编码序列，设计在ND4蛋白的后面，其作用是稳定线粒体靶向序列和ND4的表达。
Figure PCTCN2018113799-appb-000008
Figure PCTCN2018113799-appb-000009
本发明药物组合、药盒及其用途和给药方式
本发明药物组合如本发明第一方面所述，所述药物组合包括：
(A)第一药物，所述第一药物含有(a)第一活性成分，所述第一活性成分为重组腺相关病毒，所述重组腺相关病毒包含编码多肽的外源核酸，所述多肽为恢复或增强视神经或视网膜细胞功能的多肽；和(b)药学上可接受的载体；和
(B)第二药物，所述第二药物为美卓乐。
本发明药盒如本发明第二方面所述，所述药盒包括：
(A)第一制剂，所述第一制剂为重组腺相关病毒，所述重组腺相关病毒包含编码多肽的外源核酸，所述多肽为恢复或增强视神经或视网膜细胞功能的多肽；和
(B)第二制剂，所述第二制剂为美卓乐。
必要时，所述组合物还可以包括药学上可接受的载体。如本文所用，术语“药学上可接受的”的成分是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的，即有合理的效益/风险比的物质。
如本文所用，术语“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，术语“预防和/或治疗有效量”是指可对人和/或动物产生预防性效果和 /或治疗功能或活性的且可被人和/或动物所接受的量。
如本文所用，术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。该术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。
使在施用本发明的药物组合或药盒时，是将安全有效量的本发明组合物、活性成分组合或药物组合物施用于哺乳动物。具体剂量还应考虑给药途径、病人健康状况等因素，这些都是在熟练医师技能范围之内的。本发明的药物组合和药盒中的第一药物(或第一制剂)和第一药物(或第一制剂)分别以不同的途径同时或相继给药，其中包括但并不限于：口服给药，注射给药，瘤内给药，植入给药，腔内给药，肛门给药，透皮给药，内外敷；
对于本发明，优选的剂型包括：口服给药的各种剂型、植入剂、注射剂。
对于第一药物(或第一制剂)，优选的注射施用方式为眼内或玻璃体腔内注射；
对于第二药物(或第二制剂)，优选的施用方式为口服。
在本发明的优选例中，恢复或增强视神经或视网膜细胞功能意指通过治疗或改善视神经或视网膜(细胞)病变，恢复或增强视神经或视网膜细胞接受光信号并传递的能力，从而恢复或提高受试者视力。因此，视神经或视网膜相比于未用如本文描述的方法处理的视神经或视网膜，将具有增加的接收光信号并且传送此类信号的能力，增加可以是任何量。
如本文所用，恢复受试者中的视力意指，受试者相比于治疗之前，例如使用如本文描述的视力测试，显示改进的视力。恢复包括任何程度的改进，包括视力的完全恢复到完美的或接近完美的视力。
如本文所用，治疗眼部疾病意指施用如本文描述的重组腺相关病毒以改善或减轻疾病的一种或多种症状，所述眼部疾病选自下组：视神经病变、青光眼、色素性视网膜炎、黄斑变性、视网膜劈裂症、利伯氏先天性黑内障、糖尿病性视网膜病、全色盲或色盲、视网膜退化性疾病、或其组合。改善或减轻可导致外周或中央视力、和/或白天或夜间视力的改善。
与现有技术相比，本发明主要优点在于：
本发明为基因治疗前后的组合用药，与现有的技术相比，大大提高了基因治疗眼部疾病(如Leber病)的有效性和安全性，尤其是安全性，有效地减少了基因治疗眼部疾病的并发症(如葡萄膜炎等)。本发明方法对基因治疗Leber病安全性的提高是基因治疗领域的极大突破。
本发明提到的上述特征，或实施例提到的特征可以任意组合。本案说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何被提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1制备重组腺相关病毒
本发明仅以携带有ND4编码序列的重组腺相关病毒为例，但不限于此。
参考CN 102634527 B实施例1的方法制备得到重组腺相关病毒rAAV2/2-ND4。
1.1 质粒制备：由成都擎科梓熙生物技术有限公司合成如SEQ ID NO.:6所示的融合核酸。通过PCR扩增全长基因，通过EcoRI/SalI酶切在融合基因上形成粘性末端，并将融合基因嵌入有EcoRI/SalI酶切位点的腺相关病毒载体pSNaV，即pSNaV/rAAV2/2-ND4(以下简称为pAAV2-ND4)。重组子的筛选和鉴定步骤同CN 102634527 B，简述如下：取37℃培养后的LB平板，出现蓝斑和白斑，其中白色为重组克隆。挑取白色的菌落加入到含有Amp 100mg/L的LB液体培养基中，37℃，200rpm培养8h。培养好后取菌液，提取质粒，质粒提取步骤参照Biomiga说明书，使用EcoRI/SalI酶切鉴定。
1.2 细胞转染：转染前一天，将HEK293细胞接种于225cm 2细胞培养瓶中，接种密度3.0×10 7个/mL细胞，培养基为DMEM+10％牛血清，置37℃含5％CO 2的培养箱中培养过夜。转染当天换液，用新鲜的含10％牛血清的DMEM培养基继续培养。待细胞生长至80～90％时，弃去培养基，用PlasmidTrans II(VGTC)转染试剂盒进行转染pAAV2-ND4(具体转染步骤参见CN 102634527 B实施例1)。转染48h后，收取细胞。
1.3 重组腺相关病毒的收集、浓缩与纯化：
1.3.1 病毒的收集：1)准备干冰乙醇浴(或液氮)和37℃水浴；2)将产毒的细胞连同培养基一同收集到一个15ml的离心管中；3)1000rpm/min，离心3分钟，分离细胞和上清，将上清另外存放，细胞用1ml PBS重悬；4)将细胞悬浮液在干冰乙醇浴和37℃水浴中反复转移，冻融四次，冻和融各10分钟，每次融解后稍加震荡。
1.3.2 病毒的浓缩：1)10,000g离心去除细胞碎片，将离心上清转移到一个新离心管中；2)用0.45μm滤器过滤除杂质；3)加入各1/2体积的1M NaCl、10％PEG8000溶液，混合均匀，4℃过夜；4)12,000rpm离心2h，弃上清，病毒沉淀用适量的PBS溶液溶解，待完全溶解后用0.22μm滤器过滤除菌；5)加入Benzonase核酸酶消化去除残留的质粒DNA(终浓度为50U/ml)。合上管盖，颠倒几次以充分混合。在37℃孵育30分钟；6)用0.45μm过滤头过滤，取滤出液，即为浓缩的rAAV2病毒。
1.3.3 病毒的纯化：1)向病毒浓缩液中添加固体CsCl直到密度为1.41g/ml(折射率为1.372)；2)将样品加入到超速离心管中，用预先配好的1.41g/ml CsCl溶液将离心管剩余空间填满；3)在175,000g下离心24小时，以形成密度梯度。按顺序分步收集不同密度的样品，取样进行滴度测定。收集富集有rAAV2颗粒的组分；4)重复上述过程一次。将病毒装入100kDa的透析袋，4℃透析脱盐过夜。
至此，获得浓缩和纯化的重组腺相关病毒rAAV2-ND4。
实施例2基因治疗LHON治疗前、治疗和治疗后处理，以及疗效情况
一、治疗前规范：
1、研究场所
本研究是临床试验，基因诊断在医院基因诊断中心完成；眼科检查在同济医院眼科检查室用同类型设备完成；全身检查(部分)均在检验科由指定人员及指定设备完成；所有的手术都在眼科专用层流手术间进行操作；术后复查及随访由团队专门指定人员及设备完成。
2、临床试验设计
本研究采用多中心、前瞻性临床试验。受试者符合Leber遗传性视神经病变诊断标准：
2.1 患者准入规范：
1.拟接受LHON眼部基因治疗者，需经基因检测确诊为LHON患者；
2.拟接受LHON眼部基因治疗者，需至少经过三个月观察且无明显自愈倾向者；
3.拟接受LHON眼部基因治疗者，需排除其他致命性疾病病史及既往眼部病史者；
4.拟接受LHON眼部基因治疗者，需排除治疗过程中可能发生必需药物过敏者；
5.拟接受LHON眼部基因治疗者，需接受体液免疫反应实验，阳性者排除；
6.拟接受LHON眼部基因治疗者，均需签署《Leber基因治疗知情同意书》和《玻璃体腔注射知情同意书》。
2.2 围手术期规范：
1拟参与LHON眼部基因治疗活动的医护人员，术前需和患者充分建立信任关系，及时了解患者的思想动态，做好心理疏导及安抚工作；
2拟参与LHON眼部基因治疗活动的医护人员，要抱着高度的责任心和同情心参与本项医疗活动；
3拟参与LHON眼部基因治疗活动的医护人员，术前需做好手术室及医疗用品的消毒，做好术中耗材准备；
4在LHON眼部基因治疗活动中，医护人员需严格遵守手术分级制度及无菌操作原则。
2.3 术前检查
1.拟接受LHON眼部基因治疗者，签署《Leber基因治疗知情同意书》；
2.拟接受LHON眼部基因治疗者，做好行程规划，带齐身份证、医保卡及既往完整的病史资料；
3.全身检查：血液分析、尿液分析、肝肾功能、凝血功能、感染性疾病筛查、免疫全套(细胞免疫：CD3，CD4，CD8；体液免疫：IgA,IgM，IgG)；心电图，胸部透视；
4.眼部专科检查：视力、眼压、裂隙灯检查、眼底检查；眼底照相、视神经OCT、视野检查、VEP，且该系列检查6个月内不少于三次；术前眼前节照相一次；
5.体液免疫反应：AAV2体液免疫检查；
6.基因检测突变位点为mtDNA 11778点突变。
2.4 术前预处理
1确定接受LHON眼部基因治疗者，术前7天起口服激素(美卓乐)，每日一次，每次剂量为32mg/60kg；
2体液免疫反应：AAV2体液免疫检查，确定患者用药前rAAV2免疫处于低水平；
3术前一天局部应用抗生素眼药水及眼膏，冲洗泪道结膜囊。
2.5 玻璃体腔注射
1.层流手术间做好消毒措施及手术耗材准备；
2.术前常规消毒铺巾，做好面部及眼部消毒，做好术眼标记；
3.基因药物由专人保管。药物开取前，核对患者信息，并确定药物包装完整无污染，严格做好药物外包装消毒；药物抽取时，缓慢平稳操作，一次完成药物抽吸，勿反复抽吸及触碰瓶壁，避免药物污染；药物抽吸后，需半小时内完成注射。注射完成后，保留药物包装及剩余药物，-20℃保存，备查。(注意：抽药过程中需绝对无菌操作，如药物坠落地面，或抽吸针头触碰污染区，药物均需废弃，任何有污染风险的药物均不可注入患者玻璃体腔。)
4.玻璃体腔注射具体操作规范：注射前器械及药物等准备充分，助手做好消毒铺巾等常规玻璃体腔注射操作。基因药物由助手从干冰中取出，用手心握紧2-3min直至药物从固体融化为液体，整个过程注意无菌操作。然后将纱布用碘伏浸湿消毒瓶盖瓶身2遍，再用干纱布擦干，注意均为顺时针方向，防止瓶口开放，碘伏渗入。消毒完毕，打开瓶盖，手术者用胰岛素针吸取药物，体积调整为0.05ml。助手同时做好注射眼麻醉、消毒、开睑等准备，0.5％聚维酮碘冲洗结膜囊3次，按照玻璃体腔注射规范进行玻璃体腔注射。拨出针头的后立即用棉签压住注射点并按摩10秒钟，防止药物渗漏。
5.药物注射完成后，抗生素眼膏涂术眼，无菌纱布包盖术眼，患者平躺20min后返回病房；嘱勿揉眼；
6.注射完毕注意将基因药物回收。
2.6 术后用药：
1全身用药：(1)磷酸肌酸钠ivdrip，2g/60kg，Qd*3d(包括手术当天)
(2)甲强龙ivdrip，80mg/60kg，Qd*3d(包括手术当天)
(3)术后第三天，甲强龙改为美卓乐，po，40mg/60kg；Qd*4d；
术后第二周，美卓乐减量，po，32mg/60kg；Qd*7d；
术后第三周，美卓乐减量，po，24mg/60kg；Qd*7d；
术后第四周，美卓乐减量，po，16mg/60kg；Qd*7d；
术后第五周，美卓乐减量，po，8mg/60kg；Qd*7d；
术后第六周，美卓乐减量，po，6mg/60kg；Qd*7d；
术后第七周，美卓乐减量，po，4mg/60kg；Qd*7d。
2局部用药：
(1)妥布霉素地塞米松滴眼液：滴术眼，Qid*1周；
(2)妥布霉素地塞米松眼膏：滴术眼，Qn*1周；
2.7 术后检查：
1术后第一天：视力、眼压、裂隙灯、眼底；眼前节照相、眼底照相。
术后第二天：视力、眼压、裂隙灯、眼底；
术后第三天：视力、眼压、裂隙灯、眼底。
2术后检查须由专业人员操作，所有检查报告需经副主任职称以上医师审核签字后，方可告知患者；禁止非专业人员或不熟悉该领域的医护人员擅自为患者检查或为患者解答病情；
3注意：术后检查不宜过多，以不超过术前专科检查项目内容为宜；所有检查前，均需酒精擦拭仪器与患者接触部位；检查完成后，需给予抗生素眼药水点眼；禁止将污染的眼药水及眼膏滴入结膜囊。
2.8 术后复查：
1.告知所有接受LHON眼部基因治疗者，需在与医师约定的时间前往医院复诊；
2.告知所有接受LHON眼部基因治疗者，如有任何不适，随时就诊。
二、试验用药品的剂型、剂量、给药途径、给药方法、给药次数、疗程和有关合并用药的规定
1本研究所用临床级rAAV2-ND4剂型为注射剂；所述重组腺相关病毒rAAV2-ND4为实施例1制备的，含有SEQ ID NO.:6所示的融合核酸的重组腺相关病毒。
2剂量0.2ml；
3给药途径：腔内注射；
4给药方法：局麻下玻璃体腔注药0.05ml；
5给药次数：单次；
三结果
1有效性研究
疗效评价：国际视力提高标准指南显示，视力提高0.3logMAR(15个字母)即显著提高，提高0.2logMAR(10个字母)为提高。提高0.1logMAR(5个字母)以下为未提高
截至2018年6月27日为止共治疗病人145例患者。治疗后观察3天的结果显示：第一天显著提高0.3logMAR的有24例(16.55％)，提高0.2logMAR的有6例(4.14％)，总有效率20.69％，下降2例(1.38％)。第二天显著提高0.3logMAR的有35例(24.14％)，提高0.2logMAR的有12例(8.22％)，总有效率32.41％。第三天显著提高0.3logMAR的有42例(28.96％)，提高0.2logMAR的有11例(7.59％)，总有效率36.55％。
表1 145例患者治疗3天内疗效分析
Figure PCTCN2018113799-appb-000010
129例患者完成一月复查，视力显著提高0.3logMAR的有55例(42.66％)，提高0.2logMAR的有16例(12.40％)。一月复查视力提高总有效率为55.04％，视力下降9例(6.98％)。67例病人完成3月后复查，视力显著提高0.3logMAR的有37例(55.22％)，提高0.2logMAR的有8例(11.94％)，三月复查视力提高总有效率高达67.16％，下降4例(5.97％)。
表2患者治疗后复查疗效分析
Figure PCTCN2018113799-appb-000011
2安全性研究
分别检测了术后三天，一个月和三个月的眼部不良反应。术后三天除了21例患者有轻微的高眼压，无其他明显并发症。术后一个月检查，只有17例高眼压，术后三个月仅剩5例，无其他不良反应。
表3基因治疗Leber病患者出现的常见并发症统计
  术后前三天(n＝141) 术后第一月(n＝102) 术后第三月(n＝41)
前房炎症 0 0 0
玻璃体炎 0 0 0
高眼压 21 17 5
白内障摘除 0 0 0
角膜炎 0 0 0
玻璃体积血 0 0 0
过敏性结膜炎 0 0 0
眼痛 0 0 0
3.安全性结果比较
截至2018年6月5日为止共治疗病人143例，其中还有7名阿根廷国际患者，发病时间分为2年以内组和2年以上组，定期进行安全性检查。我们分别检测了术后三天，一个月和三个月的眼部不良反应。术后三天除了21例患者有轻微的高眼压，属轻度并发症，可自行恢复，无明显其他严重并发症。术后一个月检查有17例高眼压，术后三个月仅剩5例，无其他不良反应(见表1)。其中5月13日，对7名阿根廷患者进行了安全性检查，结果显示，全部安全，仅有1例轻微高眼压，药物治疗后恢复正常。法国Gensight公司也进行了类似的工作，但他们根据发病时间分为6个月以内和6个月到12个月患者进行临床治疗试验。一般来说，发病时间较短患者的视神经节细胞损坏程度较轻，基因治疗预后效果最好。随着发病时间的延长，预后也越差。他们治疗的15例病人，除了10例高眼压外，出现了2例选择性白内障摘除、2个严重的前房炎症和玻璃体炎症事件以及其他多种不良反应，如角膜炎、玻璃体积血、过敏性结膜炎、眼痛等(见表1)。美国Guy教授临床治疗15例病人，患者被分为发病时间大于12个月组和发病时间小于12个月组，治疗发生了2例葡萄膜炎，1例角膜炎，1眼痛和1例高眼压等不良反应。从上面数据可以看出，本发明的基因治疗的安全性显著高于法国Gensight公司与美国Guy教授。
表4本发明基因治疗Leber’s病患者出现的常见并发症统计及与法国Gensight公司和美国Guy教授的结果比较
Figure PCTCN2018113799-appb-000012
*属于轻度并发症(可自行恢复)，其余都属于重度并发症，需要进行干预治疗。
葡萄膜炎的病因种类繁多，每种类型的临床表现、进展规律和治疗所用药物及时间都不尽相同，因此，医生需根据患者的具体情况制定出适合患者的个体化治疗方案。
很多类型的葡萄膜炎可能合并全身感染和免疫性疾病，且需要长期治疗，有的甚至要2-3年以上，患者需要了解治疗此类病的复杂性、艰巨性和长期性，要有足够的耐心和信心，积极配合医生治疗，才能获得较好的治疗效果。
很多类型的葡萄膜炎通常需要使用激素和免疫抑制剂治疗。在西方，大约有四分之一的葡萄膜炎患者需要激素和免疫抑制剂系统治疗，即便如此，也会有35％的患者视力致残。 另外不管医生有多么好的技术和经验，治疗方案多么优化、多么个体化，在治疗过程中仍不可避免地出现一些副作用。免疫抑制剂常见的副作用有肝肾功能损害、血糖升高、高血压、不育、神经及精神系统异常、恶心、呕吐、乏力等。
高眼压属于轻度并发症，是眼科手术中最常见的并发症，一般情况下患者可以自行恢复到正常水平，还可以用降眼压眼药水后，眼压可以下降到正常水平。
综上所述，本发明治疗方法可以有效治疗眼部疾病(如Leber遗传性视神经病变)，并且没有相关并发症，如葡萄膜炎等，副作用很小，非常安全。
实施例3
将实施例2中的含有SEQ ID NO.:6所示融合核酸的重组腺相关病毒rAAV2-ND4替换为含有SEQ ID NO.:5所示融合核酸的重组腺相关病毒，其它实验方法和实验中所用的试剂与实施例2完全相同。结果表明，该治疗方法可以有效治疗眼部疾病，同时也没有相关并发症，如葡萄膜炎等，副作用很小，非常安全。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种药物组合，其特征在于，所述药物组合包括：
    (A)第一药物，所述第一药物含有(a)第一活性成分，所述第一活性成分为重组腺相关病毒，所述重组腺相关病毒包含编码多肽的外源核酸，所述多肽为恢复或增强视神经或视网膜细胞功能的多肽；和(b)药学上可接受的载体；和
    (B)第二药物，所述第二药物为美卓乐。
  2. 如权利要求1所述的药物组合，其特征在于，所述多肽选自下组：ND1、ND4、ND6、或其组合。
  3. 如权利要求1所述的药物组合，其特征在于，所述重组腺相关病毒含有融合核酸，所述融合核酸包含所述编码多肽的外源核酸和线粒体靶向肽的编码序列。
  4. 如权利要求3所述的药物组合，其特征在于，所述融合核酸从5’-3’端的结构为COX10-ND4-UTR。
  5. 如权利要求3所述的药物组合，其特征在于，所述融合核酸序列如SEQ ID NO.:5或6所示。
  6. 如权利要求1所述的药物组合，其特征在于，所述第一药物的剂型为注射剂型；和/或所述第二药物的剂型优选为口服制剂。
  7. 一种药盒，其特征在于，所述药盒包括：
    (A)第一制剂，所述第一制剂为重组腺相关病毒，所述重组腺相关病毒包含编码多肽的外源核酸，所述多肽为恢复或增强视神经或视网膜细胞功能的多肽；和
    (B)第二制剂，所述第二制剂为美卓乐。
  8. 如权利要求1所述的药物组合的用途，其特征在于，所述药物组合用于制备用于恢复或提高受试者视力和/或治疗眼部疾病的药物或制剂。
  9. 一种治疗眼部疾病的方法，其特征在于，所述方法包括步骤：给需要的对象，施用权利要求1所述的药物组合或权利要求7所述的药盒。
  10. 一种治疗方法，其特征在于，所述治疗方法包括步骤：在向需要的对象眼内施用重组腺相关病毒之前、同时和/或之后，向所述对象施用美卓乐。
  11. 如权利要求10所述的治疗方法，其特征在于，所述治疗方法包括步骤：
    (1)术前预处理，所述术前预处理包括向所需要的对象施用美卓乐；
    (2)向所述对象眼内施用重组腺相关病毒，所述重组腺相关病毒包含编码多肽的外源核酸，所述多肽为恢复或增强视神经或视网膜细胞功能的多肽；和
    (3)术后处理，所述术后处理包括向所述对象施用美卓乐。
  12. 一种美卓乐的用途，其特征在于，用于制备一制剂或组合物，所述制剂或组合物用于降低眼内施用重组腺相关病毒的副作用或并发症。
  13. 如权利要求12所述的用途，其特征在于，所述副作用或并发症包括：白内障摘 除、葡萄膜炎、玻璃体炎、角膜炎、前房炎症、玻璃体积血、眼痛、过敏性结膜炎、高眼压、或其组合。
  14. 如权利要求12所述的用途，其特征在于，所述副作用或并发症包括：白内障摘除、玻璃体炎、角膜炎、前房炎症、玻璃体积血、眼痛、过敏性结膜炎、或其组合。
  15. 如权利要求12所述的用途，其特征在于，所述降低为预防性地降低，较佳地为降低眼内施用重组腺相关病毒的副作用或并发症的发生率。
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