WO2020077756A1 - Nd4蛋白的编码序列及其应用 - Google Patents

Nd4蛋白的编码序列及其应用 Download PDF

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WO2020077756A1
WO2020077756A1 PCT/CN2018/118662 CN2018118662W WO2020077756A1 WO 2020077756 A1 WO2020077756 A1 WO 2020077756A1 CN 2018118662 W CN2018118662 W CN 2018118662W WO 2020077756 A1 WO2020077756 A1 WO 2020077756A1
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sequence
nucleic acid
protein
cells
vector
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PCT/CN2018/118662
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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 KR1020247001775A priority Critical patent/KR20240014102A/ko
Priority to KR1020217001385A priority patent/KR102627561B1/ko
Priority to CN202110786772.2A priority patent/CN113528510A/zh
Priority to CN202110786630.6A priority patent/CN113476484A/zh
Priority to SG11202012044QA priority patent/SG11202012044QA/en
Priority to MX2020013772A priority patent/MX2020013772A/es
Priority to CA3103740A priority patent/CA3103740A1/en
Priority to JP2021521870A priority patent/JP2021529001A/ja
Priority to AU2019296451A priority patent/AU2019296451B2/en
Priority to CN201980003485.0A priority patent/CN110876269B/zh
Priority to PCT/CN2019/094136 priority patent/WO2020001657A1/en
Priority to BR112020026361-3A priority patent/BR112020026361A2/pt
Priority to EP19826653.8A priority patent/EP3814492A4/en
Priority to CA3109432A priority patent/CA3109432A1/en
Priority to EP19853225.1A priority patent/EP3840785A4/en
Priority to JP2021509893A priority patent/JP7403852B2/ja
Priority to CN201980054770.5A priority patent/CN112584874A/zh
Priority to PCT/CN2019/101538 priority patent/WO2020038352A1/en
Priority to KR1020217007727A priority patent/KR20210068014A/ko
Priority to SG11202101032VA priority patent/SG11202101032VA/en
Priority to AU2019323434A priority patent/AU2019323434A1/en
Priority to US16/836,644 priority patent/US11034954B2/en
Publication of WO2020077756A1 publication Critical patent/WO2020077756A1/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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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/99—Oxidoreductases acting on NADH or NADPH (1.6) with other acceptors (1.6.99)
    • C12Y106/99003—NADH dehydrogenase (1.6.99.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the field of biological preparations, in particular to the coding sequence of ND4 protein and its application.
  • Leber hereditary optic neuropathy is a degenerative vision disorder, usually manifested as bilateral loss of central vision. The average age of onset is in the middle of 20 years, usually no pain within a few weeks to several months, until the vision of both eyes deteriorates below 0.1, which seriously affects the patient's quality of life.
  • LHON is caused by a mutation in the mitochondrial gene and is related to a mutation in one of the three mitochondrial genes of NADH ubiquinone oxidoreductase, the complex I subunit of the mitochondrial respiratory chain.
  • G3460A mutation that affects the ND1 gene the T14484C mutation that affects the ND6 gene, and the G11778A mutation that affects the ND4 gene are considered to be the main causes of LHON, and each mutation has a significant risk of permanent vision loss. All of these are related to the focal degeneration of retinal ganglion cells.
  • CN 102634527 B discloses a recombinant human NADH dehydrogenase subunit 4 protein gene (ND4 gene) and its expression vector construction method.
  • COX10 encodes a 28 amino acid peptide chain to guide the ND4 protein into mitochondria in.
  • CN104450747A discloses a recombinant adeno-associated virus-NADH dehydrogenase subunit 4 (ND4) full-length gene and a medicament for treating Leber hereditary optic neuropathy.
  • the gene consists of CAG promoter sequence, ND4 coding sequence with COX10 mitochondrial localization sequence and UTR.
  • the purpose of the present invention is to provide an expression system and preparation method of human NADH dehydrogenase subunit 4 protein with high transfection efficiency and good therapeutic effect.
  • the object of the present invention is to provide an optimized nucleic acid sequence, vector and preparation method encoding human NADH dehydrogenase subunit 4 protein.
  • nucleotide sequence encoding the human NADH dehydrogenase subunit 4 protein, and the nucleotide sequence is selected from the group consisting of:
  • nucleotide sequence has ⁇ 95% identity with the nucleotide sequence shown in SEQ ID NO.:1, preferably ⁇ 98%, more preferably ⁇ 99%;
  • the nucleotide sequence includes a DNA sequence, a cDNA sequence, or an mRNA sequence.
  • the nucleotide sequence includes a single-stranded sequence and a double-stranded sequence.
  • the nucleotide sequence includes a nucleotide sequence that is completely complementary to SEQ ID NO.:1.
  • a fusion nucleic acid comprising the nucleotide sequence encoding the human NADH dehydrogenase subunit 4 protein according to the first aspect of the present invention.
  • the fusion nucleic acid further comprises a sequence selected from the group consisting of a mitochondrial targeting peptide coding sequence, a UTR sequence, or a combination thereof.
  • the coding sequence of the mitochondrial targeting peptide includes: COX10 sequence and / or OPA1 sequence.
  • the COX10 coding sequence has the sequence shown in SEQ ID NO .: 2.
  • the coding sequence of OPA1 has the sequence shown in SEQ ID NO.:3.
  • the UTR sequence includes 3'-UTR and / or 5'-UTR, preferably 3'-UTR.
  • the UTR sequence has the sequence shown in SEQ ID NO .: 4 or 11.
  • 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 nucleotide sequence according to the first aspect of the present invention.
  • Z3 is a 3'-UTR sequence.
  • the Z1 is a COX10 coding sequence or an OPA1 coding sequence.
  • the structure of the fusion nucleic acid from the 5'-3 'end is COX10-ND4-UTR.
  • sequence of the fusion nucleic acid is shown in SEQ ID NO .: 5; wherein,
  • 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 structure of the fusion nucleic acid from the 5'-3 'end is OPA1-ND4-UTR.
  • the fusion nucleic acid sequence is shown in SEQ ID NO.:10.
  • sequence is as shown in SEQ ID NO .: 10 in the fusion nucleic acid
  • Bit 1-266 is the coding sequence of OPA1;
  • Positions 267-1646 are the nucleotide sequence encoding human NADH dehydrogenase subunit 4 protein
  • Positions 1647-2271 are 3'-UTR sequences.
  • each nucleotide linking sequence is 1-30 nt, preferably 1-15 nt, more preferably 3-6 nt.
  • the nucleotide linking sequence is derived from a nucleotide linker sequence formed by restriction enzyme digestion.
  • a vector containing the nucleotide sequence according to the first aspect of the present invention or the fusion nucleic acid according to the second aspect of the present invention is provided.
  • the vector comprises one or more promoters, the promoter is operable with the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, selectable marker , Nucleic acid restriction sites, and / or homologous recombination sites.
  • the vector is selected from the group consisting of plasmids and viral vectors.
  • the vector is selected from the group consisting of a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, or a combination thereof.
  • the vector is an AAV vector.
  • the serotype of the AAV vector is selected from: AAV2, AAV5, AAV7, AAV8, or a combination thereof.
  • the vector includes DNA virus and retrovirus vector.
  • the vector is an AAV vector containing or inserted with the nucleotide sequence as described in the first aspect of the invention or the fusion nucleic acid as described in the second aspect of the invention; preferably, the AAV vector plasmid pSNaV .
  • the backbone of the vector is adeno-associated virus vector plasmid pSNaV.
  • the vector is used to express recombinant human NADH dehydrogenase subunit 4 protein.
  • a host cell containing the vector according to the third aspect of the present invention, or a foreign nucleotide as described in the first aspect of the present invention integrated into its chromosome Sequence or fusion nucleic acid according to the second aspect of the invention.
  • the host cell is a mammalian cell, and the mammal includes human and non-human mammals.
  • the host cell is selected from the group consisting of HEK293 cells, photoreceptor cells (including cone cells and / or rod cells), other visual cells (such as biganglionic cells), (optical) nerve cells, Or a combination thereof.
  • the host cell is selected from the group consisting of rod cells, cone cells, light-giving bipolar cells, light-extracting bipolar cells, horizontal cells, ganglion cells, amacrine cells, or combination.
  • the host cell is a (retinal) ganglion cell.
  • a fifth aspect of the present invention there is provided the use of the carrier according to the third aspect of the present invention, for preparing a formulation or composition for restoring the vision of a subject and / or treating the eye disease.
  • the eye disease is a degenerative disease of the optic nerve.
  • the preparation or composition is used to treat focal degeneration of retinal ganglion cells.
  • the formulation or composition is used to treat hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).
  • hereditary optic neuropathy preferably Leber's hereditary optic neuropathy (LHON).
  • a pharmaceutical preparation comprising (a) the carrier according to the third aspect of the present invention, and (b) a pharmaceutically acceptable carrier or excipient.
  • the dosage form of the pharmaceutical preparation is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.
  • the vector is selected from the group consisting of a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, or a combination thereof.
  • the vector is an AAV vector.
  • the content of the carrier in the pharmaceutical preparation is 1 ⁇ 10 9 -1 ⁇ 10 16 , preferably 1 ⁇ 10 12 -1 ⁇ 10 13 viruses / ml.
  • the pharmaceutical preparation is used to treat ocular diseases, preferably to treat degenerative diseases of the optic nerve, and more preferably to treat focal degeneration of retinal ganglion cells.
  • the pharmaceutical preparation is used to treat hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).
  • hereditary optic neuropathy preferably Leber's hereditary optic neuropathy (LHON).
  • a method of treatment comprising applying the vector of the third aspect of the present invention to a subject in need.
  • the vector is selected from the group consisting of a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, or a combination thereof.
  • the vector is an AAV vector.
  • the carrier is introduced into the eyes of the desired subject.
  • the objects in need include human and non-human mammals.
  • the treatment method is a method for treating eye diseases.
  • the eye disease is hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).
  • LHON Leber's hereditary optic neuropathy
  • An eighth aspect of the present invention provides a method for preparing recombinant human NADH dehydrogenase subunit 4 protein, comprising the steps of: cultivating the host cell of the fourth aspect of the present invention to obtain a recombinant human NADH dehydrogenase subunit 4 protein.
  • the ninth aspect of the present invention provides a fusion protein encoded by the fusion nucleic acid according to the second aspect of the present invention.
  • Figure 1 shows the nucleotide sequence comparison of the ND4 fusion nucleic acid and the optimized ND4 fusion nucleic acid.
  • the "ND4 gene” line sequence is an unoptimized ND4 fusion nucleic acid sequence
  • the "human optimized ND4 gene” line sequence is an optimized ND4 fusion nucleic acid sequence.
  • Figure 2 shows the results of PCR nucleic acid electrophoresis to verify ND4 (lane A) and optimized ND4 (lane B) gene cloning results.
  • Figure 3 shows the fluorescence observation results of rAAV2-ND4 (A) and rAAV2-optimized ND4 (B) infected 293T cells.
  • Fig. 4 shows the expression levels of rAAV2-optimized ND4 and rAAV2-ND4 on 293T cells using ⁇ -actin as the internal reference protein.
  • Lane A is rAAV2-ND4 and lane B is rAAV2-optimized ND4.
  • Figure 5 shows the expression level of ND4 protein after ⁇ -actin as the internal reference protein, rAAV2-optimized ND4 and rAAV2-ND4 respectively infected 293T cells.
  • Fig. 6 shows the expression levels of rAAV2-optimized ND4 and rAAV2-ND4 on rabbit optic nerve cells using ⁇ -actin as the internal reference protein.
  • the lane A is rAAV2-optimized ND4 and lane B is rAAV2-ND4.
  • Figure 7 shows the expression level of ND4 protein after ⁇ -actin as the internal reference protein, rAAV2-optimized ND4 and rAAV2-ND4 respectively infect the rabbit optic nerve cells.
  • Figure 8 shows a fundus photograph taken under a rabbit eye glass microscope, where A is the injection of rAAV2-ND4 virus and B is the injection of rAAV2-optimized ND4 virus.
  • Fig. 9 shows the results of microscopic examination of HE slices in rabbit eyes, where A is the injection of rAAV2-ND4 virus and B is the injection of rAAV2-optimized ND4 virus.
  • the present inventors targeted and optimized the coding sequence of the recombinant human NADH dehydrogenase subunit 4 protein gene coding, thereby obtaining a highly efficient transcription in mammalian (such as human) cells And highly express the nucleotide sequence and fusion nucleic acid of ND4 protein, and construct a recombinant expression vector of recombinant human NADH dehydrogenase subunit 4 protein.
  • the transcription efficiency and translation efficiency of the ND4 coding sequence SEQ ID NO.:1 were significantly improved, and the expression of recombinant human NADH dehydrogenase subunit 4 protein was increased by more than 3 times.
  • the term “about” may refer to a value or composition within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • 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”.
  • Sequence identity by comparing two aligned along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) Sequence and determine the number of positions where the same residue appears. Generally, this is expressed as a percentage.
  • a predetermined comparison window which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein
  • the terms “subject” and “subject in need” refer to any mammal or non-mammal. Mammals include but are not limited to humans, vertebrates such as rodents, non-human primates, cattle, horses, dogs, cats, pigs, sheep, goats.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus of dependent viruses of the family Parvoviridae. It is the simplest single-stranded DNA-defective virus found so far and requires a helper virus (usually an adeno-associated virus). Virus) involved in replication. It encodes the cap and rep genes in the inverted repeat sequence (ITR) at both ends. ITRs play a decisive role in virus replication and packaging. The cap gene encodes a viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.
  • Recombinant adeno-associated virus vector is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, the time to express foreign genes in vivo The long-term characteristics are regarded as one of the most promising gene transfer vectors, and are widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been thoroughly understood, especially its application in various cells, tissues and in vivo experiments has accumulated a lot of data.
  • rAAV is used in gene therapy research of various diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, disease model construction, gene preparation Knock out rats and so on.
  • the vector is a recombinant AAV vector.
  • AAVs are relatively small DNA viruses that can integrate into the genome of the cells they infect in a stable and site-specific manner. They can infect a large series of cells without any effect on cell growth, morphology or differentiation, and they do not seem to involve human pathology.
  • the AAV genome has been cloned, sequenced and characterized.
  • the AAV contains about 4700 bases and contains about 145 base inverted terminal repeat (ITR) regions at each end, which serve as the origin of replication of the virus.
  • ITR inverted terminal repeat
  • the rest of the genome is divided into two important regions with capsidization functions: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.
  • AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated by reference in their entirety. The preparation of hybrid carriers is described in, for example, PCT Application No. PCT / US2005 / 027091, the disclosure content of which is incorporated herein by reference in its entirety. The use of vectors derived from AAV for in vitro and in vivo gene transfer has been described (see, eg, International Patent Application Publication Nos. WO91 / 18088 and WO93 / 09239; US Patent Nos.
  • Replication-defective recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (eg, adenovirus): the nucleic acid sequence of interest is flanked by two AAV inverted terminal repeat sequences (ITR) Plasmids in the region, and plasmids carrying AAV capsidization genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.
  • a human helper virus eg, adenovirus
  • the recombinant vector is capsidized into viral particles (for example, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 And AAV16 AAV virions). Therefore, the present disclosure includes recombinant viral particles (recombinant because they contain recombinant polynucleotides) containing any of the vectors described herein. Methods for generating such particles are known in the art and are described in US Patent No. 6,596,535.
  • the technical problem to be solved by the present invention is to overcome the technical defects in the prior art that NADH dehydrogenase subunit 4 protein transfection efficiency is not high and the therapeutic effect is not good.
  • the invention provides a targeted NADH dehydrogenase subunit 4 protein, and a preparation method and application thereof. It has been found through research that the optimized ND4 gene sequence (SEQ ID NO .: 1) of the present invention makes ND4 protein expression more efficient, and more ND4 protein plays a physiological role in the optic ganglion cells of patients.
  • the nucleotide sequence of the nucleic acid encoding human NADH dehydrogenase subunit 4 protein according to the present invention is shown in SEQ ID NO .: 1.
  • the nucleic acid encoding human NADH dehydrogenase subunit 4 protein has a total length of 1380 bp.
  • the nucleic acid encoding human NADH dehydrogenase subunit 4 protein is also called ND4 optimized gene or ND4 optimized nucleic acid.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the nucleotide is DNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the full-length nucleotide sequence of the present invention or a fragment thereof can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the related nucleotide sequences disclosed, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art can be used as Template, the relevant sequence is amplified.
  • the DNA sequence encoding the polypeptide (or fragment or derivative thereof) of the present invention can be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector or polypeptide coding sequence of the present invention.
  • the above polynucleotide, vector or host cell may be isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances present in the natural state.
  • nucleotide sequence is shown in SEQ ID NO .: 1.
  • the relevant sequence can be obtained in large quantities by the recombination method. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the propagated host cell by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the length of the fragments is short.
  • a long sequence can be obtained by synthesizing multiple small fragments and then connecting them.
  • the method of amplifying DNA / RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods.
  • the amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector or protein coding sequence of the present invention, and a method of using the host cell to express ND4 protein by recombinant technology.
  • the polynucleotide sequences of the present invention can be used to obtain host cells (such as mammalian cells) expressing the ND4 protein of the present invention. Generally, it includes the step of introducing the polynucleotide according to the first aspect of the present invention or the vector according to the third aspect of the present invention into a host cell.
  • an expression vector containing the DNA sequence encoding the polypeptide of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology and so on.
  • the DNA sequence can be effectively linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors containing the appropriate DNA sequence and the appropriate promoter or control sequence described above can be used to transform a suitable host cell so that it can express the polypeptide.
  • the host cell may be a prokaryotic cell, or a lower eukaryotic cell, or a higher eukaryotic cell, such as a mammalian cell (including human and non-human mammals). Representative examples are: animal cells such as CHO, NS0, COS7, or 293 cells.
  • animal cells such as CHO, NS0, COS7, or 293 cells.
  • the host cell is selected from the group consisting of rod cells, cone cells, light-giving bipolar cells, light-extracting bipolar cells, horizontal cells, ganglion cells, amacrine cells, or combination.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • transformation can also be carried out by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by a conventional method and express the protein encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the cultivation is carried out under conditions suitable for the growth of host cells.
  • the selected promoter is induced by an appropriate method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the polypeptide in the above method may be expressed in a cell, on a cell membrane, or secreted out of the cell. If necessary, the protein can be separated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with protein precipitation agent (salting out method)
  • centrifugation osmotic disruption
  • ultra-treatment ultra-centrifugation
  • molecular sieve chromatography gel filtration
  • adsorption layer Analysis ion exchange chromatography
  • HPLC high performance
  • the present invention changes the COX10 mitochondrial coding sequence to a nucleic acid coding sequence (as shown in SEQ ID NO: 2) and performs ND4 nucleotide sequence optimization (as shown in SEQ ID NO: 1, in the present invention is referred to as optimizing ND4 gene / Nucleic acid), this sequence is specially optimized, and the transcription efficiency and translation efficiency are significantly improved.
  • the homology of the optimized COX10 + ND4 and unoptimized COX10 + ND4 sequences is 76.16% (1115/1464).
  • an optimized coding sequence of recombinant human NADH dehydrogenase subunit 4 protein with high transcription efficiency and translation efficiency is provided.
  • the coding sequence is shown in SEQ ID NO .: 1.
  • optically ND4 coding sequence and “optimized ND4 coding gene” both refer to a nucleotide sequence used to encode recombinant human NADH dehydrogenase subunit 4 protein, and the nucleotide sequence Encoding the amino acid sequence shown in SEQ ID NO.:1.
  • the present invention also provides a fusion nucleic acid comprising the nucleic acid encoding the human NADH dehydrogenase subunit 4 protein.
  • fusion nucleic acid refers to a nucleic acid composed of two or more nucleotide sequences from different sources, or two or more nucleosides from the same source but whose natural positions are not connected to each other Nucleic acid formed by linking acid sequences.
  • the protein encoded by the fusion nucleic acid of the present invention is called a fusion protein. In the present invention, it is an ND4 optimized protein.
  • the fusion nucleic acid is operably linked to the coding sequence and / or UTR sequence of the mitochondrial targeting peptide in the nucleic acid encoding the human NADH dehydrogenase subunit 4 protein.
  • the coding sequence of the mitochondrial targeting peptide is as shown in SEQ ID NO .: 2
  • the coding sequence of the COX10, the nucleic acid encoding the human NADH dehydrogenase subunit 4 protein, and the UTR sequence are sequentially arranged from the 5 ′ end to the 3 ′ end .
  • the sequence of the fusion nucleic acid is shown in SEQ ID NO .: 5.
  • the nucleotide sequence of the fusion nucleic acid has a full length of 2889 bp, from 1 bp to 84 bp is the optimized COX10 sequence (a total of 84 bp); 85 bp to 1464 bp is the optimized ND4 gene, that is, the encoding human NADH
  • the nucleic acid of the hydrogenase 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 functions; 3'UTR is a non-coding sequence designed behind the ND4 protein, and its role is to stabilize the coding sequence of the mitochondrial targeting peptide and the expression of ND4. Among them, the homology of the optimized COX10 + ND4 and unoptimized COX10 + ND4 sequences is 76.16%.
  • sequence of the fusion nucleic acid is shown in SEQ ID NO.:10.
  • the invention also provides an expression vector for ND4 protein, which contains the optimized ND4 coding sequence of the invention.
  • sequence information By providing the sequence information, skilled artisans can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transduction into cells.
  • the nucleic acid sequence encoding the ND4 protein is provided as a vector, preferably an expression vector.
  • it can be provided as a gene therapy vector that is preferably suitable for transduction and expression in retinal target cells.
  • the vector may be viral or non-viral (eg plasmid).
  • Viral vectors include those derived from the following: adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes virus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV) including mutant forms , HIV, pox virus and SV40.
  • the viral vector is replication-defective, although it is envisaged that it may be replication-deficient, capable of replication, or conditionally replicated.
  • Viral vectors can usually maintain an extrachromosomal state without integrating into the genome of the target retinal cell.
  • a preferred viral vector for introducing ND4 protein-encoding nucleic acid sequences into retinal target cells is an AAV vector, such as a self-complementary adeno-associated virus (scAAV).
  • Selective targeting can be achieved using specific AAV serotypes (AAV serotype 2 to AAV serotype 12) or modified versions of any of these serotypes (including AAV 4YF and AAV 7m8 vectors).
  • the viral vector can be modified to delete any non-essential sequences.
  • viruses can be modified to delete all or part of the IX gene, Ela and / or Elb genes.
  • helper viruses such as adenovirus
  • replication is very inefficient.
  • the replication gene and capsid gene are provided in trans (in the pRep / Cap plasmid), and only the 2ITR of the AAV genome is retained and packaged into the virion, while the adenovirus gene Provided by adenovirus or another plasmid. Similar modifications can also be made to lentiviral vectors.
  • Viral vectors have the ability to enter cells.
  • non-viral vectors such as plasmids can be complexed with agents to facilitate the uptake of viral vectors by target cells.
  • agents include polycationic agents.
  • delivery systems such as liposome-based delivery systems can be used.
  • the carrier for use in the present invention is preferably suitable for use in vivo or in vitro, and preferably suitable for use in humans.
  • the vector will preferably contain one or more regulatory sequences to direct the expression of the nucleic acid sequence in retinal target cells. Regulatory sequences can include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operably linked to the nucleic acid sequence.
  • the vector may also include a selectable marker, for example, to determine the expression of the vector in a growth system (eg, bacterial cells) or in retinal target cells.
  • “Operably linked” means that nucleic acid sequences are functionally related to their operably linked sequences so that they are linked in such a way that they affect each other's expression or function.
  • a nucleic acid sequence operably linked to a promoter will have an expression pattern affected by the promoter.
  • the promoter mediates the expression of the nucleic acid sequence linked thereto.
  • the promoter may be constitutive or may be inducible. Promoters can direct ubiquitous expression in inner retinal cells, or neuron-specific expression. In the latter case, the promoter can direct cell type-specific expression, for example, to the optic ganglion cells. Suitable promoters will be known to those skilled in the art.
  • a suitable promoter may be selected from the group consisting of: L7, thy-1, restorer protein, calcium binding protein, human CMV, GAD-67, chicken ⁇ -actin, hSyn, Grm6, Grm6 enhancer SV40 fusion protein .
  • Targeting can be achieved using cell-specific promoters, such as Grm6-SV40 for selective targeting to optic nerve cells.
  • the Grm6 promoter is a fusion of the 200 base pair enhancer sequence of the Grm6 gene and the SV40 eukaryotic promoter.
  • the Grm6 gene encodes a specific metabolic glutamate receptor mGluR6 for optic nerve cells.
  • the preferred sources of the Grm6 gene are mice and humans.
  • Ubiquitous neuronal promoters can be used to achieve ubiquitous expression, examples of which are known and available in the art.
  • One such example is CAG.
  • the CAG promoter is then a fusion of the early CMV enhancer and the chicken ⁇ -actin promoter.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences can also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ sarcoma virus promoter, and human gene promoters, such as but not limited to the actin promoter , Myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered as part of the present invention.
  • an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off the expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.
  • ND4 protein can be used to express in mammalian cells (preferably human, more preferably human optic nerve cells or photoreceptor cells).
  • mammalian cells preferably human, more preferably human optic nerve cells or photoreceptor cells.
  • the present invention preferably uses an adeno-associated virus as an expression vector.
  • the present invention also provides a host cell for expressing ND4 protein.
  • the host cell is a mammalian cell (preferably a human, more preferably a human optic nerve cell or a photoreceptor cell) to increase the expression level of ND4 protein.
  • the present invention provides a formulation or composition comprising (a) the carrier according to the third aspect of the present invention, and (b) a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical preparation is used to treat eye diseases.
  • the pharmaceutical preparation is used to treat hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).
  • hereditary optic neuropathy preferably Leber's hereditary optic neuropathy (LHON).
  • the “active ingredient” in the pharmaceutical composition of the present invention refers to the vector of the present invention, such as a viral vector (including adeno-associated viral vector).
  • the "active ingredients", formulations and / or compositions described in the present invention can be used to treat eye diseases.
  • Safe and effective amount means that the amount of the active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects.
  • “Pharmaceutically acceptable carrier or excipient” means: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficient Low toxicity.
  • “Compatibility” here means that the components of the composition can be blended with the active ingredient of the present invention and between them without significantly reducing the efficacy of the active ingredient.
  • the composition may be liquid or solid, such as a powder, gel or paste.
  • the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.
  • the carrier can be administered to the eye by subretinal or intravitreal administration.
  • the carrier is provided as an injectable liquid.
  • the injectable liquid is provided as a capsule or syringe.
  • Examples of pharmaceutically acceptable carrier parts are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid) , Magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as ), Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate,
  • the composition may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the nucleic acid or fusion nucleic acid encoding ND4 provided by the present invention can produce ND4 protein or ND4 fusion protein in vitro or in vivo, and the fusion protein or the preparation containing the fusion protein can be applied to the preparation of a medicament for treating Leber hereditary optic neuropathy.
  • the optimized nucleic acid encoding human NADH dehydrogenase subunit 4 protein has a higher expression level, resulting in more ND4 fusion proteins being translated, and the COX10 sequence can accurately locate the ND4 fusion protein on the mitochondrial inner membrane, so there are more Many ND4 proteins were transfected into mitochondria.
  • An agent containing COX10-optimized ND4 fusion nucleic acid was injected into the vitreous cavity of the rabbit eye. The agent remained viable in the vitreous cavity and was transfected into optic nerve cells.
  • the optimized ND4 nucleic acid encodes more ND4 protein than the prior art, its transfection efficiency is higher, and it can better treat Leber's hereditary optic neuropathy.
  • the present invention mainly has the following advantages:
  • the gene sequence encoding the recombinant human NADH dehydrogenase subunit 4 protein (ND4) of the present invention has been specially optimized. Compared with the unoptimized DNA coding sequence of ND4, the transcription efficiency and translation efficiency are significantly improved. The optimized sequence ND4 protein expression was significantly increased and the biological activity was high.
  • the optimized COX10 sequence or OPA1 sequence of the present invention can accurately locate the ND4 fusion protein on the mitochondrial inner membrane, so more ND4 protein is transfected into the mitochondria.
  • the optimized ND4 encoding gene (SEQ ID NO .: 1) or fusion nucleic acid of the present invention can very effectively treat Leber's hereditary optic neuropathy and has good safety.
  • ND4 nucleotide sequence (National Biotechnology Information Center reference sequence: yp_003024035.1)
  • the present invention changes the COX10 mitochondrial coding sequence to a nucleic acid coding sequence (as shown in SEQ ID NO: 2 )
  • ND4 nucleotide sequence optimization (as shown in SEQ ID NO: 1, referred to as optimizing ND4 gene / nucleic acid in the present invention), this sequence is specially optimized, the transcription efficiency and translation efficiency are significantly improved, and the optimized COX10 +
  • the homology between ND4 and the unoptimized COX10 + ND4 sequence is 76.16%.
  • the UTR sequence (shown as SEQ ID NO: 4) is connected to the 3 'end of the optimized ND4 gene, and the sequence of the fusion gene (or fusion nucleic acid) is shown as SEQ ID NO .: 5, which is provided by Chengdu Qingke Zixi Synthesized by Biotechnology Co., Ltd.
  • pAAV2-ND4 For the preparation of the control pSNaV / rAAV2 / 2-ND4 (hereinafter referred to as pAAV2-ND4), see CN 102634527 B.
  • 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 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 culture medium is discarded and pAAV2-ND4 and pAAV2-optimized ND4 are 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. Minutes, a little shock after each melt.
  • Virus purification 1) Add solid CsCl to the virus concentrate until the density is 1.41g / ml (refractive index is 1.372); 2) Add the sample to the ultracentrifuge tube and use the pre-made 1.41g / The mlCsCl solution fills the remaining space of the centrifuge tube; 3) Centrifuge at 175,000g for 24 hours to form a density gradient. Collect samples of different densities in order and take samples for titer determination. Collect the fraction enriched with rAAV2 particles; 4) Repeat the above process once. The virus was loaded into a 100kDa dialysis bag and dialyzed against desalting at 4 ° C overnight.
  • OPA1 (sequence shown in SEQ ID NO .: 3) can also be fused with the optimized ND4 gene of the present invention, and the coding sequence of the mitochondrial targeting peptide OPA1 can optimize ND4 gene expression
  • the protein is brought into the inner mitochondrial membrane, so as to achieve targeted expression of the protein mitochondria.
  • rAAV2-ND4-EGFP The virus solutions rAAV2-ND4-EGFP and rAAV2-optimized ND4-EGFP were prepared, and the experimental method was the same as in Example 1. Among them, rAAV2-ND4-EGFP is rAAV2-ND4 virus with EGFP tag, rAAV2-optimized ND4-EGFP is rAAV2-optimized ND4 virus with EGFP tag.
  • the frozen 293T cells After recovering the frozen 293T cells, they were subcultured and grown to about 90% of the T75 flask. The cells were digested with trypsin, the cell pellet was taken, and the cell density was resuspended in DMEM complete medium at 5 ⁇ 10 4 cells / mL. Use 96-well plates to plant cells, add 100 ⁇ L of cell suspension to each well, about 5000 cells / well.
  • rAAV2-ND4 and rAAV2-optimized ND4 (1 ⁇ 10 10 vg / 0.05mL) and PBS were punctured the flat part of the ciliary body at 3 mm from the limbus to enter the vitreous cavity. After intravitreal injection, slit lamp, fundus photography and HE staining were performed. After 30 days of injection, RT-PCR and immunoblotting were performed in each group.
  • RNA and reverse transcription of 293T cells transfected with rAAV2-ND4 and rAAV2-Optimized-ND4 and rabbit optic nerve cells were extracted, and the total RNA was extracted using TRIZOL kit and synthesized by reverse transcription.
  • ⁇ -actin-S CGAGATCGTGCGGGACAT (the sequence is shown in SEQ ID NO: 6);
  • ⁇ -actin-A CAGGAAGGAGGGCTGGAAC (the sequence is shown in SEQ ID NO: 7);
  • ND4-S GCCAACAGCAACTACGAGC (sequence shown in SEQ ID NO: 8);
  • ND4-A TGATGTTGCTCCAGCTGAAG (the sequence is shown in SEQ ID NO: 9);
  • Real-time PCR detection was performed on the Real-time PCR Detection System instrument. Add 12.5 ⁇ L of SYBR Green mix, 8 ⁇ L of ddH 2 O, 1 ⁇ L of each primer to a 0.2 mL PCR reaction tube, 2.5 ⁇ L of cDNA sample, and 25 ⁇ L of the total system. Each sample is used to amplify both the target gene and the internal reference gene ⁇ -actin, and the amplification of each gene is repeated three times. In order to reduce the error during the actual sample loading, the reagents shared in each PCR reaction tube can be added together and then packed separately. After the sample is added, fluorescence quantitative PCR is performed.
  • the relative quantitative method is used to study the difference in gene expression. This method does not need to make a standard curve. With ⁇ -actin as the internal reference gene, the analysis software that comes with the instrument can automatically generate expression values and calculate relative expression.
  • the ND4 protein of 293T cells transfected with rAAV2-ND4 and rAAV2-optimized-ND4 and rabbit nerve cells were extracted, and then subjected to 10% polyacrylamide gel electrophoresis, and the spots were transferred to a polyfluoride membrane (Bio-Rad, Her- cules, CA, USA), used for immunoassay, using ⁇ -actin as the internal reference gene, using automatic image analysis equipment (Li-Cor; Lincoln, NE, USA) to observe and analyze the bands on the film, and the integral of each protein band
  • the optical density is integrated by the normalization method to obtain the corresponding optical density value of the same sample.
  • Statistical analysis is performed using SPSS 19.0 statistical software for statistical analysis.
  • the results of infection of 293T cells by rAAV2-optimized ND4 and rAAV2-ND4 are shown in Figures 6 and 7, respectively.
  • the average expression value of the rAAV2-ND4 group of rabbit optic nerve cells was 0.16, while the average expression value of the rAAV2-optimized ND4 group was 0.48, and the rAAV2-optimized ND4 group was about 3 times that of the rAAV2-ND4 group. There was a significant difference between the two groups (p ⁇ 0.01).
  • the protein level of rAAV2-optimized ND4 group is significantly higher than that of the unoptimized group, which indicates that in terms of translation efficiency, the optimized ND4 encoding nucleotide sequence of the present invention (SEQ ID NO .: 5 85-1464) and the corresponding fusion nucleic acid (SEQ ID NO .: 5 1-2889) translation efficiency is higher.
  • the rabbits in the two groups were examined for slit lamp and intraocular pressure at 1, 3, 7, and 30 days after surgery. All rabbits had no obvious abnormalities, no conjunctival hyperemia, secretions, no endophthalmitis, and no increase in intraocular pressure.
  • the fundus photography of one month after the operation is shown in FIG. 8, where FIG. 8A is the result of fundus photography injected with rAAV2-ND4, and FIG. 8B is the result of fundus photography injected with rAAV2-optimized ND4. It can be seen from the figure that all rabbits have no obvious complications or damage to the retinal blood vessels and optic nerve. It shows that the formal standard intravitreal injection will not cause obvious inflammation or other complications and is safe.
  • the rabbits of the two groups were taken intraocular pressure and fundus photography at 7 days and 30 days after surgery, and the eyeballs were harvested and fixed with eyeball fixation solution. After dehydration, they were embedded in paraffin.
  • the pathological slicer was longitudinally cut along the optic nerve. After further dehydration, it was dyed with hematoxylin and eosin dye solution, and dehydrated and mounted again.
  • FIG. 9A is the HE staining result of rAAV2-ND4 injection
  • FIG. 9B is the HE staining result of rAAV2-optimized ND4 injection. It can be seen from the figure that the retinal ganglion fiber layer of all rabbits is not damaged, and ganglion cells are not reduced. It showed that the formal standard intravitreal injection did not produce retinal toxicity and nerve damage and was safe.
  • the experimental method is the same as that in Embodiment 1-6, and the fusion nucleic acid shown in SEQ ID NO .: 5 is replaced with the fusion nucleic acid shown in SEQ ID NO .: 10 in this embodiment. It was found that compared with the unoptimized ND4 coding sequence, the optimized ND4 coding nucleotide sequence of the present invention (SEQ ID NO: 10 position 267-1646) and the corresponding fusion nucleic acid (SEQ ID NO: 10 1-2271) ND4 transcription efficiency and translation efficiency are significantly improved, the expression level is also significantly higher, can effectively treat Leber hereditary optic neuropathy, and has good safety.

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Abstract

公开了编码人NADH脱氢酶亚单位4蛋白的核酸及其应用，其核苷酸序列如SEQ ID NO.:1所示。还公开了一种融合核酸，其包含所述的编码人NADH脱氢酶亚单位4蛋白的核酸。进一步公开了一种重组表达载体，其包含上述核酸或融合核酸。还公开了一种转化体，其在宿主中导入上述核酸或融合核酸。所述的编码人NADH脱氢酶亚单位4蛋白的核酸表达量更高，因此可在线粒体中获得更多的人NADH脱氢酶亚单位4蛋白，能较好地治疗Leber遗传性视神经病变。

Description

ND4蛋白的编码序列及其应用 技术领域
本发明涉及生物制剂领域，尤其涉及ND4蛋白的编码序列及其应用。
背景技术
Leber遗传性视神经病变(Leber hereditary optic neuropathy,LHON)是一种退行性视力障碍，通常表现为中央视力的双侧丧失。平均发病年龄在20岁中期，通常在数周至数月内没有疼痛，直到双眼视力恶化到0.1以下，严重影响患者的生活质量。LHON是由于线粒体基因突变引起的，与NADH泛醌氧化还原酶、即线粒体呼吸链的复合体I亚基的三个线粒体基因之一的突变有关。研究表明，影响ND1基因的G3460A突变、影响ND6基因的T14484C突变和影响ND4基因的G11778A突变被认为是LHON的主要原因，并且每种突变都具有永久性视力丧失的显著风险。所有这些都与视网膜神经节细胞的局灶性退化有关。
两个主要的LHON突变G3460A和T14484C导致患者血小板中分离的线粒体NADH脱氢酶活性降低80％。然而，从G11778A细胞中分离出的线粒体显示复合物I和呼吸链中大多数其他成分的活性接近正常。对中国LHON患者而言，G11778A位点突变患者占90％。在11778位点的突变，使人NADH脱氢酶亚单位4蛋白(ND4蛋白)的精氨酸转变成组氨酸，导致功能障碍、视神经损伤和Leber遗传性视神经病变，其发病率高、预后差。
LHON治疗的主要问题产生于将DNA输送到细胞器的障碍。现有技术CN 102634527 B公开了一种重组人NADH脱氢酶亚单位4蛋白的基因(ND4基因)及其表达载体构建方法，由COX10的编码28个氨基酸的肽链，引导ND4蛋白进入到线粒体中。CN 104450747 A公开了一种用于治疗Leber遗传性视神经病变的重组腺相关病毒-NADH脱氢酶亚单位4(ND4)基因全长以及药剂。该基因其由CAG启动子序列、带COX10的线粒体定位序列的ND4的编码序列和UTR组成。将CN 102634527 B的药剂，或CN 104450747 A的含有CAG-Cox10-ND4的药剂注入眼玻璃体腔中，用于治疗Leber遗传性视神经病变，该等药剂能够在玻璃体腔内保持活力，并转染到视神经细胞，该蛋白N前端的信号肽定向引导该蛋白进入线粒体，成熟的ND4蛋白发挥作用。但是，该技术还存在转染效率不高，治疗效果不佳的缺点。
因此，本领域亟需开发一种转染效率高，治疗效果好的人NADH脱氢酶亚单位4蛋白的表达体系及制备方法。
发明内容
本发明的目的是提供一种转染效率高，治疗效果好的人NADH脱氢酶亚单位4蛋白的表达体系及制备方法。
本发明的目的是提供一种编码人NADH脱氢酶亚单位4蛋白的优化核酸序列、载体及 制备方法。
本发明的第一方面，提供了一种核苷酸序列，所述核苷酸序列编码人NADH脱氢酶亚单位4蛋白，且所述核苷酸序列选自下组：
(a)所述核苷酸序列如SEQ ID NO.:1所示；
(b)所述核苷酸序列与SEQ ID NO.:1所示的核苷酸序列有≥95％相同性，优选地≥98％，更优选地≥99％；和
(c)与(a)或(b)所述的核苷酸序列互补的核苷酸序列。
在另一优选例中，所述核苷酸序列包括DNA序列、cDNA序列、或mRNA序列。
在另一优选例中，所述核苷酸序列包括单链序列和双链序列。
在另一优选例中，所述核苷酸序列包括与SEQ ID NO.:1完全互补的核苷酸序列。
本发明的第二方面，提供了一种融合核酸，所述融合核酸包含如本发明第一方面所述的编码人NADH脱氢酶亚单位4蛋白的核苷酸序列。
在另一优选例中，所述融合核酸还包含选自下组的序列：线粒体靶向肽的编码序列、UTR序列、或其组合。
在另一优选例中，所述线粒体靶向肽的编码序列包括：COX10序列和/或OPA1序列。
在另一优选例中，所述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OPA1的编码序列具有如SEQ ID NO.:3所示的序列。
在另一优选例中，所述UTR序列包括3’-UTR和/或5’-UTR，较佳地为3’-UTR。
在另一优选例中，所述UTR序列具有如SEQ ID NO.:4或11所示的序列。
在另一优选例中，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为如本发明第一方面所述的核苷酸序列；和
Z3为3’-UTR序列。
在另一优选例中，所述的Z1为COX10的编码序列或OPA1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，所述融合核酸的序列如SEQ ID NO.:5所示；其中，
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，所述融合核酸从5’-3’端的结构为OPA1-ND4-UTR。较佳地，所 述融合核酸序列如SEQ ID NO.:10所示。
在另一优选例中，所述序列如SEQ ID NO.:10所示的融合核酸中，
第1-266位为OPA1的编码序列；
第267-1646位为编码人NADH脱氢酶亚单位4蛋白的核苷酸序列；
第1647-2271位为3’-UTR序列。
在另一优选例中，各个核苷酸连接序列的长度为1-30nt，较佳地1-15nt，更佳地3-6nt。
在另一优选例中，所述的核苷酸连接序列来源于限制性内切酶酶切形成的核苷酸接头序列。
本发明的第三方面，提供了一种载体，所述载体含有如本发明第一方面所述的核苷酸序列或本发明第二方面所述的融合核酸。
在另一优选例中，所述载体包含一个或多个启动子，所述启动子可操作地与所述核酸序列、增强子、转录终止信号、多腺苷酸化序列、复制起点、选择性标记、核酸限制性位点、和/或同源重组位点连接。
在另一优选例中，所述的载体选自下组：质粒、病毒载体。
在另一优选例中，所述的载体选自下组：慢病毒载体、腺病毒载体、腺相关病毒载体、或其组合。较佳地，所述载体为AAV载体。
在另一优选例中，所述AAV载体的血清型选自：AAV2、AAV5、AAV7、AAV8、或其组合。
在另一优选例中，所述的载体包括DNA病毒、逆转录病毒载体。
在另一优选例中，所述载体为含有或插入有如本发明第一方面所述的核苷酸序列或本发明第二方面所述的融合核酸的AAV载体；较佳地为AAV载体质粒pSNaV。
在另一优选例中，所述载体的骨架为腺相关病毒载体质粒pSNaV。
在另一优选例中，所述载体用于表达重组人NADH脱氢酶亚单位4蛋白。
本发明的第四方面，提供了一种宿主细胞，所述宿主细胞含有本发明第三方面所述的载体，或其染色体中整合有外源的如本发明第一方面所述的核苷酸序列或本发明第二方面所述的融合核酸。
在另一优选例中，所述宿主细胞为哺乳动物细胞，所述哺乳动物包括人和非人哺乳动物。
在另一优选例中，所述宿主细胞选自下组：HEK293细胞、感光细胞(包括锥状细胞和/或杆状细胞)、其他视觉细胞(如双节细胞)、(视)神经细胞、或其组合。
在另一优选例中，所述宿主细胞选自下组：视杆细胞、视锥细胞、给光双极细胞、撤光双极细胞、水平细胞、神经节细胞、无长突细胞、或其组合。较佳地，所述宿主细胞 为(视网膜)神经节细胞。
本发明的第五方面，提供了如本发明第三方面所述的载体的用途，用于制备一制剂或组合物，所述制剂或组合物用于恢复受试者视力和/或治疗眼部疾病。
在另一优选例中，所述眼部疾病为视神经退化性疾病。
在另一优选例中，所述制剂或组合物用于治疗视网膜神经节细胞的局灶性退化。
在另一优选例中，所述制剂或组合物用于治疗遗传性视神经病变，较佳地为Leber遗传性视神经病变(LHON)。
本发明的第六方面，提供了一种药物制剂，所述的制剂含有(a)本发明第三方面所述的载体，以及(b)药学上可接受的载体或赋形剂。
在另一优选例中，所述药物制剂的剂型选自下组：冻干制剂、液体制剂、或其组合。
在另一优选例中，所述的载体选自下组：慢病毒载体、腺病毒载体、腺相关病毒载体、或其组合。较佳地，所述载体为AAV载体。
在另一优选例中，所述药物制剂中载体的含量为1×10 9-1×10 16，较佳地1×10 12-1×10 13个病毒/毫升。
在另一优选例中，所述药物制剂用于治疗眼部疾病，较佳地治疗视神经退化性疾病，更佳地治疗视网膜神经节细胞的局灶性退化。
在另一优选例中，所述药物制剂用于治疗遗传性视神经病变，较佳地为Leber遗传性视神经病变(LHON)。
本发明的第七方面，提供了一种治疗方法，所述方法包括将本发明第三方面所述的载体施用于需要的对象。
在另一优选例中，所述的载体选自下组：慢病毒载体、腺病毒载体、腺相关病毒载体、或其组合。较佳地，所述载体为AAV载体。
在另一优选例中，将所述载体引入到需要的对象的眼睛内。
在另一优选例中，所述需要的对象包括人和非人哺乳动物。
在另一优选例中，所述治疗方法为治疗眼部疾病的方法。
在另一优选例中，所述眼部疾病为遗传性视神经病变，较佳地为Leber遗传性视神经病变(LHON)。
本发明的第八方面，提供了一种重组人NADH脱氢酶亚单位4蛋白的制备方法，包括步骤：培养本发明第四方面所述的宿主细胞，从而得到重组人NADH脱氢酶亚单位4蛋白。
本发明的第九方面，提供了一种如本发明第二方面所述的融合核酸编码的融合蛋白。
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ND4融合核酸与经优化的ND4融合核酸的核苷酸序列对比。其中“ND4基因”行序列为未优化的ND4融合核酸序列，“人优化ND4基因”行序列为经优化的ND4融合核酸序列。
图2显示了PCR核酸电泳验证ND4(泳道A)和优化ND4(泳道B)基因克隆结果。
图3显示了rAAV2-ND4(A)和rAAV2-优化ND4(B)感染293T细胞荧光观察结果。
图4显示了以β-actin为内参蛋白，免疫印迹比较293T细胞rAAV2-优化ND4和rAAV2-ND4在蛋白水平上的表达量，其中泳道A为rAAV2-ND4，泳道B为rAAV2-优化ND4。
图5显示了以β-actin为内参蛋白，rAAV2-优化ND4和rAAV2-ND4分别感染293T细胞后ND4蛋白水平上的表达量。
图6显示了以β-actin为内参蛋白，免疫印迹比较兔视神经细胞rAAV2-优化ND4和rAAV2-ND4在蛋白水平上的表达量，其中泳道A为rAAV2-优化ND4，泳道B为rAAV2-ND4。
图7显示了以β-actin为内参蛋白，rAAV2-优化ND4和rAAV2-ND4分别感染兔视神经细胞后ND4蛋白水平上的表达量。
图8显示了兔眼玻切镜下眼底拍照，其中A为注射rAAV2-ND4病毒，B为注射rAAV2-优化ND4病毒。
图9显示了兔眼HE切片镜检结果，其中A为注射rAAV2-ND4病毒，B为注射rAAV2-优化ND4病毒。
具体实施方式
本发明人经过广泛而深入的研究，对重组人NADH脱氢酶亚单位4蛋白基因编码序列进行了针对性优化设计，从而获得了一种特别适合在哺乳动物(如人)细胞中进行高效转录和高效表达ND4蛋白的核苷酸序列和融合核酸，并构建了重组人NADH脱氢酶亚单位4蛋白的重组表达载体。经过特殊优化后的ND4编码序列(SEQ ID NO.:1)的转录效率和翻译效率显著提高，重组人NADH脱氢酶亚单位4蛋白表达量提高了3倍以上。在此基础上，发明人完成了本发明。
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。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
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”。
序列同一性通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
如本文使用的，术语“受试者”、“需要的对象”指任何哺乳动物或非哺乳动物。哺乳动物包括但不限于人类、脊椎动物诸如啮齿类、非人类灵长类、牛、马、狗、猫、猪、绵羊、山羊。
术语“人NADH脱氢酶亚单位4蛋白”、“ND4(蛋白)”、“多肽”、“本发明多肽”和“本发明蛋白”可互换使用。
腺相关病毒
腺相关病毒(adeno-associated virus，AAV),也称腺伴随病毒,属于微小病毒科依赖病毒属,是目前发现的一类结构最简单的单链DNA缺陷型病毒，需要辅助病毒(通常为腺病毒)参与复制。它编码两个末端的反向重复序列(ITR)中的cap和rep基因。ITRs对于病毒的复制和包装具有决定性作用。cap基因编码病毒衣壳蛋白，rep基因参与病毒的复制和整合。AAV能感染多种细胞。
重组腺相关病毒载体(rAAV)源于非致病的野生型腺相关病毒，由于其安全性好、宿主细胞范围广(分裂和非分裂细胞)、免疫源性低，在体内表达外源基因时间长等特点，被视为最有前途的基因转移载体之一，在世界范围内的基因治疗和疫苗研究中得到广泛应用。经过10余年的研究，重组腺相关病毒的生物学特性己被深入了解，尤其是其在各种细胞、组织和体内实验中的应用效果方面已经积累了许多资料。在医学研究中，rAAV被用于多种疾病的基因治疗的研究(包括体内、体外实验)；同时作为一种有特点的基因转移载体，还广泛用于基因功能研究、构建疾病模型、制备基因敲除鼠等方面。
在本发明一个优选的实施例中，载体为重组AAV载体。AAV是相对较小的DNA病毒，其可以稳定和位点特异性方式整合到它们所感染的细胞的基因组中。它们能够感染一大系列的细胞而不对细胞生长、形态或分化产生任何影响，并且它们似乎并不涉及人体病理学。AAV基因组己被克隆、测序及表征。AAV含有大约4700碱基并在每个末端包含约145个碱基的反向末端重复序列(ITR)区域，其作为病毒的复制起点。该基因组的其余被分成两个带有衣壳化功能的重要区域：包含涉及病毒复制和病毒基因表达的rep基因的基因组左边部分；以及包含编码病毒衣壳蛋白的cap基因的基因组右边部分。
AAV载体可采用本领域的标准方法制备。任何血清型的腺相关病毒均是合适的。用于纯化载体的方法可见于例如美国专利No.6566118、6989264和6995006，它们的公开内容整体以引用方式并入本文。杂合载体的制备在例如PCT申请No.PCT/US2005/027091中有所描述，该申请的公开内容整体以引用方式并入本文。用于体外和体内转运基因的衍生自AAV的载体的使用己有描述(参见例如国际专利申请公布No.WO91/18088和WO93/09239；美国专利No.4,797,368、6,596,535和5,139,941，以及欧洲专利No.0488528，它们均整体以引用方式并入本文)。这些专利公布描述了其中rep和/或cap基因缺失并被所关注的基因替换的各种来源于AAV的构建体，以及这些构建体在体外(进入培养的细胞中)或体内(直接进入生物体)转运所关注的基因的用途。复制缺陷重组AAV可通过将以下质粒共转染进被人类辅助病毒(例如腺病毒)感染的细胞系而制备：所含的所关注核酸序列的侧翼为两个AAV反向末端重复序列(ITR)区域的质粒，和携带AAV衣壳化基因(rep和cap基因)的质粒。然后通过标准技术纯化所产生的AAV重组体。
在一些实施方案中，重组载体被衣壳化到病毒粒子(例如包括但不限于AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15和AAV16的AAV病毒粒子)中。因此，本公开包括含有本文所述的任何载体的重组病毒粒子(因其包含重组多核苷酸而为重组的)。产生这样的粒子的方法是本领域己知的，并在美国专利No.6,596,535中有所描述。
核酸编码序列
本发明的要解决的技术问题是克服现有技术中NADH脱氢酶亚单位4蛋白转染效率不高、治疗效果不佳的技术缺陷。本发明提供一种靶向性NADH脱氢酶亚单位4蛋白及其制备方法和应用。经研究发现，本发明优化的ND4基因序列(SEQ ID NO.:1)，使ND4蛋白表达效率更高，有更多的ND4蛋白在患者视神经节细胞发挥生理作用。
本发明所述的编码人NADH脱氢酶亚单位4蛋白的核酸，其核苷酸序列如SEQ ID NO.:1所示。所述编码人NADH脱氢酶亚单位4蛋白的核酸，其全长为1380bp。在本发明中，所述编码人NADH脱氢酶亚单位4蛋白的核酸又称作ND4优化基因或ND4优化核酸。
本发明的多核苷酸可以是DNA形式或RNA形式。在另一优选例中，所述核苷酸为DNA。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
本发明的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据已公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。目前，已经可以完全通过化学合成来得到编码本发明多肽(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种 现有的DNA分子(或如载体)和细胞中。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或多肽编码序列经基因工程产生的宿主细胞。上述多核苷酸、载体或宿主细胞可以是分离的。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多核苷酸和多肽是没有分离纯化的，但同样的多核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
在本发明较佳的实施方式中，所述核苷酸序列如SEQ ID NO.:1所示。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的基因。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术利用所述宿主细胞表达ND4蛋白的方法。
通过常规的重组DNA技术，可利用本发明的多核苷酸序列获得表达本发明ND4蛋白的宿主细胞(如哺乳动物细胞)。一般来说包括步骤：将本发明第一方面所述的多核苷酸或本发明第三方面所述的载体转导入宿主细胞内。
本领域的技术人员熟知的方法能用于构建含本发明多肽的编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达多肽。
宿主细胞可以是原核细胞，或是低等真核细胞，或是高等真核细胞，如哺乳动物细胞(包括人和非人哺乳动物)。代表性例子有：CHO、NS0、COS7、或293细胞的动物细胞等。在本发明的一个优选实施方式中，选择HEK细胞、感光细胞(包括锥状细胞和/或杆状细胞)、其他视觉细胞(如双节细胞)、神经细胞为宿主细胞。在另一优选例中，所述宿主细胞选自下组：视杆细胞、视锥细胞、给光双极细胞、撤光双极细胞、水平细胞、神经节细胞、无长突细胞、或其组合。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生 物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的蛋白质。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
序列优化
本发明改变了COX10的线粒体编码序列为核酸编码序列(如SEQ ID NO：2所示)且进行ND4核苷酸序列优化(如SEQ ID NO：1所示，在本发明中简称优化ND4基因/核酸)，此序列是经过特殊优化，转录效率和翻译效率显著提高，优化的COX10+ND4和未优化的COX10+ND4序列的同源性为76.16％(1115/1464)。
在本发明中，提供了优化的、转录效率和翻译效率高的重组人NADH脱氢酶亚单位4蛋白的编码序列，所述编码序列如SEQ ID NO.:1所示。
如本文所用，所述“优化的ND4编码序列”、“优化ND4编码基因”均指一种用于编码重组人NADH脱氢酶亚单位4蛋白的核苷酸序列，所述的核苷酸序列编码SEQ ID NO.:1所示的氨基酸序列。
Figure PCTCN2018118662-appb-000001
Figure PCTCN2018118662-appb-000002
融合核酸
本发明还提供了一种融合核酸，其包含所述的编码人NADH脱氢酶亚单位4蛋白的核酸。
如本文所用，“融合核酸”指由两个或两个以上不同来源的核苷酸序列连接而成的核酸，或者由同一来源但其天然位置并不互相连接的两个或两个以上核苷酸序列连接而成的核酸。本发明的融合核酸所编码的蛋白称作融合蛋白，在本发明中即ND4优化蛋白。
优选地，所述融合核酸在所述的编码人NADH脱氢酶亚单位4蛋白的核酸可操作性地连接有线粒体靶向肽的编码序列和/或UTR序列。优选地，当所述的编码人NADH脱氢酶亚单位4蛋白的核酸连接了线粒体靶向肽的编码序列时，所述线粒体靶向肽的编码序列为如SEQ ID NO.:2所示的COX10基因的线粒体靶向肽的编码序列(简称COX10序列)，或如SEQ ID NO.:3所示的OPA1序列；当所述的编码ND4蛋白的核酸连接了UTR序列时，所述UTR序列如SEQ ID NO.:4或SEQ ID NO.:11所示。
Figure PCTCN2018118662-appb-000003
Figure PCTCN2018118662-appb-000004
Figure PCTCN2018118662-appb-000005
Figure PCTCN2018118662-appb-000006
在另一优选例中，在所述融合核酸中，所述COX10的编码序列、所述编码人NADH脱氢酶亚单位4蛋白的核酸、所述UTR序列由5’端至3’端依次排列。
在另一优选例中，所述融合核酸的序列如SEQ ID NO.:5所示。具体地，所述融合核酸的核苷酸序列其全长为2889bp，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肽的编码序列和ND4的表达。其中，优化的COX10+ND4和未优化的COX10+ND4序列的同源性为76.16％。
Figure PCTCN2018118662-appb-000007
Figure PCTCN2018118662-appb-000008
在另一优选例中，所述融合核酸的序列如SEQ ID NO.:10所示。
Figure PCTCN2018118662-appb-000009
表达载体和宿主细胞
本发明还提供了一种用于ND4蛋白的表达载体，它含有本发明的优化ND4编码序列。
通过提供的序列信息，熟练的技术人员可以使用可用的克隆技术以产生适于转导进入细胞的核酸序列或载体。
优选地，编码ND4蛋白的核酸序列作为载体，优选地表达载体被提供。优选地，其可作为优选地适用于在视网膜靶细胞中转导和表达的基因治疗载体被提供。载体可以是病毒的或非病毒的(例如质粒)。病毒载体包括源自以下的那些病毒载体:腺病毒、包括突变的形式的腺相关病毒(AAV)、逆转录病毒、慢病毒、疱疹病毒、牛痘病毒、MMLV、GaLV、猿猴免疫缺陷病毒(SIV)、HIV、痘病毒和SV40。优选地，病毒载体是复制缺陷的(replication defective)，尽管设想其可以是复制缺乏的(replication deficient)、能够复制或条件性复制的。病毒载体通常可以保持染色体外状态而不整合进入靶视网膜细胞的基因组。用于向视网膜靶细胞引入编码ND4蛋白的核酸序列的优选的病毒载体是AAV载体，例如自身互补的腺相关病毒(scAAV)。使用特定的AAV血清型(AAV血清型2到AAV血清型12)或这些血清型中的任何一个的修饰的版本(包括AAV 4YF和AAV 7m8载体)可以实现选择性靶向。
病毒载体可被修饰以缺失任何非必需的序列。例如，AAV中，病毒可被修饰以缺失全部或部分的IX基因、Ela和/或Elb基因。对于野生型AAV，没有辅助病毒诸如腺病毒的存在，复制是非常低效率的。对于重组的腺相关病毒，优选地，复制基因和衣壳基因以反式被提供(在pRep/Cap质粒中)，并且仅AAV基因组的2ITR被保留并且包装进入病毒体，同时需要的腺病毒基因被被腺病毒或另一个质粒提供。也可对慢病毒载体做出类似的修饰。
病毒载体具有进入细胞的能力。然而，非病毒载体诸如质粒可与剂复合以有利于病毒载体被靶细胞的摄取。此类剂包括聚阳离子剂。可选地，递送系统诸如基于脂质体的递送系统可被使用。用于在本发明中使用的载体优选地适于在体内或体外使用，并且优选地适于在人类中使用。
载体将优选地包含一个或多个调节序列以指导核酸序列在视网膜靶细胞中的表达。调节序列可以包括与核酸序列可操作地连接的启动子、增强子、转录终止信号、多腺苷酸化序列、复制起点、核酸限制性位点、和同源重组位点。载体还可包括选择性标记，例如来确定载体在生长系统(例如细菌细胞)中或在视网膜靶细胞中的表达。
“可操作地连接”意指，核酸序列在功能上与其可操作地连接的序列相关，以使得它们以使得它们影响彼此的表达或功能的方式连接。例如，与启动子可操作地连接的核酸序列将具有被启动子影响的表达模式。
启动子介导与其连接的核酸序列的表达。启动子可以是组成型的或可以是诱导型的。启动子可以指导在内视网膜细胞中遍在的表达，或神经元特异的表达。在后一种情况中，启动子可以指导细胞类型特异的表达，例如对给视神经节细胞。合适的启动子将是本领域技术人员己知的。例如，合适的启动子可以选自由以下组成的组:L7、thy-1、恢复蛋白、 钙结合蛋白、人类CMV、GAD-67、鸡β肌动蛋白、hSyn、Grm6、Grm6增强子SV40融合蛋白。使用细胞特异的启动子可以实现靶向，例如Grm6-SV40用于选择性靶向给视神经细胞。Grm6启动子是Grm6基因的200碱基对增强子序列和SV40真核启动予的融合体，Grm6基因编码给视神经细胞特异的代谢型谷氨酸受体mGluR6。Grm6基因的优选的来源是小鼠和人类。使用泛-神经元的启动子可以实现遍在的表达，其实例在本领域是己知的并且可得的。一个此类实例是CAG。CAG启动于是CMV早期增强子和鸡β肌动蛋白启动子的融合体。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
许多表达载体可应用ND4蛋白在哺乳动物细胞(较佳地为人，更佳地为人视神经细胞或感光细胞)表达。本发明优选用腺相关病毒作为表达载体。
本发明还提供了一种宿主细胞，用于表达ND4蛋白。优选地，所述宿主细胞为哺乳动物细胞(较佳地为人，更佳地为人视神经细胞或感光细胞)，提高ND4蛋白的表达量。
制剂和组合物
本发明提供一种制剂或组合物，所述制剂或组合物含有(a)本发明第三方面所述的载体，以及(b)药学上可接受的载体或赋形剂。
在另一优选例中，所述药物制剂用于治疗眼部疾病。
在另一优选例中，所述药物制剂用于治疗遗传性视神经病变，较佳地为Leber遗传性视神经病变(LHON)。
本发明所述药物组合物中的“活性成分”是指本发明所述的载体(vector)，例如病毒载体(包括腺相关病毒载体)。本发明所述的“活性成分”、制剂和/或组合物可用于治疗眼部疾病。“安全有效量”指的是：活性成分的量足以明显改善病情或症状，而不至于产生严重的副作用。“药学上可接受的载体或赋形剂(excipient)”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的活性成分以及它们之间相互掺 和，而不明显降低活性成分的药效。
组合物可以是液体或固体，例如粉末、凝胶或糊剂。优选地，组合物是液体，优选地可注射液体。合适的赋形剂将是本领域技术人员己知的。
在本发明中，所述载体可通过视网膜下或玻璃体内施用向眼睛施用。在任一种施用模式中，优选地，载体作为可注射液体被提供。优选地，可注射液体作为胶囊或注射器被提供。
药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
Figure PCTCN2018118662-appb-000010
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
本发明提供的编码ND4的核酸或融合核酸，可以体外或体内生产ND4蛋白或ND4融合蛋白，所述融合蛋白或者含所述融合蛋白的制剂可应用于制备治疗Leber遗传性视神经病变的药物。
经优化的编码人NADH脱氢酶亚单位4蛋白的核酸表达量更高，从而翻译出更多的ND4融合蛋白，而COX10序列可以准确地将ND4融合蛋白定位到线粒体内膜上，因此有更多的ND4蛋白转染到在线粒体中。将含有COX10-优化ND4融合核酸的药剂注入兔眼玻璃体腔中，该药剂在玻璃体腔内保持活力，并转染到视神经细胞。优化ND4核酸编码比现有技术更多的ND4蛋白，其转染效率更高，能较好地治疗Leber遗传性视神经病变。
与现有技术相比，本发明主要具有以下优点：
1.本发明重组人NADH脱氢酶亚单位4蛋白(ND4)编码基因序列进行了特殊优化。与ND4的未优化的DNA编码序列相比，转录效率和翻译效率显著提高。优化后的序列ND4蛋白表达量显著提高、生物活性高。
2.本发明优化后的COX10序列或OPA1序列可以准确地将ND4融合蛋白定位到线粒体内膜上，因此有更多的ND4蛋白转染到在线粒体中。
3.本发明优化的ND4编码基因(SEQ ID NO.:1)或融合核酸能够非常有效地治疗Leber遗传性视神经病变，并且安全性好。
下面结合具体实施，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，否则百分比和份数按重量计算。
实验器材
凝胶成像系统：Gene Genius公司；TaqDNA聚合酶：生工生物工程(上海)有限公司；Marker：生工生物工程(上海)有限公司；6×DNA Loading Dye：生工生物工程(上海)有限公司；PCR产物纯化回收试剂盒：生工生物工程(上海)有限公司；KpnI/SalI酶：生工生物工程(上海)有限公司；Lipofectamine 2000试剂盒：美国Invitrogen公司。
实施例1质粒的构建和重组腺相关病毒的制备
1.1质粒制备：获取人的ND4核苷酸序列后(美国国家生物技术信息中心参考序列：yp_003024035.1)，本发明改变了COX10的线粒体编码序列为核酸编码序列(如SEQ ID NO：2所示)且进行ND4核苷酸序列优化(如SEQ ID NO：1所示，在本发明中简称优化ND4基因/核酸)，此序列是经过特殊优化，转录效率和翻译效率显著提高，优化的COX10+ND4和未优化的COX10+ND4序列的同源性为76.16％。并在优化ND4基因的3’端连接UTR序列(如SEQ ID NO：4所示)，其融合基因(或称融合核酸)的序列如SEQ ID NO.:5所示，由成都擎科梓熙生物技术有限公司合成。通过PCR扩增全长基因(图2)，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-优化ND4(以下简称为pAAV2-优化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。对照pSNaV/rAAV2/2-ND4(以下简称为pAAV2-ND4)制备参见CN 102634527 B。
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和pAAV2-优化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和rAAV2-优化ND4。
如本领域技术人员所知，除COX10以外，OPA1(序列如SEQ ID NO.:3所示)也可与本发明的优化ND4基因融合，线粒体靶向肽的编码序列OPA1可将优化ND4基因表达的蛋白带入到线粒体内膜上，从而实现蛋白的线粒体靶向表达。
实施例2 rAAV2感染293T细胞实验
制备病毒液rAAV2-ND4-EGFP和rAAV2-优化ND4-EGFP，实验方法同实施例1。其中rAAV2-ND4-EGFP为含有EGFP标签的rAAV2-ND4病毒，rAAV2-优化ND4-EGFP为含有EGFP标签的rAAV2-优化ND4病毒。
将冻存的293T细胞复苏后进行传代培养，长满至T75培养瓶的90％左右，胰酶消化细胞，取细胞沉淀，使用DMEM完全培养基重悬细胞密度为5×10 4个/mL。使用96孔板种植细胞，每孔加入100μL细胞悬液，约5000个/孔。37℃、5％CO 2环境下培养至细胞长满至孔板的50％左右，按照MOI＝10 4分别加入病毒液rAAV2-ND4-EGFP和rAAV2-优化ND4-EGFP(2×10 10vg/0.02uL)以及0.02uL PBS，48h后进行荧光显微镜观察及RT-PCR检测和免疫印迹。
48h后进行荧光显微镜观察如图3，荧光照相显示，EGFP成功地被表达，表明以rAAV为载体，携带EGFP基因转染293T细胞内可以正常表达，证明rAAV2-ND4-EGFP和rAAV2-优化ND4-EGFP重组基因可以表达。
实施例3兔眼玻璃体腔注射rAAV2实验
取12只兔子平分为3组，分别用病毒液rAAV2-ND4和rAAV2-优化ND4(1×10 10vg/0.05mL)和PBS在距角膜缘外3mm处穿刺睫状体平坦部进入玻璃体腔内，进行玻璃体腔注射后进行裂隙灯、眼底照相检查和HE染色，注射30天，各组分别进行RT-PCR检测和免疫印迹。
实施例4RT-PCR检测ND4的表达
分别提取转染rAAV2-ND4和rAAV2-优化-ND4的293T细胞及兔视神经细胞的RNA、反转录，利用TRIZOL试剂盒提取总RNA并反转录合成cDNA模板。用NCBI的保守结构域分析软件分析ND4的保守结构，确保所设计引物的扩增片段位于非保守区；然后根据荧光定量PCR的引物设计原则，用primer premier 5设计引物：
β-actin-S：CGAGATCGTGCGGGACAT(序列如SEQ ID NO:6所示)；
β-actin-A：CAGGAAGGAGGGCTGGAAC(序列如SEQ ID NO:7所示)；
ND4-S：GCCAACAGCAACTACGAGC(序列如SEQ ID NO:8所示)；
ND4-A：TGATGTTGCTCCAGCTGAAG(序列如SEQ ID NO:9所示)；
优化ND4-S：GCCTGACCCTGATCCTGAAC(序列如SEQ ID NO:12所示)
优化ND4-A：GTGCGCTCGTAGTTGCTGTT(序列如SEQ ID NO:13所示)
荧光定量PCR的反应体系和反应程序：
在Real-time PCR Detection System仪器上进行荧光定量PCR。在0.2mL的PCR反应管中加入SYBR Green mix12.5μL、ddH 2O 8μL、引物各1μL，cDNA样品2.5μL，总体系25μL。每个样品既要用于扩增目的基因又要扩增内参基因β-actin，各个基因的扩增都做三个重复。实际加样时，为减小误差，各PCR反应管中共有的试剂可加在一起然后分装。加样完毕，进行荧光定量PCR。
按照95℃预变性10min，94℃变性10s，60℃退火20s，72℃延伸20s，共40个循环的反应程序进行扩增，并于每个循环的延伸阶段采集荧光信号。反应结束后做融解曲线分析。
采用相对定量方法研究基因表达量的差异，该方法无需制作标准曲线，以β-actin为内参基因，仪器自带的分析软件即可自动生成表达数值，计算相对表达。
结果显示，在mRNA水平，293T细胞rAAV2-ND4组和rAAV2-优化ND4组比对照组的mRNA相对表达量分别提高为89.99倍和108.21倍，两组比较有显著性差异(p＜0.05)；兔视神经细胞rAAV2-ND4组和rAAV2-优化ND4组比对照组的相对表达量分别为75.29倍和78.21倍，两组比较无显著性差异(p＞0.05)。
该结果出乎意料地表明，本发明的经优化的ND4的编码核苷酸序列(SEQ ID NO.:5的第85-1464位)和相应的融合核酸(SEQ ID NO.:5的第1-2889位)，居然意外地提高了转录效率，从而使得rAAV2-优化ND4组基因水平上表达明显比rAAV2-ND4组提高，提高幅度为约20％。该结果表明，在转录效率方面，rAAV2-优化ND4组的转录效率显著更高。
实施例5免疫印迹检测ND4的表达
分别提取转染rAAV2-ND4和rAAV2-优化-ND4的293T细胞及兔神经细胞的ND4蛋白，然后进行10％聚丙烯酰胺凝胶电泳，点转移到聚偏氟膜上(Bio-Rad,Her-cules,CA, USA)，用于免疫检测，以β-actin为内参基因，用自动图像分析仪器(Li-Cor；Lincoln,NE,USA)对薄膜上条带进行观察分析，各蛋白带的积分光密度用归一化法积分，得到相同的样本对应的光学密度值，统计分析采用SPSS 19.0统计软件进行统计学分析。
rAAV2-优化ND4和rAAV2-ND4分别感染293T细胞的结果如图4和图5所示。ND4蛋白表达分析293T细胞rAAV2-ND4组的平均表达值0.36，而rAAV2-优化ND4组的平均表达值是1.65，rAAV2-优化ND4组约为rAAV2-ND4组的4.6倍，两组比较有显著性差异(p＜0.01)。
rAAV2-优化ND4和rAAV2-ND4分别感染293T细胞的结果如图6和图7所示。兔视神经细胞rAAV2-ND4组的平均表达值0.16，而rAAV2-优化ND4组的平均表达值是0.48，rAAV2-优化ND4组约为rAAV2-ND4组的3倍，两组比较有显著性差异(p＜0.01)。
可以看出，rAAV2-优化ND4组的蛋白水平上表达明显比未优化组提高，这表明，在翻译效率方面，本发明的经优化的ND4的编码核苷酸序列(SEQ ID NO.:5的第85-1464位)和相应的融合核酸(SEQ ID NO.:5的第1-2889位)的翻译效率更高。
实施例6兔子眼压和眼底照相
2组兔子分别于术后1、3、7、30天进行裂隙灯、眼压的检查。所有兔子均无明显异常，无结膜充血、分泌物，无眼内炎，眼压均无升高。术后一个月的眼底照相显示参见图8，其中图8A为注射rAAV2-ND4的眼底照相结果，图8B为注射rAAV2-优化ND4的眼底照相结果。由图可见，所有兔子的视网膜血管和视神经均无明显并发症或损害。表明正规标准的玻璃体腔注射不会发生明显的炎症反应或其他并发症，是安全的。
实施例7兔子眼球HE切片
2组兔子分别于术后7天、30天进行眼压和眼底照相后摘取眼球，用眼球固定液固定，脱水后进行石蜡包埋，病理切片机沿着视神经方向纵切。进一步脱水后先后用苏木素和伊红染液染色，再次脱水封片。显微镜镜检结果参见图9，其中图9A为注射rAAV2-ND4的HE染色结果，图9B为注射rAAV2-优化ND4的HE染色结果。由图可见，所有兔子的视网膜神经节纤维层未受损，神经节细胞未见减少。表明正规标准的玻璃体腔注射没有产生视网膜毒性和神经损伤，是安全的。
实施例8
将序列如SEQ ID NO.:5中的COX10序列(第1-84位)替换为OPA1线粒体靶向肽序列(SEQ ID NO.:3)，实验方法同实施例1-6。结果发现，相比未优化的ND4编码序列，本发明的经优化的ND4的编码核苷酸序列和相应的融合核酸的ND4转录效率和翻译效率均显著提高，表达量也明显更高,能够有效地治疗Leber遗传性视神经病变，并且安全性好。
实施例9
将序列如SEQ ID NO.:5中的COX10序列(第1-84位)替换为OPA1线粒体靶向肽序列(SEQ ID NO.:3)，第1465-2889位替换为如SEQ ID NO.:11所示的UTR序列，得到结构为OPA1-ND4-UTR的融合核酸，序列如SEQ ID NO.:10所示。
实验方法同实施例1-6，将其中SEQ ID NO.:5所示的融合核酸替换为本实施例SEQ ID NO.:10所示的融合核酸。结果发现，相比未优化的ND4编码序列，本发明的经优化的ND4的编码核苷酸序列(SEQ ID NO.:10的第267-1646位)和相应的融合核酸(SEQ ID NO.:10的第1-2271位)的ND4转录效率和翻译效率均显著提高，表达量也明显更高,能够有效地治疗Leber遗传性视神经病变，并且安全性好。
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 (17)

  1. 一种核苷酸序列，其特征在于，所述核苷酸序列编码人NADH脱氢酶亚单位4蛋白，且所述核苷酸序列选自下组：
    (a)所述核苷酸序列如SEQ ID NO.:1所示；
    (b)所述核苷酸序列与SEQ ID NO.:1所示的核苷酸序列有≥95％相同性，优选地≥98％，更优选地≥99％；和
    (c)与(a)或(b)所述的核苷酸序列互补的核苷酸序列。
  2. 一种融合核酸，其特征在于，所述融合核酸包含如权利要求1所述的核苷酸序列。
  3. 如权利要求2所述的融合核酸，其特征在于，所述融合核酸还包含选自下组的序列：线粒体靶向肽的编码序列、UTR序列、或其组合。
  4. 如权利要求3所述的融合核酸，其特征在于，所述线粒体靶向肽的编码序列包括COX10的编码序列和/或OPA1的编码序列。
  5. 如权利要求4所述的融合核酸，其特征在于，所述COX10的编码序列具有如SEQ ID NO.:2所示的序列；所述OPA1的编码序列具有如SEQ ID NO.:3所示的序列；和/或所述UTR序列具有如SEQ ID NO.:4或11所示的序列。
  6. 如权利要求2所述的融合核酸，其特征在于，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为如权利要求1所述的核苷酸序列；和
    Z3为3’-UTR序列。
  7. 如权利要求2所述的融合核酸，其特征在于，所述融合核酸具有如SEQ ID NO.:5或10所示的序列。
  8. 一种载体，其特征在于，所述载体含有如权利要求1所述的核苷酸序列或权利要求2所述的融合核酸。
  9. 如权利要求8所述的载体，其特征在于，所述载体为腺相关病毒载体，较佳地，所述载体的骨架为腺相关病毒载体质粒pSNaV。
  10. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求8所述的载体，或其染色体中整合有外源的如权利要求1所述的核苷酸序列或权利要求2所述的融合核酸。
  11. 如权利要求10所述的宿主细胞，其特征在于，所述宿主细胞选自下组：HEK293细胞、感光细胞(包括锥状细胞和/或杆状细胞)、其他视觉细胞(如双节细胞)、(视)神经 细胞、或其组合。
  12. 如权利要求8所述的载体的用途，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恢复受试者视力和/或治疗眼部疾病。
  13. 如权利要求12所述的用途，其特征在于，所述制剂或组合物用于治疗遗传性视神经病变，较佳地为Leber遗传性视神经病变(LHON)。
  14. 一种药物制剂，其特征在于，所述的制剂含有(a)权利要求8所述的载体，以及(b)药学上可接受的载体或赋形剂。
  15. 一种治疗方法，其特征在于，所述方法包括将权利要求8所述的载体施用于需要的对象。
  16. 一种重组人NADH脱氢酶亚单位4蛋白的制备方法，其特征在于，包括步骤：培养权利要求10所述的宿主细胞，从而得到重组人NADH脱氢酶亚单位4蛋白。
  17. 一种如权利要求3-7中任一所述的融合核酸编码的融合蛋白。
PCT/CN2018/118662 2018-06-29 2018-11-30 Nd4蛋白的编码序列及其应用 WO2020077756A1 (zh)

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KR1020217001385A KR102627561B1 (ko) 2018-06-29 2019-07-01 레버 유전성 시신경병증의 치료를 위한 조성물 및 방법
CN202110786772.2A CN113528510A (zh) 2018-06-29 2019-07-01 治疗遗传性视神经病变的组合物和方法
CN202110786630.6A CN113476484A (zh) 2018-06-29 2019-07-01 治疗遗传性视神经病变的组合物和方法
SG11202012044QA SG11202012044QA (en) 2018-06-29 2019-07-01 Compositions and methods for treating leber's hereditary optic neuropathy
MX2020013772A MX2020013772A (es) 2018-06-29 2019-07-01 Composiciones y métodos para el tratamiento de la neuropatía óptica hereditaria de leber.
CA3103740A CA3103740A1 (en) 2018-06-29 2019-07-01 Compositions and methods for treating leber's hereditary optic neuropathy
JP2021521870A JP2021529001A (ja) 2018-06-29 2019-07-01 レーベル遺伝性視神経症を治療するための組成物及び方法
AU2019296451A AU2019296451B2 (en) 2018-06-29 2019-07-01 Compositions and methods for treating leber's hereditary optic neuropathy
CN201980003485.0A CN110876269B (zh) 2018-06-29 2019-07-01 治疗遗传性视神经病变的组合物和方法
PCT/CN2019/094136 WO2020001657A1 (en) 2018-06-29 2019-07-01 Compositions and methods for treating leber's hereditary optic neuropathy
BR112020026361-3A BR112020026361A2 (pt) 2018-06-29 2019-07-01 Composições e métodos para tratar neuropatia óptica hereditária de leber
EP19826653.8A EP3814492A4 (en) 2018-06-29 2019-07-01 COMPOSITIONS AND METHODS OF TREATING LEBER'S HEREDITARY OPTIC NEUROPATHY
EP19853225.1A EP3840785A4 (en) 2018-08-20 2019-08-20 COMPOSITIONS AND METHODS FOR THE TREATMENT OF LEBERIAN OPTIC ATROPHY
AU2019323434A AU2019323434A1 (en) 2018-08-20 2019-08-20 Compositions and methods for treating leber's hereditary optic neuropathy
CA3109432A CA3109432A1 (en) 2018-08-20 2019-08-20 Compositions and methods for treating leber's hereditary optic neuropathy
JP2021509893A JP7403852B2 (ja) 2018-08-20 2019-08-20 レーベル遺伝性視神経症を治療するための組成物及び方法
CN201980054770.5A CN112584874A (zh) 2018-08-20 2019-08-20 用于治疗莱伯氏遗传性视神经病变的组合物和方法
PCT/CN2019/101538 WO2020038352A1 (en) 2018-08-20 2019-08-20 Compositions and methods for treating leber's hereditary optic neuropathy
KR1020217007727A KR20210068014A (ko) 2018-08-20 2019-08-20 레버 유전성 시신경병증의 치료를 위한 조성물 및 방법
SG11202101032VA SG11202101032VA (en) 2018-08-20 2019-08-20 Compositions and methods for treating leber's hereditary optic neuropathy
US16/836,644 US11034954B2 (en) 2018-06-29 2020-03-31 Compositions and methods for treating leber's hereditary optic neuropathy
US17/181,849 US11352645B2 (en) 2018-08-20 2021-02-22 Compositions and methods for treating Leber's hereditary optic neuropathy
US17/317,295 US20220340895A1 (en) 2018-06-29 2021-05-11 Compositions and methods for treating leber's hereditary optic neuropathy
US17/320,388 US11332741B1 (en) 2018-06-29 2021-05-14 Compositions and methods for treating leber's hereditary optic neuropathy
AU2021204690A AU2021204690A1 (en) 2018-06-29 2021-07-05 Compositions and methods for treating Leber's hereditary optic neuropathy
US17/726,833 US20220259619A1 (en) 2018-08-20 2022-04-22 Compositions and methods for treating leber's hereditary optic neuropathy
JP2023029170A JP2023078173A (ja) 2018-06-29 2023-02-28 レーベル遺伝性視神経症を治療するための組成物及び方法
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