WO2024083013A1 - 人线粒体动力蛋白样gtp酶及应用 - Google Patents

人线粒体动力蛋白样gtp酶及应用 Download PDF

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WO2024083013A1
WO2024083013A1 PCT/CN2023/124008 CN2023124008W WO2024083013A1 WO 2024083013 A1 WO2024083013 A1 WO 2024083013A1 CN 2023124008 W CN2023124008 W CN 2023124008W WO 2024083013 A1 WO2024083013 A1 WO 2024083013A1
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seq
vector
opa1
nucleic acid
cells
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French (fr)
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李斌
李秋棠
刘婷
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武汉纽福斯生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N9/14—Hydrolases (3)

Definitions

  • the present invention relates to the field of biological preparations, and in particular to human mitochondrial dynamin-like GTPase and its application.
  • ADOA Autosomal dominant optic atrophy
  • ADOA is caused by mutations in genes encoding inner mitochondrial membrane proteins and loci. Foreign scholars have determined that these genes are mainly OPA1 to OPA8. It has been preliminarily confirmed that about 75% of ADOA patients are related to heterozygous and dominant mutations carried by OPA1, among which mutations at exons 740 and 2794 are the most common.
  • OPA1 is a protein encoded by a nuclear gene, with a gene sequence length of ⁇ 40kb.
  • the molecular structure of OPA1 includes a GTPase domain (GTPase), two hydrophobic repeat sequences (HR) and a mitochondrial localization sequence (MTS).
  • OPA1 protein is located in the inner membrane of mitochondria, controls energy metabolism and apoptosis, maintains the integrity of cristae and mtDNA, and has GTPase activity.
  • the pathogenesis of ADOA is that the mutated OPA1 encodes a variety of truncated proteins, which usually lack a complete GTPase domain, which is equivalent to the loss of one allele, which will greatly reduce the amount of OPA1, resulting in the so-called haploinsufficiency, thereby causing mitochondrial dysfunction. Fragmented mitochondria cannot normally supply ATP to optic nerve cells, and RGC cells gradually undergo apoptosis, leading to ADOA in patients.
  • ADOA currently has no treatment method and is one of the world's recognized hereditary optic neuropathy.
  • gene therapy for ADOA has become possible, but the difficulty lies in the multiple forms of OPA1 exon splicing and its multiple forms of mutation, which result in a variety of pathogenic protein types and mutation forms, making it impossible to treat all patients with a single gene drug.
  • the technical problem to be solved by the present invention is to provide human mitochondrial dynamin-like GTPase and its application.
  • the present invention provides a nucleic acid encoding a human type II mitochondrial dynamin-like GTPase, which is at least one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.
  • the nucleic acid encoding human type II mitochondrial dynamin-like GTPase is derived from a nucleic acid encoding Four wild-type variants of human type II mitochondrial dynamin-like GTPase (variant 1, variant 2, variant 4, variant 7; wherein the variant 1 nucleic acid is shown in SEQ ID NO: 1, the variant 2 nucleic acid is shown in SEQ ID NO: 5, the variant 4 nucleic acid is shown in SEQ ID NO: 9, and the variant 7 nucleic acid is shown in SEQ ID NO: 13), wherein the nucleic acids shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 are obtained after sequence optimization of variant 1; the nucleic acids shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 are obtained after sequence optimization of variant 2; the nucleic acids shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 are obtained after sequence optimization of variant 4; the nucleic acids shown in SEQ ID
  • the present invention screened out SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 15 through nucleic acid sequence stability and protein expression analysis, and the nucleic acid sequence stability was significantly better than other sequences.
  • the virus titer obtained after transfection of cells with plasmid vectors containing the four sequences was high, among which the nucleic acid shown in SEQ ID NO: 15 had a higher titer for virus packaging than other sequences.
  • the expression level of the optimized nucleic acid encoding human type II mitochondrial dynamin-like GTPase is higher, thereby translating more OPA1 protein, and the MIS sequence in the optimized OPA1 protein can accurately enter the GTP functional protein into the mitochondria, so that more GTPase is transfected into the mitochondria.
  • the agent containing the nucleic acid of the present invention is injected into the vitreous cavity of the rabbit eye, and the agent maintains vitality in the vitreous cavity and is transfected into the optic nerve cells.
  • the optimized OPA1 nucleic acid encoding more OPA1 protein than the prior art can better treat autosomal dominant optic atrophy (ADOA).
  • the present invention provides an expression module, which comprises a promoter, a terminator and at least one of the nucleic acids described in the present invention.
  • the promoter is selected from human CMV, GAD-67, chicken ⁇ -actin, hSyn, Grm6, Grm6-SV40.
  • Targeted expression can be achieved using a cell-specific promoter, for example, Grm6-SV40 is used for selective targeting to optic nerve cells.
  • the Grm6 promoter is a fusion of a 200 base pair enhancer sequence of the Grm6 gene and the SV40 eukaryotic promoter.
  • the Grm6 gene encodes a metabolic glutamate receptor mGluR6 specific to optic nerve cells.
  • the preferred sources of the Grm6 gene are mice and humans.
  • Ubiquitous expression can be achieved using a pan-neuronal promoter, examples of which are known in the art.
  • the present invention uses a CMV promoter for the expression of the human type II mitochondrial dynein-like GTPase.
  • the expression module also includes an expression module formed by combining a single or multiple nucleic acids of the present invention in series, fusion expression or other feasible ways, and the present invention is not limited to this.
  • the present invention also provides a transcription unit of the fusion protein, which refers to a DNA sequence starting from a promoter and ending at a terminator.
  • the promoter and the terminator may also include a regulatory segment on both sides or between them, and the regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, a transmembrane signal peptide and a homologous recombination site operably connected to the nucleic acid sequence, such as an enhancer of the promoter, an ITR sequence, polyA, a MIS signal peptide, etc.
  • the ITR sequence includes 3'ITR and/or 5'ITR; the polyA includes SV40, hGH, BGH and rbGlob; the MIS signal peptide, which directs the protein into the mitochondria, is located at the N-terminus of OPA1, and after it is hydrolyzed by protease, the mature OPA1 variants 1, 2, 4, and 7 proteins enter the mitochondria to exert their effects.
  • the present invention provides a vector comprising:
  • a vector backbone and the nucleic acid of the present invention are provided.
  • the vectors described in the present invention may be derived from plants, animals, microorganisms, or viral vectors, which are not limited in the present invention.
  • the viral vectors may be derived from adenovirus, adeno-associated virus (AAV), retrovirus, Viruses, lentiviruses, herpes viruses, vaccinia viruses, MMLV, GaLV, simian immunodeficiency viruses (SIV), HIV, poxviruses and SV40.
  • Viral vectors can generally remain in an extrachromosomal state without integrating into the genome of target retinal cells.
  • a preferred viral vector for introducing a nucleic acid sequence encoding OPA1 protein into retinal target cells is an AAV vector, such as a self-complementary adeno-associated virus (scAAV).
  • AAV vector such as a self-complementary adeno-associated virus (scAAV).
  • scAAV self-complementary adeno-associated virus
  • the use of specific AAV serotypes includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV2.7M8 or AAV2-TYF mutants or combinations thereof, or modified versions of any of these serotypes (including AAV 4YF and AAV7m8 vectors), which can achieve selective targeting.
  • the recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence or element that is essential for the expression of an operably connected coding gene in a specific host organism.
  • the nucleic acid sequence or element necessary for the expression in a virus, microorganism or mammalian cell includes a promoter, a ribosome binding site and possible other sequences.
  • Known eukaryotic cells utilize promoters, enhancers and terminators.
  • plasmid and “vector” can sometimes be interchanged and used, because plasmid is the most commonly used vector form at present.
  • the present invention is intended to include such other forms of expression vectors, which play an equivalent role, which are known or will become known in the art, including but not limited to: plasmid, phage particle, viral vector and/or only potential genome insert.
  • the nucleic acid encoding the fusion protein provided by the present invention can be constructed in various eukaryotic expression vectors.
  • the present invention provides a host cell, which comprises any one or more of the following I) to III):
  • the host provided by the present invention may be derived from plants, animals, microorganisms or viruses, which are not limited in the present invention.
  • the present invention uses a vector constructed by recombinant DNA technology to transform or transfect host cells, so that the transformed host cells have the ability to replicate protein-encoding vectors or express desired proteins.
  • the transformation method includes: chemical transformation and electrotransformation; the transfection method includes calcium phosphate coprecipitation, artificial liposome method, and viral transfection.
  • the viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.
  • the host is transfected with adeno-associated virus.
  • the host cell of the present invention is a mammalian cell, specifically selected from 293T cells, photoreceptor cells, binocular cells, optic nerve cells, or a combination thereof.
  • the host cell of the present invention is selected from rod cells, cone cells, light-giving bipolar cells, light-withdrawing bipolar cells, horizontal cells, ganglion cells, amacrine cells, or a combination thereof.
  • the present invention provides a virus, which is produced by co-transfecting or transforming a host cell with the vector of the present invention and a packaging plasmid.
  • the virus includes at least one of adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes virus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, poxvirus and SV40.
  • AAV adeno-associated virus
  • retrovirus retrovirus
  • lentivirus lentivirus
  • herpes virus vaccinia virus
  • MMLV MMLV
  • GaLV GaLV
  • HIV poxvirus
  • SV40 simian immunodeficiency virus
  • the present invention provides a method for preparing a recombinant human type II mitochondrial dynamin-like GTPase, which comprises: culturing the host cell of the present invention to obtain the recombinant human type II mitochondrial dynamin-like GTPase.
  • the present invention provides the use of any one of the following a) to f) in a drug for restoring vision or treating eye diseases:
  • the application of the present invention is to effectively increase the ATP content produced by cells and/or inhibit mitochondrial apoptosis by using the vector or virus of the present invention at a safe and effective drug dose, thereby restoring vision or treating eye diseases.
  • the content of the vector in the drug is 1 ⁇ 10 9 -1 ⁇ 10 16 viruses/ml, preferably 1 ⁇ 10 11 -1 ⁇ 10 13 viruses/ml, and more preferably 2 ⁇ 10 11 -1 ⁇ 10 12 viruses/ml.
  • the safe and effective dose refers to the amount of active ingredient sufficient to significantly improve the condition or symptoms without causing serious side effects.
  • “Pharmaceutically acceptable carrier or excipient” refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity.
  • “Compatibility” here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.
  • the active ingredient of the present invention refers to a replicable vector, or a liposome particle containing the vector, or a virus with biological activity, or a recombinant human type II mitochondrial dynamin-like GTPase.
  • the eye disease of the present invention includes at least one of autosomal dominant optic atrophy or retinal ganglion cell apoptosis caused by OPA1 mutation.
  • the present invention provides a drug for restoring vision or treating eye diseases, wherein the raw materials thereof include at least one of the following A) to F):
  • Recombinant human type II mitochondrial dynamin-like GTPase prepared by the preparation method of the present invention or a mixture containing the same.
  • the drug also includes lipid nanoparticles containing the nucleic acid, recombinant nucleic acid or mRNA.
  • the preparation process of the lipid nanoparticles includes encapsulating the nucleic acid, recombinant nucleic acid or mRNA using a transfection reagent in an animal body to form a vaccine in the form of lipid nanoparticles containing the nucleic acid or mRNA for tumor prevention and treatment.
  • the medicine of the present invention also includes a pharmaceutically acceptable excipient or carrier.
  • the dosage form of the drug includes but is not limited to a lyophilized preparation, a liquid preparation, or a combination thereof.
  • the present invention provides a method for increasing the expression and/or activity of mitochondrial dynamin-like GTPase, which comprises introducing the vector described in the present invention and/or administering the drug described in the present invention; the method can effectively increase the ATP content produced by cells and/or inhibit mitochondrial apoptosis; and continuously upregulate the expression and/or activity of GTPase.
  • the present invention provides a method for restoring vision or treating an eye disease, which comprises administering the drug of the present invention.
  • the method can effectively increase the expression and/or activity of mitochondrial dynamin-like GTPase in the eyeball.
  • the expression and/or activity of mitochondrial dynamin-like GTPase in the eyeball can last for up to 6 months, preferably up to 3 months.
  • the treatment method can effectively increase the retinal ATP content and/or inhibit mitochondrial apoptosis.
  • the drug administration method of the present invention can include injection, oral administration or gene gun.
  • the present invention relates to the field of biological preparations, and in particular to human mitochondrial dynamin-like GTPases and their applications.
  • the present invention optimizes the gene sequences encoding four variants of recombinant human type II mitochondrial dynamin-like GTPases, and experiments on sequence stability and protein expression show that the gene sequences encoding human mitochondrial dynamin-like GTPases provided by the present invention are GTPase nucleotide sequences are stable, safe, and have high protein expression, and have good prospects for treating eye diseases such as ADOA.
  • Figure 1 shows an AAV-OPA1 vector map, wherein A is a schematic diagram of a codon-optimized OPA1-iso1-opt plasmid vector; B is a schematic diagram of a codon-optimized OPA1-iso2-opt plasmid vector; C is a schematic diagram of a codon-optimized OPA1-iso4-opt plasmid vector; and D is a schematic diagram of a codon-optimized OPA1-iso7-opt plasmid vector.
  • Figure 2 shows the enzyme digestion identification of AAV-OPA1 recombinant plasmid, the red box shows the band with abnormal enzyme digestion, and M is Trans5000Marker; wherein A is the stability detection of the enzyme digestion band of the three codon-optimized sequences opt1 to opt3 of variant iso1; B is the stability detection of the enzyme digestion band of the three codon-optimized sequences opt1 to opt3 of variant iso2; C is the stability detection of the enzyme digestion band of the three codon-optimized sequences opt1 to opt3 of variant iso4; D is the stability detection of the enzyme digestion band of the three codon-optimized sequences opt1 to opt3 of variant iso7;
  • FIG3 shows a comparison of AAV virus yields of different variants of AAV-OPA1 and their respective optimized plasmid packaging
  • FIG4 shows electrophoresis identification of AAV viral genomes of different variants of AAV-OPA1 and their respective optimized plasmid packaging
  • FIG5 shows the protein expression detection of optimized plasmids of various AAV-OPA1 variants
  • FIG. 6 shows a comparison of the mitochondrial repair functions of optimized vectors of various AAV-OPA1 variants.
  • the present invention provides human mitochondrial dynamin-like GTPase and applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.
  • the nucleotide sequence of wild-type OPA1-iso1 is:
  • the nucleotide sequence of iso1-opt1 is:
  • the nucleotide sequence of iso1-opt2 is:
  • the nucleotide sequence of iso-opt3 is:
  • the nucleotide sequence of wild-type OPA1-iso2 is:
  • the nucleotide sequence of iso2-opt1 is:
  • the nucleotide sequence of iso2-opt2 is:
  • the nucleotide sequence of iso2-opt3 is:
  • the nucleotide sequence of wild-type OPA1-iso4 is:
  • the nucleotide sequence of iso4-opt1 is:
  • the nucleotide sequence of iso4-opt2 is:
  • the nucleotide sequence of iso4-opt3 is:
  • the nucleotide sequence of wild-type OPA1-iso7 is:
  • the nucleotide sequence of iso7-opt1 is:
  • the nucleotide sequence of iso7-opt2 is:
  • the nucleotide sequence of iso7-opt3 is:
  • the amino acid sequence of wild-type OPA1-iso1 is:
  • the amino acid sequence of OPA1-iso2 is:
  • the amino acid sequence of OPA1-iso4 is:
  • the amino acid sequence of OPA1-iso7 is:
  • the term “comprising” or “including (comprising)” may be open, semi-closed and closed. In other words, the term also includes “consisting essentially of” or “consisting of”.
  • the term "subject”, “subject in need” refers to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cows, horses, dogs, cats, pigs, sheep, goats.
  • OPA1 is a transmembrane protein with GTPase activity located in the inner membrane of mitochondria.
  • the protein includes an N-terminal transmembrane region with a mitochondrial membrane insertion sequence (mitochondrial import sequence, MIS), a transmembrane region (TR) and a hydrophobic region (HR), a GTPase domain, a central dynamin domain and a C-terminal with a GTPase effector domain (GED).
  • mitochondrial membrane insertion sequence mitochondrial membrane insertion sequence
  • TR transmembrane region
  • HR hydrophobic region
  • GTPase domain a GTPase domain
  • GED GTPase effector domain
  • the function of the MIS polypeptide of OPA1 is to guide the GTP protein into the mitochondria.
  • the biosynthesis of OPA1 is regulated at two levels: transcription and translation.
  • OPA1 has many spliceosomes in mammals.
  • the OPA1 mRNA precursor is selectively spliced at exons 4, 4b, and 5b, which can produce eight tissue-specific mRNA variants to encode OPA1, and these different forms of OPA1 spliceosomes have different functions.
  • variant 1 is most abundant in the macula of the retina.
  • variants 1, 2, 4, and 7 can produce long and short forms of OPA1 protein through post-translational hydrolysis.
  • the long OPA1 protein is localized to the inner membrane of mitochondria and participates in the fusion and fission of the inner membrane of mitochondria; the short OPA1 protein is located in the mitochondrial membrane space, responsible for maintaining the integrity of cristae and mtDNA, and controlling energy metabolism and apoptosis.
  • ADOA is caused by mutations in genes encoding inner mitochondrial membrane proteins and loci. Foreign researchers have determined that these genes are mainly OPA1-OPA8. It has been preliminarily confirmed that about 75% of ADOA patients are related to heterozygous and dominant mutations carried by OPA1, among which mutations at exons 740 and 2794 are the most common.
  • the pathogenesis of ADOA is that the mutated OPA1 encodes a variety of truncated proteins, which usually lack a complete GTPase domain. The loss of one allele will cause a significant decrease in the amount of OPA1, resulting in so-called haploinsufficiency, which will cause mitochondrial dysfunction. Fragmented mitochondria cannot normally supply ATP to optic nerve cells, and RGC cells gradually die, leading to ADOA in patients.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus Dependinovirus of the family Parvoviridae. It is the simplest type of single-stranded DNA defective virus discovered so far, and requires a helper virus (usually adenovirus) to participate in replication. It encodes the cap and rep genes in the inverted repeat sequences (ITR) at both ends. ITRs play a decisive role in the replication and packaging of the virus. The cap gene encodes the viral capsid protein, and the rep gene is involved in the replication and integration of the virus. AAV can infect a variety of cells.
  • Recombinant adeno-associated virus vector which is derived from non-pathogenic wild-type adeno-associated virus, is regarded as one of the most promising gene transfer vectors due to its good safety, wide range of host cells (dividing and non-dividing cells), low immunogenicity, and long-term expression of foreign genes in vivo. It has been widely used in gene therapy and vaccine research around the world. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially its application effects in various cells, tissues and in vivo experiments have been accumulated.
  • rAAV has been used in the study of gene therapy for a variety of 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, construction of disease models, preparation of gene knockout mice, etc.
  • the vector is a recombinant AAV vector.
  • AAV is a relatively small DNA virus that can be integrated 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 be involved in human pathology.
  • the AAV genome has been cloned, sequenced and characterized. AAV contains approximately 4700 bases and an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of viral replication. The rest of the genome is divided into two important regions with encapsidation function: 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.
  • ITR inverted terminal repeat
  • 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 herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT/US2005/027091, the disclosures of which are incorporated herein by reference in their entirety. The use of vectors derived from AAV for transporting genes in vitro and in vivo has been described (see, for example, International Patent Application Publication Nos. WO91/18088 and WO93/09239; U.S. Patent Nos.
  • Replication-deficient recombinant AAV can be prepared by co-transfecting a cell line infected with a human helper virus (e.g., adenovirus) with a plasmid containing a nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions and a plasmid carrying the AAV encapsidation genes (rep and cap genes).
  • a human helper virus e.g., adenovirus
  • ITR inverted terminal repeat
  • the recombinant vector is encapsidated into a virion (e.g., AAV virions including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16).
  • a virion e.g., AAV virions including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16.
  • a virion e.g., AAV virions including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15,
  • the technical problem to be solved by the present invention is to overcome the technical defects of the prior art that the expression of recombinant human type II mitochondrial dynamin-like GTPase is not high and the therapeutic effect is not good.
  • the purpose of the present invention is to provide an optimized gene sequence of recombinant human type II mitochondrial dynamin-like GTPase.
  • the function of the recombinant human type II mitochondrial dynamin-like GTPase gene is to guide the GTP protein into the mitochondria to play its physiological function; 289 to 2772bp, encoding a peptide chain of 828 amino acids, is a GTP functional protein, and the last 3bp is a stop codon.
  • the optimized OPA1 gene sequence of the present invention makes the OPA1 protein expression more efficient, and more OPA1 protein plays a physiological role in the patient's retinal ganglion cells.
  • the nucleic acid encoding human type II mitochondrial dynamin-like GTPase of the present invention has a nucleotide sequence as shown in SEQ ID NOs: 2-4, SEQ ID NOs: 6-8, SEQ ID NOs: 10-12, and SEQ ID NOs: 14-16.
  • the nucleotide sequence has ⁇ 95% identity with the nucleotide sequence shown in SEQ ID NOs: 2-4, SEQ ID NOs: 6-8, SEQ ID NOs: 10-12, and SEQ ID NOs: 14-16, preferably ⁇ 98%, and more preferably ⁇ 99%.
  • the nucleic acid encoding human type II mitochondrial dynamin-like GTPase is also referred to as OPA1 optimized gene or OPA1 optimized nucleic acid.
  • the polynucleotide of the present invention can be in the form of DNA or RNA.
  • the nucleotide is DNA.
  • DNA forms include cDNA, genomic DNA or artificially synthesized DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the nucleotide sequence of the present invention encodes the amino acid sequence shown in SEQ ID NOs: 17-20.
  • the OPA1-MIS signal peptide directs the protein into the mitochondria, and after being hydrolyzed by protease, the mature OPA1 isoform 1, 2, 4, and 7 proteins enter the mitochondria to exert their effects.
  • the nucleic acid sequence can be DNA, RNA, cDNA or PNA.
  • the nucleic acid sequence can be genomic, recombinant or synthetic.
  • the nucleic acid sequence can be isolated or purified.
  • the nucleic acid sequence can be single-stranded or double-stranded.
  • the nucleic acid sequence will encode a light-sensitive protein as described herein.
  • the nucleic acid sequence can be derived by cloning, for example, using standard molecular cloning techniques including restriction enzyme cutting, ligation, gel electrophoresis, such as described in Sambrook et al. Molecular Cloning: A laboratory manual, Cold Spring Harbour Laboratory Press).
  • the nucleic acid sequence may be isolated, for example, isolated using PCR technology.
  • the nucleic acid sequence can be synthetic, for example, produced by direct chemical synthesis.
  • the nucleic acid sequence can be provided as a naked nucleic acid, or it can be provided in a complex with a protein or lipid.
  • the full-length nucleotide sequence of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • primers can be designed based on the disclosed relevant nucleotide sequence, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence.
  • a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence.
  • the DNA sequence encoding the polypeptide of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • 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 encoding sequence of the present invention.
  • the above polynucleotide, vector or host cell may be isolated.
  • isolated means that a substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in their natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist with them in their natural state.
  • nucleotide sequence is as shown in SEQ ID NOs: 3, 8, 12, 15 Show.
  • the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter.
  • a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.
  • the method of using PCR technology to amplify DNA/RNA 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 DNA/RNA fragments amplified 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, a host cell produced by genetic engineering using the vector or protein coding sequence of the present invention, and a method of expressing OPA1 protein using the host cell by recombinant technology.
  • the polynucleotide sequence of the present invention can be used to obtain a host cell (such as a mammalian cell) expressing the OPA1 protein of the present invention by conventional recombinant DNA technology, which generally includes the steps of: transducing the polynucleotide of the first aspect of the present invention or the vector of the third aspect of the present invention into the host cell.
  • a host cell such as a mammalian cell
  • OPA1 protein of the present invention by conventional recombinant DNA technology, which generally includes the steps of: transducing the polynucleotide of the first aspect of the present invention or the vector of the third aspect of the present invention into the host cell.
  • expression vectors containing the coding DNA sequence of 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 recombination technology, etc.
  • the DNA sequence can be effectively linked to an appropriate promoter in the 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 a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.
  • selectable marker genes such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the polypeptide.
  • the host cell can 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 include: animal cells such as CHO, NS0, COS7, or 293 cells.
  • 293T cells, photoreceptor cells (including cone cells and/or rod cells), other visual cells (such as binocular cells), and nerve cells are selected as host cells.
  • the host cell is selected from the following group: rod cells, cone cells, light-applying bipolar cells, light-withdrawing bipolar cells, horizontal cells, ganglion cells, amacrine cells, or a combination thereof.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2 . If necessary, transformation can also be carried out by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the protein encoded by the gene of the present invention.
  • the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the promoter selected is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel chromatography), and purification. filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • an optimized coding sequence of a recombinant human type II mitochondrial dynamin-like GTPase that is particularly suitable for expression in mammalian cells is provided.
  • the coding sequence is shown in SEQ ID NOs: 2-4, SEQ ID NOs: 6-8, SEQ ID NOs: 10-12, and SEQ ID NOs: 14-16, which are sequence optimizations of variant 1, variant 2, variant 4, and variant 7 of OPA1, respectively.
  • the "optimized OPA1 coding sequence” and “optimized OPA1 coding gene” both refer to a nucleotide sequence used to encode a recombinant human type II mitochondrial dynamin-like GTPase, and the nucleotide sequence encodes the amino acid sequence shown in SEQ ID NOs: 17-20.
  • the wild-type DNA coding sequence (unoptimized DNA coding sequence) of the recombinant human type II mitochondrial dynamin-like GTPase is shown as SEQ ID NO: 1, 5, 9, 13.
  • the expression level of the unoptimized wild-type DNA coding sequence is very low (the four levels are equivalent).
  • the present invention optimizes sequence fragments that affect gene expression and protein localization, including but not limited to, codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, hidden splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.
  • codon usage preference such as hairpin structures
  • secondary structures that are not conducive to expression such as hairpin structures
  • changes in GC content CpG dinucleotide content
  • secondary structure of mRNA hidden splicing sites
  • early polyadenylation sites early polyadenylation sites
  • internal ribosome entry sites and binding sites negative CpG islands
  • RNA unstable regions repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect
  • SEQ ID NO1 wild Homo sapiens:OPA1 isoform1;
  • SEQ ID NO2 OPA1 isoform1-opt1;
  • SEQ ID NO4 OPA1 isoform1-opt3;
  • SEQ ID NO5 wild Homo sapiens:OPA1 isoform2;
  • SEQ ID NO9 wild Homo sapiens: OPA1 isoform4;
  • SEQ ID NO12 OPA1 isoform4-opt3;
  • SEQ ID NO13 Wild Homo sapiens: OPA1 isoform 7;
  • SEQ ID NO17 OPA1-isoform1 amino acid sequence
  • SEQ ID NO18 OPA1-isoform2 amino acid sequence
  • SEQ ID NO19 OPA1-isoform4 amino acid sequence
  • SEQ ID NO20 OPA1-isoform7 amino acid sequence.
  • the present invention also provides a fusion nucleic acid, which comprises the nucleic acid sequence encoding human type II mitochondrial dynamin-like GTPase as described in the first aspect of the present invention.
  • fusion nucleic acid refers to a nucleic acid formed by the connection of two or more nucleotide sequences from different sources, or a nucleic acid formed by the connection of two or more nucleotide sequences from the same source but not linked to each other in their natural positions.
  • the present invention also provides an expression vector for OPA1 protein, which contains the optimized OPA1 coding sequence of the present invention.
  • nucleic acid sequences or vectors suitable for transduction into cells Given the sequence information, a skilled artisan can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transduction into cells.
  • the nucleic acid sequence encoding the OPA1 protein is provided as a vector, preferably an expression vector.
  • it can be provided as a gene therapy vector preferably suitable for transduction and expression in retinal target cells.
  • the vector can be viral or non-viral (e.g., a plasmid).
  • Viral vectors include those derived from: adenovirus, adeno-associated virus (AAV) including mutated forms, retrovirus, lentivirus, herpes virus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, poxvirus, and SV40.
  • the viral vector is replication defective, although it is envisioned that it may be replication deficient, capable of replication or conditional replication.
  • the viral vector can generally maintain an extrachromosomal state without integrating into the genome of the target retinal cell.
  • the preferred viral vector for introducing the nucleic acid sequence encoding the OPA1 protein into the retinal target cell is an AAV vector, such as a self-complementary adeno-associated virus (scAAV).
  • scAAV self-complementary adeno-associated virus
  • 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 sequence.
  • the virus in AAV, can be modified to delete all or part of the IX gene, Ela and/or Elb gene.
  • a helper virus such as adenovirus
  • replication is very inefficient.
  • adeno-associated viruses preferably, replication genes and capsid genes are provided in trans (in pRep/Cap plasmids), and only the 2ITR of the AAV genome is retained and packaged into virions, while the required adenoviral genes are 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 used in the present invention is preferably suitable for use in vivo or in vitro, and is 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 the retinal target cells. Regulatory sequences may include promoters, enhancers, transcription termination signals, polyadenylation sequences, replication origins, nucleic acid restriction sites, and homologous recombination sites operably linked to the nucleic acid sequence.
  • the vector may also include a selective marker, for example to determine expression of the vector in a growth system (e.g., bacterial cells) or in retinal target cells.
  • operably linked means that a nucleic acid sequence is functionally related to a sequence to which it is operably linked, such that they are linked in a manner such that they affect the expression or function of each other.
  • 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 connected thereto.
  • the promoter may be constitutive or may be inducible.
  • the promoter may direct ubiquitous expression in inner retinal cells, or neuron-specific expression. In the latter case, the promoter may direct cell type-specific expression, such as to optic ganglion cells.
  • suitable promoters will be known to those skilled in the art.
  • suitable promoters may be selected from the group consisting of: L7, thy-1, recovery protein, calcium-binding protein, human CMV, GAD-67, chicken b-actin, hSyn, Grm6, Grm6 enhancer SV40 fusion protein.
  • Targeting may be achieved using a cell-specific promoter, such as Grm6-SV40 for selective targeting to optic nerve cells.
  • the Grm6 promoter is a 200 base pair enhancer sequence of the Grm6 gene
  • the Grm6 gene is a fusion of the SV40 eukaryotic promoter, and encodes the metabotropic glutamate receptor mGluR6 specific to optic nerve cells.
  • the preferred sources of the Grm6 gene are mice and humans.
  • Ubiquitous expression can be achieved using a pan-neuronal promoter, examples of which are known and available in the art.
  • One such example is CAG.
  • the CAG promoter is a fusion of the CMV early enhancer and the chicken b-actin promoter.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level.
  • Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter avian leukemia virus promoter
  • Epstein-Barr Epstein-Barr
  • Rous sarcoma virus promoter Rous sarcoma virus promoter
  • human gene promoters such as but not limited to
  • 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 expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • OPA1 protein can be expressed 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.
  • adeno-associated virus is preferably used as an expression vector.
  • the present invention also provides a method for constructing a recombinant adeno-associated virus vector carrying a recombinant human type II mitochondrial dynamin-like GTPase gene.
  • the method can quickly and easily construct a recombinant adeno-associated virus vector carrying a recombinant human type II mitochondrial dynamin-like GTPase gene, and package it to obtain a complex defective adeno-associated virus vector.
  • the present invention also provides a host cell for expressing OPA1 protein.
  • the host cell is a mammalian cell (preferably a human cell, more preferably a human optic nerve cell or a photoreceptor cell), and the expression level of OPA1 protein is increased.
  • the present invention provides a preparation or composition, which contains (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 autosomal dominant optic atrophy (ADOA).
  • ADOA autosomal dominant optic atrophy
  • the “active ingredient” in the pharmaceutical composition of the present invention refers to the vector described in the present invention, such as a viral vector (including an adeno-associated viral vector).
  • the "active ingredient”, preparation and/or composition described in the present invention can be used to treat eye diseases.
  • Safe and effective amount means: 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” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity.
  • “Compatibility” here means that the components in the composition can be mixed with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient.
  • the composition may be a 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 vector may be administered to the eye via subretinal or intravitreal administration.
  • the vector is provided as an injectable liquid.
  • the injectable liquid is provided as a capsule or a syringe.
  • Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl Sodium 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, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), 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, ethyl Sodium cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, ethyl Sodium cellulose, cellulose acetate,
  • the composition may comprise a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension or emulsion, 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 OPA1 provided by the present invention can produce OPA1 protein or OPA1 fusion protein in vitro or in vivo, and the fusion protein or the preparation containing the fusion protein can be used to prepare a drug for treating autosomal dominant optic atrophy (ADOA).
  • ADOA autosomal dominant optic atrophy
  • the optimized nucleic acid encoding human type II mitochondrial dynamin-like GTPase has a higher expression level, thereby translating more OPA1 protein, and the MIS sequence in the optimized OPA1 protein can accurately enter the GTP functional protein into the mitochondria, so that more GTPase is transfected into the mitochondria.
  • the agent containing the nucleic acid of the present invention is injected into the vitreous cavity of the rabbit eye, and the agent remains active in the vitreous cavity and is transfected into the optic nerve cells.
  • the optimized OPA1 nucleic acid encoding more OPA1 protein than the prior art can better treat autosomal dominant optic atrophy (ADOA).
  • the coding sequences of different variants of wild-type OPA1 were codon-optimized and the corresponding AAV vector plasmids were constructed ( Figure 1), wherein the vector backbone contained AAV2 5’ITR, CMV promoter, CMV enhancer, SV40 polyA sequence and AAV2 3’ITR.
  • a in Figure 1 is the codon-optimized OPA1-iso1 vector;
  • B in Figure 1 is the codon-optimized OPA1-iso2 vector;
  • C in Figure 1 is the codon-optimized OPA1-is3 vector; and
  • D in Figure 1 is the codon-optimized OPA1-iso3 vector.
  • Plasmids containing codon-optimized OPA1 sequences and wild-type OPA1 sequences were transformed into Escherichia coli Stbl3 strains and coated on LB plates supplemented with corresponding antibiotics. After plasmids were extracted, the stability of the plasmids was identified.
  • variant 1 iso1-opt1, iso1-opt2, and iso1-opt3
  • iso1-opt1 is shown in SEQ ID NO: 2
  • iso1-opt2 is shown in SEQ ID NO: 3
  • iso1-opt3 is shown in SEQ ID NO: 4
  • the stability of the recombinant plasmid significantly affects the application value of the target DNA it carries.
  • This experiment screened out recombinant plasmids that can be stably passaged in different variant sequences by optimizing the codon composition and eliminating potential splicing sites and secondary structures.
  • Example 2 AAV virus packaging and detection
  • the three optimized sequence plasmids of different OPA1 variants were transfected into 293T cells respectively, and multiple virus packaging was completed. The virus titer was then tested to evaluate the virus yield.
  • HEK293T cells with a polymerization degree of more than 90% were passaged at a ratio of 1:3.
  • the virus mixture was purified by iodixanol density gradient centrifugation and then concentrated using an ultrafiltration tube.
  • EASY Dilution Add 100 ⁇ l of EASY Dilution to the lysed virus sample to dilute it 10 times, and then use EASY Dilution to perform a 10-fold series gradient dilution to 100 times, 1000 times, 10000 times, and 100000 times.
  • AAV-iso1-opt2 is the virus with the highest genome concentration in variant No. 1 (A in Figure 4), while AAV-iso2-opt3 (B in Figure 4), AAV-iso4-opt3 (C in Figure 4) and AAV-iso7-opt2 (D in Figure 4) are the viruses with the highest genome concentrations in variant No. 2, variant No. 4 and variant No. 7, respectively.
  • HEK293 cells were transfected with 2 ⁇ g of pAAV-OPA1-unoptimized, pAAV-iso1-opt2, pAAV-iso2-opt3, pAAV-iso4-opt3, and pAAV-iso7-opt2 plasmids. After 48 hours of culture, cells were collected for WB detection of opa1 protein and internal reference GAPDH expression. The WB results are shown in Figure 5. The expression level of the optimized vector was significantly better than that of the unoptimized vector. After grayscale analysis of the WB bands using ImageJ software, the relative expression levels of OPA1 protein between the groups were calculated (see the table below). The amount of OPA1 protein in the optimized vector was 2 to 3 times higher than that in the unoptimized vector.
  • OPA1-null MEF cells have mitochondrial fragmentation and aerobic respiration dysfunction due to the lack of OPA1 protein, so the growth of the cells in galactose medium (using aerobic respiration for energy) is significantly slower than that in glucose medium (using glycolysis for energy).
  • OPA1-null MEF cells take OPA1-null MEF cells in the logarithmic growth phase and trypsinize and adjust the cells. The density was 1E4 cells/ml and inoculated into a 96-well plate, 100 microliters per well, a total of 14 groups, 3 replicates per group, and cultured overnight at 37°C incubator.
  • the culture medium was replaced with galactose or glucose culture medium according to the grouping, and the cells were lysed after 72 hours of continuous culture, and the OD value was detected with CellTiter-Lumi TM luminescence detection reagent, and the growth rate ratio of the cells in the two culture media was calculated.
  • the experimental results are shown in Figure 6. Compared with the uninfected group, infection with each optimized vector of AAV-OPA1 can restore the mitochondrial aerobic respiration function of OPA1-null MEF cells. Compared with the unoptimized vector, the optimized vector significantly increased the growth rate of cells in galactose medium.

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Abstract

一种人线粒体动力蛋白样GTP酶及应用。对编码重组人Ⅱ型线粒体动力蛋白样GTP酶的四个变体的基因序列进行了优化，通过序列稳定性及蛋白表达情况实验表明，提供的编码人线粒体动力蛋白样GTP酶核苷酸序列稳定性好，蛋白表达量高，有较好地治疗ADOA等眼部疾病的前景。

Description

人线粒体动力蛋白样GTP酶及应用
本申请要求于2022年10月17日提交中国专利局、申请号为202211267366.6、发明名称为“人线粒体动力蛋白样GTP酶及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物制剂领域，尤其涉及人线粒体动力蛋白样GTP酶及应用。
背景技术
常染色体显性视神经萎缩ADOA(autosomal dominant optic atrophy)是原发性遗传性视神经病变的最常见形式，发病比例约为1:12 000至1:50 000，并在幼年时期隐匿发病。其临床特征表现为轻度至中度逐渐丧失视力，色觉缺陷，中心视野缺损和颞视盘苍白。起初是乳头状血管束的损伤，其次是视神经的上升萎缩和视神经髓鞘的丧失。
ADOA由编码内线粒体膜蛋白和基因座的基因突变引起，国外学者已确定这类基因主要为OPA1～OPA8，其中已初步确认75﹪左右的ADOA患者与OPA1携带的杂合与显性突变有关，其中外显子740、2794位点突变最常见。OPA1是核基因编码的蛋白质，基因序列长度≥40kb，OPA1分子结构包括1个GTP酶结构域(GTPase),2个疏水重复序列(HR)和1个线粒体定位序列(MTS)。OPA1蛋白定位于线粒体内膜，控制能量代谢和凋亡，维持嵴和mtDNA完整性，具有GTP酶活性。ADOA的发病机制是：突变的OPA1编码形成多种截短形式蛋白，这些蛋白通常缺乏完整的GTP酶结构域，等同于缺失一个等位基因，将使OPA1的量大幅下降，出现所谓的单倍体不足，从而使线粒体功能出现障碍。碎片化的线粒体的无法为视神经细胞正常供应ATP，RGC细胞逐渐凋亡，从而导致患者发生ADOA。
ADOA目前尚无方法治疗，是世界公认的遗传性视神经病变之一。随着基因治疗的发展，ADOA的基因治疗成为可能，其中的难点在于OPA1外显子剪接的多种形式以及其突变的多种形式，这就造成多样的致病蛋白类型及突变形式，无法用某一种基因药物治疗所有患者。
国外的研究中已有人对OPA1突变导致ADOA的发病机制进行过研究，分析了OPA1转录和基因产物的多种形式，探索了用于重组OPA1蛋白的更优良纯化方式，并通过家族病史的调查分析寻求如何筛选、鉴定及诊断是否携带缺陷性OPA1，同时还展望了OPA1突变导致的ADOA的预前基因诊断及治疗。目前也已经存在人线粒体动力蛋白样GTP酶基因的重组应用腺相关病毒载体的报道及生产应用。但其编码人II型线粒体动力蛋白样GTP酶的序列单一，核酸序列不稳定，导致其作用效果相对低下，因此本领域亟需开发一种治疗视神经疾病效果好的人线粒体动力蛋白样GTP酶。
发明内容
有鉴于此，本发明要解决的技术问题在于提供人线粒体动力蛋白样GTP酶及应用。
本发明提供了编码人Ⅱ型线粒体动力蛋白样GTP酶的核酸，其如SEQ ID NO：2，SEQ ID NO：3，SEQ ID NO：4，SEQ ID NO：6，SEQ ID NO：7，SEQ ID NO：8，SEQ ID NO：10，SEQ ID NO：11，SEQ ID NO：12，SEQ ID NO：14，SEQ ID NO：15，或SEQ ID NO：16所示中的至少一种。
本发明中，所述的编码人Ⅱ型线粒体动力蛋白样GTP酶的核酸，其来源于编码 人Ⅱ型线粒体动力蛋白样GTP酶的四个野生型变体(变体1、变体2、变体4、变体7；其中，变体1核酸如SEQ ID NO：1所示、变体2核酸如SEQ ID NO：5所示、变体4核酸如SEQ ID NO：9、变体7核酸如SEQ ID NO：13所示)，其中，如SEQ ID NO：2，SEQ ID NO：3，SEQ ID NO：4所示的核酸是变体1经序列优化后获得；如SEQ ID NO：6，SEQ ID NO：7，SEQ ID NO：8所示的核酸是变体2经序列优化后获得；如SEQ ID NO：10，SEQ ID NO：11，SEQ ID NO：12所示的核酸是变体4经序列优化后获得；如SEQ ID NO：14，SEQ ID NO：15，或SEQ ID NO：16所示的核酸是变体7经序列优化后获得。
在一些具体的实施例中，本发明通过核酸序列稳定性和蛋白表达分析，筛选得出如SEQ ID NO：3、SEQ ID NO：8、SEQ ID NO：12、SEQ ID NO：15具有较高的核酸序列稳定性和蛋白表达量，核酸序列稳定性显著优于其他序列，且包含所述四种序列的质粒载体转染细胞后制得的病毒滴度高，其中SEQ ID NO：15所示的核酸用于病毒包装的滴度高于其他序列。
本发明中，经优化的编码人Ⅱ型线粒体动力蛋白样GTP酶的核酸表达量更高，从而翻译出更多的OPA1蛋白，而优化后的OPA1蛋白中的MIS序列可以准确地将GTP功能蛋白进入到线粒体内，因此有更多的GTP酶转染到在线粒体中。将含有本发明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OPA1核酸编码比现有技术表达更多的OPA1蛋白，能较好地治疗常染色体显性视神经萎缩(ADOA)。
本发明提供了表达模块，其包括启动子，终止子和本发明所述的核酸中的至少一种。
一些实施例中，所述启动子选自人CMV、GAD-67、鸡β肌动蛋白、hSyn、Grm6、Grm6-SV40。使用细胞特异的启动子可以实现靶向表达，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。在一些具体的实施例中，本发明采用CMV启动子用于所述人Ⅱ型线粒体动力蛋白样GTP酶的表达。
本发明中，所述的表达模块还包括单个或多个本发明所述的核酸以串联、融合表达或其他可行方式进行组合形成的表达模块，本发明对此不做限定。
本发明中还提供了融合蛋白的转录单元，所述转录单元是指启动子开始至终止子结束的DNA序列。启动子和终止子两侧或之间还可包括调控片段，所述调控片段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、跨膜信号肽和同源重组位点，例如启动子的增强子，ITR序列、polyA、MIS信号肽等。进一步的，所述ITR序列包括3'ITR和/或5'ITR；所述polyA包括SV40、hGH、BGH和rbGlob；所述MIS信号肽，定向引导该蛋白进入线粒体，其位于OPA1的N端，在其被蛋白酶水解后，成熟的OPA1变体1、2、4、7蛋白进入线粒体发挥作用。
本发明提供了载体，其包括：
载体骨架和本发明所述的核酸；
或载体骨架和本发明所述的表达模块。
进一步的，本发明所述的载体的来源包括植物、动物、微生物、或病毒载体，本发明对此不做限定。所述病毒载体来源包括：腺病毒、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。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OPA1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血清型包括AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV2.7M8或AAV2-TYF突变型或其组合，或这些血清型中的任何一个的修饰的版本(包括AAV 4YF和AAV7m8载体)，其可实现选择性靶向。
本发明所述重组的载体，是指重组的核酸载体，是一种重组DNA分子，其包含期望的编码序列和对可操作连接的编码基因在具体宿主生物内的表达所必不可少的合适的核酸序列或元件。对病毒、微生物或哺乳动物细胞中的表达必需的核酸序列或元件包括启动子，核糖体结合位点及可能的其它序列。已知真核细胞利用启动子，增强子以及终止子。一经转化进入合适的宿主，载体可以独立于宿主基因组进行复制和发挥作用，或者，在一些情况下，自己整合进入基因组。在本说明书中，“质粒”和“载体”有时可以交换通用，因为质粒是当前最普遍使用的载体形式。然而，本发明意图包括表达载体的这样的其它形式，其发挥等价作用，其在本领域是已知的或将变为已知的，包括但不限于：质粒，噬菌体颗粒，病毒载体和/或仅为潜在的基因组插入物。具体实施例中，编码本发明提供的融合蛋白的核酸可构建于各种真核表达载体中。
本发明提供了宿主细胞，其包括如下I)～III)所示中的任意一种或多种：
I)、染色体整合本发明所述的核酸；
II)、染色体整合本发明所述的表达模块；
III)、转染或转化本发明所述的载体。
本发明提供的宿主，来源包括植物、动物、微生物或病毒，本发明对此不做限定。本发明使用重组DNA技术构建的载体转化或转染宿主细胞，这样转化的宿主细胞有能力复制编码蛋白质的载体或表达期望蛋白质。
本发明中，所述转化的方法包括：化学转化和电转化；所述转染的方法包括磷酸钙共沉淀、人工脂质体法、病毒转染。所述的病毒转染包括腺病毒转染、腺相关病毒转染、慢病毒转染等。本发明一些实施例中，利用腺相关病毒对所述宿主进行转染。
进一步的，在一些具体实施例中，本发明所述的宿主细胞为哺乳动物细胞，具体的选自293T细胞、感光细胞、双节细胞、视神经细胞、或其组合。在一些具体实施例中，本发明所述的宿主细胞选自视杆细胞、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。
本发明提供了病毒，其由本发明所述的载体与包装质粒共转染或转化宿主细胞后产生。
进一步的，所述的病毒包括腺病毒、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中的至少一种。在一些具体的实施例中，本发明所述的病毒为腺相关病毒。
本发明提供了重组人Ⅱ型线粒体动力蛋白样GTP酶的制备方法，其包括：培养本发明所述的宿主细胞，得到重组人Ⅱ型线粒体动力蛋白样GTP酶。
本发明提供了如下a)～f)所示中的任意一种在恢复视力或治疗眼部疾病药物中的应用：
a)、本发明所述的核酸；
b)、本发明所述的表达模块；
c)、本发明所述的载体；
d)、本发明所述的宿主；
e)、本发明所述的病毒；
f)、本发明所述的制备方法制得的重组人Ⅱ型线粒体动力蛋白样GTP酶或含有其的培养物。
本发明所述的应用为通过使用安全有效药物剂量的本发明所述的载体或病毒来有效增加细胞产生的ATP含量，和/或抑制线粒体凋亡，从而恢复视力或治疗眼部疾病。进一步的，所述药物中载体的含量为1×109-1×1016个病毒/毫升，较佳地1×1011-1×1013个病毒/毫升,更佳地2×1011-1×1012个病毒/毫升。
所述安全有效剂量指的是：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。
本发明所述的活性成分指可复制的载体，或含有所述载体的脂质体颗粒，或具有生物活性的病毒，或重组人Ⅱ型线粒体动力蛋白样GTP酶。
进一步的，本发明所述眼部疾病包括所述眼部疾病包括由OPA1突变导致的常染色体显性视神经萎缩或视网膜神经节细胞凋亡中的至少一种。。
本发明提供了恢复视力或治疗眼部疾病的药物，其原料包括如下A)～F)所示中的至少一种：
A)、本发明所述的核酸；
B)、本发明所述的表达模块；
C)、本发明所述的载体；
D)、本发明所述的宿主；
E)、本发明所述的病毒；
F)、本发明所述的制备方法制得的重组人Ⅱ型线粒体动力蛋白样GTP酶或含有其的混合物。
进一步的，所述的药物还包括含有所述核酸、重组核酸或mRNA的脂质纳米颗粒。所述的脂质纳米颗粒的制备过程包括利用动物体内转染试剂对所述的核酸、重组核酸或mRNA进行包裹，形成含有所述核酸或mRNA脂质纳米颗粒形式的疫苗用于肿瘤的防治。
更进一步的，本发明所述的药物还包括药学上可接受的辅料或载体。
本发明中，所述的药物的剂型包括但不限于冻干制剂、液体制剂、或其组合。
本发明提供了一种提高线粒体动力蛋白样GTP酶的表达和/或活性的方法，其包括导入本发明所述的载体，和/或施用本发明所述的药物；其能有效增加细胞产生的ATP含量，和/或抑制线粒体凋亡；持续上调GTP酶的表达和/或活性。
本发明提供了恢复视力或治疗眼部疾病的方法，其为给予本发明所述的药物。所述方法能有效提高眼球线粒体动力蛋白样GTP酶的表达和/或活性。所述眼球线粒体动力蛋白样GTP酶的表达和/或活性长达6个月，优选地长达3个月。
所述治疗方法能有效增加视网膜ATP含量，和/或抑制线粒体凋亡。本发明所述药物给予方式可以包括注射、口服或基因枪。
本发明涉及生物制剂领域，尤其涉及人线粒体动力蛋白样GTP酶及应用。本发明对编码重组人Ⅱ型线粒体动力蛋白样GTP酶的四个变体的基因序列进行了优化，通过序列稳定性及蛋白表达情况实验表明，本发明提供的编码人线粒体动力蛋白样 GTP酶核苷酸序列稳定性好，安全性好、蛋白表达量高，有较好地治疗ADOA等眼部疾病的前景。
附图说明
图1示AAV-OPA1载体图谱，其中，A为密码子优化的OPA1-iso1-opt质粒载体示意图；B为密码子优化的OPA1-iso2-opt质粒载体示意图；C为密码子优化的OPA1-iso4-opt质粒载体示意图；D为密码子优化的OPA1-iso7-opt质粒载体示意图；
图2示AAV-OPA1重组质粒的酶切鉴定，红色方框示酶切异常的条带，M为Trans5000Marker；其中，A为变体iso1三种密码子优化后的序列opt1～opt3酶切条带稳定性检测；B为变体iso2三种密码子优化后的序列opt1～opt3酶切条带稳定性检测；C为变体iso4三种密码子优化后的序列opt1～opt3酶切条带稳定性检测；D为变体iso7三种密码子优化后的序列opt1～opt3酶切条带稳定性检测；
图3示AAV-OPA1不同变体及各自不同的优化质粒包装的AAV病毒产量比较；
图4示AAV-OPA1不同变体及各自不同的优化质粒包装的AAV病毒基因组电泳鉴定；
图5示AAV-OPA1各变体优化质粒的蛋白表达检测；
图6示AAV-OPA1各变体优化载体的修复线粒体功能比较。
具体实施方式
本发明提供了人线粒体动力蛋白样GTP酶及应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明中涉及序列如下：
野生OPA1-iso1的核苷酸序列为：

iso1-opt1核苷酸序列为：

iso1-opt2核苷酸序列为：
iso-opt3的核苷酸序列为：
野生OPA1-iso2的核苷酸序列为：

iso2-opt1的核苷酸序列为：

iso2-opt2的核苷酸序列为：

iso2-opt3的核苷酸序列为：
野生OPA1-iso4的核苷酸序列为：

iso4-opt1的核苷酸序列为：

iso4-opt2的核苷酸序列为：

iso4-opt3的核苷酸序列为：

野生OPA1-iso7的核苷酸序列为：

iso7-opt1的核苷酸序列为：
iso7-opt2的核苷酸序列为：

iso7-opt3的核苷酸序列为：

野生OPA1-iso1的氨基酸序列为：

OPA1-iso2氨基酸序列为：
OPA1-iso4氨基酸序列为：
OPA1-iso7氨基酸序列为：

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”。
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。
OPA1(optic atrophy 1)
如本文所用，术语“(重组)人Ⅱ型线粒体动力蛋白样GTP酶”、“视神经萎缩症蛋白1”、“OPA1(蛋白)”、“hOPA1(蛋白)”、“多肽”、“本发明多肽”和“本发明蛋白”具有相同的意义，在本文可互换使用。OPA1是位于线粒体内膜具有GTPase活性的跨膜蛋白，该蛋白包括N-末端跨膜区具有线粒体膜插入序列(mitochondrial import sequence,MIS)、跨膜区域(TR)和疏水区域(HR)，GTPase结构域，中枢动力蛋白结构域和C-末端具有GTPase效应结构域(GTPase effector domain,GED)。OPA1的MIS多肽的功能是引导GTP蛋白进入到线粒体中。OPA1的生物合成，受到转录和翻译2个水平的调控。在哺乳动物中OPA1有许多的剪接体。OPA1mRNA前体在外显子4、4b和5b处选择性剪接，可产生有组织特异性的八种mRNA变体来编码OPA1，而且这些不同形式的OPA1剪接体有不同的功能。例如在视网膜黄斑区1号变体的丰度最高。八种变体中1、2、4、7号变体均可通过翻译后水解产生长型和短型两种形式的OPA1蛋白。长型OPA1蛋白定位于线粒体内膜，参与线粒体内膜融合与分裂；短型OPA1蛋白位于线粒体膜间隙，负责维持嵴和mtDNA完整性，控制能量代谢和凋亡。
ADOA，由编码内线粒体膜蛋白和基因座的基因突变引起，国外学者已确定这类基因主要为OPA1-OPA8，其中已初步确认75％左右的ADOA患者与OPA1携带的杂合与显性突变有关，其中外显子740、2794位点突变最常见。ADOA的发病机制是：突变的OPA1编码形成多种截短形式蛋白，这些蛋白通常缺乏完整的GTP酶结构域， 等同于缺失一个等位基因，将使OPA1的量大幅下降，出现所谓的单倍体不足，从而使线粒体功能出现障碍。碎片化的线粒体的无法为视神经细胞正常供应ATP，RGC细胞逐渐凋亡，从而导致患者发生ADOA。
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)，其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重组人Ⅱ型线粒体动力蛋白样GTP酶表达量不高、治疗效果不佳的技术缺陷。本发明的目的是提供一种重组人Ⅱ型线粒体动力蛋白样GTP酶优化基因序列。重组人Ⅱ型线粒体动力蛋白样GTP酶基因，其功能是引导GTP蛋白进入到线粒体中，发挥其生理功能；289到2772bp，编码828个氨基酸的肽链，是GTP功能蛋白，最后3bp为终止密码子。经研究发现，本发明优化的OPA1基因序列使OPA1蛋白表达效率更高，有更多的OPA1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Ⅱ型线粒体动力蛋白样GTP酶的核酸，其核苷酸序列如SEQ ID NOs:2-4、SEQ ID NOs:6-8、SEQ ID NOs:10-12、SEQ ID NOs:14-16所示。优选地，所述核苷酸序列与SEQ ID NOs:2-4、SEQ ID NOs:6-8、SEQ ID NOs:10-12、SEQ ID NOs:14-16所示的核苷酸序列有≥95％相同性，优选地≥98％，更优选地≥99％。在本发明中，所述编码人Ⅱ型线粒体动力蛋白样GTP酶的核酸又称作OPA1优化基因或OPA1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。本发明所述的核苷酸序列编码SEQ ID NOs:17-20所示的氨基酸序列。OPA1-MIS信号肽，定向引导该蛋白进入线粒体，被蛋白酶水解后，成熟的OPA1 isoform 1、2、4、7蛋白进入线粒体发挥作用。
核酸序列可以是DNA、RNA、cDNA或PNA。核酸序列可以是基因组的、重组的或合成的。核酸序列可以是分离的或纯化的。核酸序列可以是单链或双链的。优选地，核酸序列将编码如本文描述的光敏蛋白。核酸序列可以通过克隆衍生，例如使用包括限制性酶切、连接、凝胶电泳的标准的分子克隆技术，例如在Sambrook等Molecular Cloning:A laboratory manual,Cold Spring Harbour Laboratory Press)中描述的。核酸序列可是分离的，例如使用PCR技术分离的。分离意指从任何杂质和从被自然地发现与其来源中的核酸序列缔合的其他核酸序列和/或蛋白中分离核酸序列。优选地，其还将不含细胞材料、培养基或来自纯化/生产过程的其他化学物质。核酸序列可以是合成的，例如通过直接的化学合成产生。核酸序列可以作为裸露的核酸被提供，或可与蛋白或脂质复合提供。
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 NOs:3、8、12、15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OPA1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OPA1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293T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CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。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
在本发明中，提供了优化的、特别适合在哺乳动物细胞中表达的重组人Ⅱ型线粒体动力蛋白样GTP酶的编码序列，所述编码序列如SEQ ID NOs:2-4、SEQ ID NOs:6-8、SEQ ID NOs:10-12、SEQ ID NOs:14-16所示,分别是对OPA1的1号变体，2号变体，4号变体以及7号变体进行序列优化。
如本文所用，所述“优化的OPA1编码序列”、“优化OPA1编码基因”均指一种用于编码重组人Ⅱ型线粒体动力蛋白样GTP酶的核苷酸序列，所述的核苷酸序列编码SEQ ID NOs:17-20所示的氨基酸序列。
在本发明中，所述重组人Ⅱ型线粒体动力蛋白样GTP酶的野生DNA编码序列(未优化的DNA编码序列)如SEQ ID NO:1,5,9,13所示。所述未优化的野生DNA编码序列的表达量很低(四者水平相当)。
本发明优化了影响基因表达和蛋白定位的序列片段，这些序列片段包括但不限于，密码子使用偏好性，消除不利于表达的二级结构(如发夹结构)，改变GC含量，CpG二核苷酸含量，mRNA的二级结构，隐蔽剪接位点，早期多聚腺苷化位点，内部核糖体进入位点和结合位点，负CpG岛，RNA不稳定区，重复序列(直接重复、反向重复等)和可能影响克隆的限制性位点。通过分析和试验筛选，最终得到如SEQ ID NOs:2-4、6-8、10-12、14-16所示的特别优化的DNA编码序列。这些序列是经过特殊优化，表达量显著提高。
SEQ ID NO1：野生Homo sapiens:OPA1 isoform1；
SEQ ID NO2：OPA1 isoform1-opt1；
SEQ ID NO3：OPA1 isoform1-opt2；
SEQ ID NO4：OPA1 isoform1-opt3；
SEQ ID NO5：野生Homo sapiens:OPA1 isoform2；
SEQ ID NO6：OPA1 isoform2-opt1；
SEQ ID NO7：OPA1 isoform2-opt2；
SEQ ID NO8：OPA1 isoform2-opt3；
SEQ ID NO9：野生Homo sapiens:OPA1 isoform4；
SEQ ID NO10：OPA1 isoform4-opt1；
SEQ ID NO11：OPA1 isoform4-opt2；
SEQ ID NO12：OPA1 isoform4-opt3；
SEQ ID NO13：野生Homo sapiens:OPA1 isoform7；
SEQ ID NO14：OPA1 isoform7-opt1；
SEQ ID NO15：OPA1 isoform7-opt2；
SEQ ID NO16：OPA1 isoform7-opt3；
SEQ ID NO17：OPA1-isoform1氨基酸序列；
SEQ ID NO18：OPA1-isoform2氨基酸序列；
SEQ ID NO19：OPA1-isoform4氨基酸序列；
SEQ ID NO20：OPA1-isoform7氨基酸序列。
融合核酸
本发明还提供了一种融合核酸，其包含本发明第一方面所述的编码人Ⅱ型线粒体动力蛋白样GTP酶的核酸序列。
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。
表达载体和宿主细胞
本发明还提供了一种用于OPA1蛋白的表达载体，它含有本发明的优化OPA1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OPA1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OPA1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b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b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OPA1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。
本发明还提供一种重组人Ⅱ型线粒体动力蛋白样GTP酶基因的重组应用腺相关病毒载体的构建方法，该方法能快速，简便地构建携带重组人Ⅱ型线粒体动力蛋白样GTP酶基因的重组腺相关病毒载体，并包装获得复杂缺陷腺相关病毒载体。
本发明还提供了一种宿主细胞，用于表达OPA1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OPA1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，较佳地为常染色体显性视神经萎缩(ADOA)。
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(如吐温)、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OPA1的核酸或融合核酸，可以体外或体内生产OPA1蛋白或OPA1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常染色体显性视神经萎缩(ADOA)的药物。
经优化的编码人Ⅱ型线粒体动力蛋白样GTP酶的核酸表达量更高，从而翻译出更多的OPA1蛋白，而优化后的OPA1蛋白中的MIS序列可以准确地将GTP功能蛋白进入到线粒体内，因此有更多的GTP酶转染到在线粒体中。将含有本发明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OPA1核酸编码比现有技术表达更多的OPA1蛋白，能较好地治疗常染色体显性视神经萎缩(ADOA)。
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。本发明采用的试材皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1 rAAV2-OPA1-iso2-opt,iso1-opt,iso4-opt,iso7-opt质粒稳定性比较
一.载体构建
将野生型OPA1不同变体(iso1，iso2，iso4，iso7)的编码序列进行了密码子优化并构建了相应的AAV载体质粒(图1)，其中，载体骨架包含AAV2 5’ITR，CMV启动子，CMV增强子，SV40polyA序列和AAV2 3’ITR。图1中的A为密码子优化后OPA1-iso1载体；图1中的B为密码子优化后OPA1-iso2载体；图1中的C为密码子优化后OPA1-is3载体；图1中的D为密码子优化后OPA1-iso3载体。将包含有密码子优化的OPA1序列和野生型OPA1序列的质粒(200ng/μl)分别转化大肠杆菌Stbl3菌株并涂布添加了对应抗生素的LB平板，提取质粒后对质粒稳定性进行鉴定。
二.质粒转化大肠杆菌：
按以下反应体系混合均匀，于冰上放置20min，室温放置10min，加入500μl无抗LB 37℃，200rpm摇床培养40min，然后5000rpm离心3min，吸弃500μl上清，将余下液体重悬菌体沉淀并于相应抗性LB固体平板均匀涂布。将平板置于37℃培养箱培养过夜。
三.质粒DNA的小量提取：
1.挑取单菌落于适量的LA(添加的抗生素取决于质粒抗性)液体培养基中，37℃，200rpm摇过夜(16～20h)；
2.将培养液分装于1.5mL EP管中，每管分装1mL，12000rpm离心3min，颠倒EP管弃去上清；
3.加入100μL溶液I，涡旋混匀至菌体充分悬浮；加入200μL溶液II，轻柔颠倒混匀，至液体澄清而粘稠；加入150μL溶液III，迅速上下颠倒混匀，冰上放置5min后，12000rpm离心10min；
4.吸取上清于新的EP管中，加入2倍体积的无水乙醇沉淀DNA，颠倒混匀，在-20℃放置30min后12000rpm离心10min，弃上清，加入70％酒精180μL，12000rpm离心2～3min，弃上清；
5.室温晾干质粒DNA，加入TE溶解，可室温放置，短期保存置于4℃，长期保存置于-20℃。
四.质粒DNA的酶切验证：
按照以下体系配制反应溶液，置于37℃孵育1～4h，取适量体积上样电泳检测。
分别提取质粒双酶切后电泳观察条带大小，发现每种变体的不同优化序列质粒稳定性表现不一致。其中，1号变体(iso1)的3种优化序列iso1-opt1，iso1-opt2，iso1-opt3(iso1-opt1如SEQ ID NO:2所示；iso1-opt2如SEQ ID NO:3所示，iso1-opt3如SEQ ID NO:4所示)相比较，经过多次传代后，发现iso1-opt2非特异性条带的比例相对最少(图2中的A)，说明iso1-opt2序列的质粒稳定性最优。同理，经过同样的培养传代步骤和酶切鉴定，发现2号变体(iso2)(图2中的B)，4号变体(iso4)(图2中的C)以及7号变体(iso7)(图2中的D)稳定性最高的序列分别为iso2-opt3(如SEQ ID NO:8所示)，iso4-opt3(如SEQ ID NO:12所示)，iso7-opt2(如SEQ ID NO:15所示)。重组质粒的稳定性显著地影响携带的目的DNA的应用价值，本实验通过优化密码子组成，消除潜在的剪接位点和二级结构，在不同的变体序列中均筛选出了能稳定传代的重组质粒。
实施例2 AAV病毒包装及检测
为了比较同一变体的3种优化序列包装病毒的效率，将OPA1不同变体的3种优化序列质粒分别转染293T细胞，完成了多个病毒包装，随后对病毒进行滴度检测，以此评估病毒产量。
一.病毒包装
1.聚合度90％以上的HEK293T细胞按1：3比例传代。
2.转质粒前1～2h左右，换成无血清培养基，用转染试剂将目的基因质粒和辅助质粒转入HEK293T中。
3.质粒转化24h后，换新的无血清培养基。
4.转染72h收毒。带着培养基，吹下细胞，离心；然后分别收获培养基上清与细胞沉淀。用PEG8000沉淀培养基上清中的病毒，沉淀过夜后收集病毒沉淀。
5.将病毒的混合液用碘克沙醇密度梯度离心进行纯化，然后用超滤管进行浓缩。
二.病毒滴度检测
1.病毒裂解
表1样本处理反应体系
按表1配置反应液，再加入20μl待测样本，56℃孵育1小时，裂解病毒，然后90℃孵育10min。
2.将引物按照要求加入适量的EASY Dilution稀释到100μΜ，再将两个引物按体积比1:1混合成50μΜ。
3.病毒样品稀释
在裂解后的病毒样品中加入100μl的EASY Dilution稀释10倍，再用EASY Dilution对其进行10倍系列梯度稀释，稀释至100倍、1000倍、10000倍、100000倍。
4.标准曲线用标准品的制备
取原液2×1012copies/ml的质粒，用注射用水(或EASY Dilution)按照比例稀释为6个梯度作为标准品的模板：2×1011copies/ml，2×1010copies/ml，2×109copies/ml，2×108copies/ml，2×107copies/ml，2×106copies/ml。
5.qPCR体系mix配制
每个样品做3个平行

6.按以下qPCR反应条件上机，结束后分析结果
病毒包装完成后，对各个变体的3种病毒进行滴度检测，发现每种变体的3种病毒产量之间无显著差异(图3)。
随后我们将病毒裂解，将所得基因组DNA进行电泳检测，比较每种变体的3种病毒之间的纯度是否存在区别。
三.基因组电泳检测
1.病毒样本处理
(1)取1支1.5ml EP管，加入10μl病毒样品、10μlPBS、1μl10％SDS、1μl0.5M EDTA、1μl Proteinase K，混匀后56℃孵育1h，95℃10min。
(2)待其冷却至室温，向其中加入4.6μl～5μl 5×Gel Loading Dye purple混匀备用。
2.配置TAE琼脂糖凝胶，配置3×SuperRed染色液，取10mL 5M NaCl加到490mL H2O中，混匀后即为0.1M NaCl溶液。取适量体积的0.1M NaCl溶液，将SuperRed10,000×储液稀释3333倍至3×SuperRed。
3.根据病毒滴度计算，取5E9～4E10的病毒，加入1×loading调整体积至10～20μl，加入上样孔；DNA marker取1～2μl加入上样孔。将电压设置为60V，电泳120min。
4.将凝胶小心地放入铝盒中，缓慢加入足量的3×染色液浸没凝胶。室温振荡染色1h左右，染色完成后拍照观察。
结合滴度检测与基因组电泳检测结果显示，虽然ITR滴度区别不大，但基因组电泳显示不同优化序列的完整基因组片段存在显著差异。其中AAV-iso1-opt2是1号变体中基因组浓度最高的病毒(图4中的A)，而AAV-iso2-opt3(图4中的B)，AAV-iso4-opt3(图4中的C)和AAV-iso7-opt2(图4中的D)分别是2号变体，4号变体和7号变体中基因组浓度最高的病毒。以上结果表明病毒包装过程中出现了部分包装的情况，即两端ITR携带不完整的基因组包裹进了病毒衣壳蛋白，因此导致ITR滴度与实际电泳浓度出现不一致的情况。而每种变体中我们均筛选出了最大限度完整包装的序列，且与实施例1中的质粒稳定性结果保持高度一致。
实施例3 AAV-OPA1各变体优化载体的体外表达检测
pAAV-OPA1-未优化、pAAV-iso1-opt2、pAAV-iso2-opt3、pAAV-iso4-opt3、pAAV-iso7-opt2质粒2μg转染HEK293细胞，培养48小时后收集细胞WB检测opa1蛋白以及内参GAPDH表达情况。WB结果如图5所示，优化后载体的表达水平显著优于未优化载体。用ImageJ软件对WB条带将进行灰度分析后，计算出各组间OPA1蛋白的相对表达水平(见下表)，优化载体的OPA1蛋白量较于未优化载体提高了2～3倍。
实施例4 AAV-OPA1各变体优化载体回复OPA1-null MEF细胞生长速率体外功能检测
OPA1-null MEF细胞由于OPA1蛋白的缺乏导致线粒体片段化，有氧呼吸功能障碍，因此该细胞在半乳糖培养基(利用有氧呼吸供能)中的生长比在葡萄糖培养基(利用糖酵解供能)中显著变慢。取OPA1-null MEF对数生长期细胞，胰酶消化调整细胞 密度为1E4个/ml并接种至96孔板，每孔100微升，共14组，每组3个复孔，37℃培养箱培养过夜。细胞铺板18小时后，按照实验分组感染AAV-GFP、AAV-OPA1未优化、AAV-iso1-opt2、AAV-iso2-opt3、AAV-iso4-opt3以及AAV-iso7-opt2病毒(MOI＝1E5)，未感染组加入相同体积的DMEM，将细胞培养板放回37℃培养箱培养。感染24小时后，将培养基按分组更换为半乳糖或葡萄糖培养基，继续培养72小时后裂解细胞，并用CellTiter-LumiTM发光法检测试剂检测OD值，计算细胞在两种培养基中的生长率比值。实验结果如图6所示，与未感染组相比，感染AAV-OPA1各优化载体均可以恢复OPA1-null MEF细胞的线粒体有氧呼吸功能。与未优化载体相比，优化后载体更加显著地提高了细胞在半乳糖培养基中的生长速率。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (12)

  1. 编码人II型线粒体动力蛋白样GTP酶的核酸，其如SEQ ID NO：2，SEQ ID NO：3，SEQ ID NO：4，SEQ ID NO：6，SEQ ID NO：7，SEQ ID NO：8，SEQ ID NO：10，SEQ ID NO：11，SEQ ID NO：12，SEQ ID NO：14，SEQ ID NO：15，或SEQ ID NO：16所示中的至少一种。
  2. 表达模块，其包括启动子，终止子和权利要求1所述的核酸。
  3. 载体，其包括
    载体骨架和权利要求1所述的核酸；
    或载体骨架和权利要求2所述的表达模块。
  4. 根据权利要求3所述的载体，其特征在于，所述载体为病毒载体。
  5. 宿主细胞，其包括如下I)～III)所示中的任意一种或多种：
    I)、染色体整合如权利要求1所述的核酸；
    II)、染色体整合如权利要求2所述的表达模块；
    III)、转染或转化如权利要求3或4所述的载体。
  6. 根据权利要求5所述的宿主细胞，其特征在于，所述宿主细胞为哺乳动物细胞；所述哺乳动物细胞选自293T细胞、感光细胞、双节细胞、视神经细胞、或其组合。
  7. 病毒，其由权利要求3或4所述的载体与包装质粒共转染或转化宿主细胞后产生。
  8. 根据权利要求7所述的病毒，其特征在于，所述的病毒为腺相关病毒。
  9. 重组人Ⅱ型线粒体动力蛋白样GTP酶的制备方法，其包括：培养如权利要求5或6所述的宿主细胞，获得重组人Ⅱ型线粒体动力蛋白样GTP酶。
  10. 如下a)～f)所示中的任意一种在恢复视力或治疗眼部疾病药物中的应用：
    a)、如权利要求1所述的核酸；
    b)、如权利要求2所述的表达模块；
    c)、如权利要求3或4所述的载体；
    d)、如权利要求5或6所述的宿主；
    e)、如权利要求7或8所述的病毒；
    f)、如权利要求9所述的制备方法制得的重组人Ⅱ型线粒体动力蛋白样GTP酶或含有其的培养物。
  11. 根据权利要求10所述的应用，其特征在于，所述眼部疾病包括由OPA1突变导致的常染色体显性视神经萎缩或视网膜神经节细胞凋亡中的至少一种。
  12. 恢复视力或治疗眼部疾病的药物，其原料包括如下A)～F)所示中的至少一项：
    A)、如权利要求1所述的核酸；
    B)、如权利要求2所述的表达模块；
    C)、如权利要求3或4所述的载体；
    D)、如权利要求5或6所述的宿主；
    E)、如权利要求7或8所述的病毒；
    F)、如权利要求9所述的制备方法制得的重组人Ⅱ型线粒体动力蛋白样GTP酶或含有其的培养物。
PCT/CN2023/124008 2022-10-17 2023-10-11 人线粒体动力蛋白样gtp酶及应用 WO2024083013A1 (zh)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014124282A1 (en) * 2013-02-08 2014-08-14 The Trustees Of The University Of Pennsylvania Enhanced aav-mediated gene transfer for retinal therapies
CN110049769A (zh) * 2016-12-04 2019-07-23 阿拉维·霍拉萨尼·默哈达姆·马塞尔·维克托 治疗与线粒体应激相关的疾病的方法
CN110857440A (zh) * 2018-08-23 2020-03-03 武汉纽福斯生物科技有限公司 重组人ⅱ型线粒体动力蛋白样gtp酶基因序列及其应用
WO2020088548A1 (zh) * 2018-10-30 2020-05-07 天津誉美医药科技有限公司 治疗视网膜色素变性疾病的基因治疗载体
CN111621502A (zh) * 2019-02-28 2020-09-04 武汉纽福斯生物科技有限公司 视网膜劈裂蛋白的编码序列、其表达载体构建及其应用
CN111926021A (zh) * 2020-07-29 2020-11-13 武汉纽福斯生物科技有限公司 重组人norrin胱氨酸结生长因子表达载体及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014124282A1 (en) * 2013-02-08 2014-08-14 The Trustees Of The University Of Pennsylvania Enhanced aav-mediated gene transfer for retinal therapies
CN110049769A (zh) * 2016-12-04 2019-07-23 阿拉维·霍拉萨尼·默哈达姆·马塞尔·维克托 治疗与线粒体应激相关的疾病的方法
CN110857440A (zh) * 2018-08-23 2020-03-03 武汉纽福斯生物科技有限公司 重组人ⅱ型线粒体动力蛋白样gtp酶基因序列及其应用
WO2020088548A1 (zh) * 2018-10-30 2020-05-07 天津誉美医药科技有限公司 治疗视网膜色素变性疾病的基因治疗载体
CN111118016A (zh) * 2018-10-30 2020-05-08 上海市第一人民医院 治疗视网膜色素变性疾病的基因治疗载体
CN111621502A (zh) * 2019-02-28 2020-09-04 武汉纽福斯生物科技有限公司 视网膜劈裂蛋白的编码序列、其表达载体构建及其应用
CN111926021A (zh) * 2020-07-29 2020-11-13 武汉纽福斯生物科技有限公司 重组人norrin胱氨酸结生长因子表达载体及其应用

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