WO2020038473A1 - 重组人ⅱ型线粒体动力蛋白样gtp酶基因序列及其应用 - Google Patents

重组人ⅱ型线粒体动力蛋白样gtp酶基因序列及其应用 Download PDF

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WO2020038473A1
WO2020038473A1 PCT/CN2019/102352 CN2019102352W WO2020038473A1 WO 2020038473 A1 WO2020038473 A1 WO 2020038473A1 CN 2019102352 W CN2019102352 W CN 2019102352W WO 2020038473 A1 WO2020038473 A1 WO 2020038473A1
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vector
nucleic acid
nucleotide sequence
sequence
gtpase
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PCT/CN2019/102352
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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 CA3110153A priority Critical patent/CA3110153A1/en
Priority to MX2021002135A priority patent/MX2021002135A/es
Priority to BR112021002566-9A priority patent/BR112021002566A2/pt
Priority to JP2021509904A priority patent/JP7285022B2/ja
Priority to EP19851390.5A priority patent/EP3851535A4/en
Priority to SG11202101236WA priority patent/SG11202101236WA/en
Priority to KR1020217008407A priority patent/KR102612148B1/ko
Priority to AU2019323501A priority patent/AU2019323501B2/en
Publication of WO2020038473A1 publication Critical patent/WO2020038473A1/zh
Priority to US17/182,903 priority patent/US11459584B2/en

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Definitions

  • the invention relates to the field of biological preparations, in particular to a recombinant human type II mitochondrial dynein-like GTPase gene sequence and application thereof.
  • ADOA Autosomal dominant optic atrophy
  • ADOA is caused by mutations in genes encoding internal mitochondrial membrane proteins and loci. Foreign scholars have determined that such genes are mainly OPA1-OPA8. Among them, it has been initially confirmed that the heterozygous and dominant mutations carried by OPA1 in patients with ADOA at about 75 %, Among them, mutations in exons 740 and 2794 are the most common.
  • OPA1 protein is located on the inner mitochondrial inner membrane, controls energy metabolism and apoptosis, maintains the integrity of tadpoles and mtDNA, and has GTPase activity.
  • the pathogenesis of ADOA is: Mutated OPA1 codes to form a variety of truncated forms of proteins.
  • ADOA is currently untreated and is one of the world's recognized hereditary optic neuropathies. With the development of gene therapy, ADOA gene therapy became possible. The difficulty lies in the multiple forms of OPA1 exon splicing and the multiple forms of mutations. This has caused a variety of pathogenic protein types and mutant forms, which cannot be used. A certain gene drug treats all patients.
  • the object of the present invention is to provide a human mitochondrial dynein-like GTPase expression system and a preparation method with good therapeutic effect.
  • the object of the present invention is to provide an optimized nucleic acid sequence encoding a human mitochondrial dynein-like GTPase, a vector and a preparation method.
  • nucleotide sequence encoding a human type II mitochondrial dynein-like GTPase, and the nucleotide sequence is selected from the following group:
  • nucleotide sequence is ⁇ 95% identical to the nucleotide sequence shown in SEQ ID NO :: 1, preferably ⁇ 98%, and 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.
  • nucleotide sequence is shown as SEQ ID NO.:1, wherein
  • Bits 1-288 are the coding sequence of the MIS
  • Positions 289-2772 are the coding sequences of the GTPase domain, central dynein domain, and GTPase effector domain;
  • Positions 2773-2775 are stop codons.
  • the sequence of SEQ ID NO.:1 includes: the MIS coding sequence, as shown in SEQ ID No.:1 at positions 1-288; and the GTPase domain, central dynein domain, and GTPase
  • the coding sequence of the effector domain is shown in SEQ ID No .: 1 at positions 289-2772.
  • the sequence of SEQ ID NO.:2 includes: MIS coding sequence, as shown in SEQ ID ID NO.:2 at positions 1-288; and the GTPase domain, central motor protein domain, and GTPase
  • the coding sequence of the effector domain is shown in positions 289-2772 of SEQ ID NO.:2.
  • the present invention also provides a nucleotide sequence (SEQ ID No .: 1 or 2 in SEQ ID No .: 1-288) that separately encodes the MIS, and a GTPase domain, a central dynein domain, and a GTPase effector domain that separately encode the MIS.
  • Nucleotide sequence positions 289-2772 or 289-2775 in SEQ ID No .: 1 or 2).
  • a fusion nucleic acid comprising the nucleotide sequence according to the first aspect of the present invention.
  • the fusion nucleic acid further comprises a sequence selected from the group consisting of a UTR sequence, a promoter sequence, or a combination thereof.
  • the UTR sequence includes 3'-UTR and / or 5'-UTR.
  • the UTR sequence contains a ployA sequence with a stable structure.
  • the fusion nucleic acid has the structure of Formula I from the 5 ′ end to the 3 ′ end:
  • Each "-" is independently a bond or a nucleotide linking sequence
  • Z1 is none, or 5'-UTR sequence
  • Z2 is a nucleotide sequence according to the first aspect of the invention.
  • Z3 is a 3'-UTR sequence.
  • the Z1 is a 5′-UTR sequence.
  • each nucleotide linking sequence is 1-30 nt, preferably 1-15 nt, and 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, and the promoters are operatively linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, and selectable marker. , Nucleic acid restriction sites, and / or homologous recombination sites.
  • the vector is selected from the group consisting of a plasmid and a viral vector.
  • the vector is selected from the group consisting of a lentiviral vector, an adenoviral 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 a DNA virus and a retrovirus vector.
  • the vector is an AAV vector containing or inserted with 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; preferably the AAV vector plasmid pSNaV .
  • the vector is used to express a recombinant human type II mitochondrial dynein-like GTPase.
  • a host cell containing the vector according to the third aspect of the present invention, or an exogenous nucleotide according to the first aspect of the present invention integrated into a chromosome thereof. Sequence or a fusion nucleic acid according to the second aspect of the invention.
  • the host cell is a mammalian cell, and the mammal includes a human and a non-human mammal.
  • the host cell is selected from the group consisting of 293T cells, photoreceptor cells (including cone cells and / or rod cells), other visual cells (such as biganglion cells), (optical) nerve cells, Or a combination.
  • the host cell is selected from the group consisting of rod cells, cone cells, light-donating bipolar cells, light-releasing bipolar cells, horizontal cells, ganglion cells, amacrine cells, or combination.
  • the host cell is a (retinal) ganglion cell.
  • the carrier according to the third aspect of the present invention for preparing a preparation or a composition for restoring a subject's vision and / or treating an eye. disease.
  • the eye disease is retinopathy.
  • the preparation or composition is used to treat hereditary optic neuropathy, preferably autosomal dominant optic atrophy (ADOA).
  • ADOA autosomal dominant optic atrophy
  • the preparation or composition is used for treating retinal ganglion cell apoptosis.
  • a pharmaceutical preparation containing (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 lentiviral vector, an adenoviral 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 1 -1 ⁇ 10 13 viruses / ml, and more preferably 2 ⁇ 10 11 -1 ⁇ 10 12 viruses / ml.
  • the pharmaceutical preparation is used to treat ocular diseases, preferably to treat retinal ganglion cell apoptosis.
  • the pharmaceutical preparation is used for treating hereditary optic neuropathy, preferably autosomal dominant optic atrophy (ADOA).
  • ADOA autosomal dominant optic atrophy
  • the pharmaceutical preparation can significantly improve the expression and / or activity of ocular mitochondrial dynein-like GTPase.
  • a seventh aspect of the present invention there is provided a method for long-term increasing the expression and / or activity of a mitochondrial dynein-like GTPase, the method comprising introducing the vector according to the third aspect of the present invention, and / or administering the first
  • the pharmaceutical preparation according to six aspects.
  • the method can effectively increase the content of ATP produced by cells, and / or inhibit mitochondrial apoptosis.
  • the method can continuously upregulate the expression and / or activity of GTPase.
  • a treatment method which comprises applying a carrier according to the third aspect of the present invention to a subject in need.
  • the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, or a combination thereof.
  • the vector is an AAV vector.
  • the carrier is introduced into the eyes of a desired subject.
  • the required objects include humans and non-human mammals.
  • the treatment method is a method for treating ocular diseases.
  • the eye disease is hereditary optic neuropathy, preferably autosomal dominant optic atrophy (ADOA).
  • ADOA autosomal dominant optic atrophy
  • the treatment method can effectively improve the expression and / or activity of mitochondrial dynein-like GTPase in the eyeball.
  • the treatment method can effectively increase the expression and / or activity of the mitochondrial dynein-like GTPase of the eyeball for up to 6 months, preferably up to 3 months.
  • the treatment method can effectively increase retinal ATP content and / or inhibit mitochondrial apoptosis.
  • a method for preparing a recombinant human type II mitochondrial dynein-like GTPase comprising the steps of: culturing a host cell according to the fourth aspect of the present invention, thereby obtaining a recombinant human type II mitochondrial dynein-like protein. GTPase.
  • an ADOA dominant genetic optic neuropathy cell model in which the expression or viability of the OPA1 gene is reduced.
  • the cells include retinal ganglion cells (RGC).
  • the OPA1 gene is not expressed or active at all.
  • a method for preparing an ADOA dominant genetic optic neuropathy cell model includes the steps of: providing a cell to perform point mutation on the OPA1 gene in the cell, thereby obtaining OPA1 gene points Mutant cells; point-monitor positive monoclonal cells were obtained by screening.
  • the OPA1 gene point mutation refers to a Q (glutamine) 285Stop point mutation.
  • the cells include RGC cells.
  • a non-human mammalian model of ADOA dominant genetic optic neuropathy in which the OPA1 gene expression and / or viability in GRC cells is reduced.
  • the OPA1 gene has a point mutation, and specifically, a Q (glutamine) 285Stop point mutation exists.
  • a method for preparing a non-human mammalian model of ADOA dominant genetic optic neuropathy including the following steps:
  • step (b) Using the cells having the point mutation of the OPA1 gene obtained in step (a) to prepare an animal model having the point mutation of the OPA1 gene.
  • the OPA1 gene point mutation is heterozygous or homozygous.
  • the glutamine site at position 285 of the OPA1 protein is mutated to stop transcription.
  • the non-human mammal is a rodent or a primate, and preferably includes a mouse, a rat, a rabbit, and a monkey.
  • step (b) the steps include:
  • the method includes:
  • step (2) using the OPA1 point-mutated mouse embryonic stem cell clone obtained in step (1) to prepare a chimeric mouse;
  • step (3) Mating and breeding the chimeric mouse obtained in step (2) and normal wild-type mice, and screening the offspring to obtain OPA1 point mutation heterozygous mice, which is the Q285Stop point mutation mouse model of OPA1.
  • the heterozygous mice are bred and bred, so that the offspring can be screened to obtain homozygous mice with OPA1 point mutations.
  • Figure 1 shows an open reading frame sequence comparison between the optimized nucleotide sequence and the original human type II mitochondrial dynein-like GTPase gene sequence.
  • the homology of the two is 72.25% (2005/2775), which is indicated by "
  • the upper sequence is optimized for the open reading frame nucleotide sequence
  • the lower sequence is the original human type II mitochondrial dynein-like GTPase gene sequence ( Unoptimized wild coding sequence).
  • Figure 2 shows a schematic diagram of the protein structure of OPA1 isforms 2 transcripts.
  • Figure 3 shows the correct clone selected from the recombinant clone with a target band of about 3000 bp, M: protein marker; lane 1: correct rAAV2 / 2-hOPA1 isoform recombinant clone; lane 2: positive control; lane 3: negative Contrast.
  • Figure 4 shows a schematic diagram of the structure of the recombinant adeno-associated virus plasmid pSNaV / rAAV2 / 2-hOPA1.
  • Figure 5 shows the results of Coomassie Brilliant Blue staining of rAAV2 / 2-hOPA1 isoform 2 purity by SDS-PAGE electrophoresis.
  • lane 1 protein marker
  • lane 2 rAAV2 / 2-hOPA1 isoform 2.
  • Figure 6 shows the fundus photographs of rabbit eyes under glass microscope, where A is the group injected with rAAV2 / 2-ZsGreen (control group), and B is the group injected with rAAV2 / 2-optimized hOPA1 isoform 2 (experimental group A).
  • Figure 7 shows immunofluorescence detection of rabbit eyeball sections.
  • Experimental group A was injected with rAAV2 / 2-optimized hOPA1 isoform2 group
  • experimental group B was injected with rAAV2 / 2-original hOPA1 isoform 2 group.
  • Figure 8 shows the OCT photo taken under a rabbit eye glass.
  • A is the rAAV2 / 2-ZsGreen group (control group)
  • B is the rAAV2 / 2-optimized hOPA1 isoform group 2 (experimental group A).
  • Figure 9 shows the retina in a rabbit eyeball HE section observed under a microscope.
  • A is the rAAV2 / 2-ZsGreen group (control group)
  • B is the rAAV2 / 2-optimized hOPA1 isoform group 2 (experimental group A).
  • Figure 10 shows the results of real-time fluorescent PCR detection of hOPA1 protein in rabbit eyeball retina injected with different plasmids.
  • Experimental group A was injected with rAAV2 / 2-optimized hOPA1 isoform 2 group
  • experimental group B was injected with rAAV2 / 2-
  • the original hOPA1 isoform 2 group, the control group was the rAAV2 / 2-ZsGreen group.
  • Figure 11 shows the results of western blot analysis of hOPA1 protein in rabbit eyeball retina injected with different plasmids.
  • Experimental group A was injected with rAAV2 / 2-optimized hOPA1 isoform 2.
  • Experimental group B was injected with rAAV2 / 2-original hOPA1 isoform 2
  • the control group was the rAAV2 / 2-ZsGreen group.
  • Figure 12 shows the construction of a mouse model of ADOA dominant genetic optic neuropathy.
  • Figure 13 shows genotyping of ADOA dominant genetic optic neuropathy point mutation model mice.
  • Figure 14 shows the change of ATP content in the retina of mice after administration by HPLC.
  • Figure 15 shows the mitochondrial morphology observed by transmission electron microscope TEM.
  • the present inventors have targeted and optimized the coding sequence of the recombinant human type II mitochondrial dynein-like GTPase (OPA1) gene, thereby obtaining a kind of DNA that is particularly suitable for use in mammalian (such as human) cells. Efficient transcription and efficient expression of the nucleotide sequence of OPA1 protein (SEQ ID NO.:1), and a recombinant expression vector of recombinant human type II mitochondrial dynein-like GTPase was constructed.
  • OPA1 recombinant human type II mitochondrial dynein-like GTPase
  • the experimental results show that, compared to the unoptimized coding sequence, the specially optimized OPA1 coding sequence (SEQ ID NO.:1) has slightly improved transcription efficiency, and the expression level has been increased by more than 5 times, which is very suitable for mammals ( Especially in human) cells, it can effectively treat eye diseases such as ADOA.
  • the inventors have completed the present invention.
  • the term “about” may refer to a value or composition within an acceptable error range for a particular value or composition as 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 (comprising)” 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 may 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 identical residues occur. Generally, this is expressed as a percentage.
  • the measurement of the sequence identity of a nucleotide sequence is a method well known to those skilled in the art.
  • 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.
  • OPA1 is a transmembrane protein with GTPase activity on the inner mitochondrial membrane.
  • the protein includes a N-terminal transmembrane region with a mitochondrial membrane insertion sequence (MIS), a transmembrane region (TR), and a hydrophobic region (HR).
  • MIS mitochondrial membrane insertion sequence
  • TR transmembrane region
  • HR hydrophobic region
  • GTPase effector domain GED
  • GED GTPase effector domain
  • ADOA is caused by mutations in genes encoding internal mitochondrial membrane proteins and loci. Foreign scholars have determined that such genes are mainly OPA1-OPA8. Among them, it has been initially confirmed that the heterozygous and dominant mutations carried by OPA1 in patients with ADOA at about 75 %, Among them, mutations in exons 740 and 2794 are the most common.
  • OPA1 protein is located on the inner mitochondrial inner membrane, controls energy metabolism and apoptosis, maintains the integrity of tadpoles and mtDNA, and has GTPase activity.
  • the pathogenesis of ADOA is: mutated OPA1 codes to form a variety of truncated forms of proteins.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus Parvoviridae-dependent virus. It is the simplest single-stranded DNA-deficient virus found in the class and requires an auxiliary virus (usually an adenovirus). Virus) is involved in replication. It encodes the cap and rep genes in an inverted repeat (ITR) at both ends. ITRs are decisive for 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, a wide range of host cells (dividing and non-dividing cells), and low immunogenicity, it can express foreign genes in vivo. It is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated viruses have been thoroughly understood, and in particular, a lot of data has been accumulated on its application effects in various cells, tissues and in vivo experiments. In medical research, rAAV is used for gene therapy research of various diseases (including in vivo and in vitro experiments); as a characteristic gene transfer vector, it is also widely used in gene function research, disease model construction, and gene preparation. Knock out rats and so on.
  • the vector is a recombinant AAV vector.
  • AAVs are relatively small DNA viruses that can be integrated into the genome of the cells they infect in a stable and site-specific manner. They are able to infect a large array 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. AAV contains approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of replication of the virus. The rest of the genome is divided into two important regions with capsidation: 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. Any serotype of adeno-associated virus is suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6566118, 6989264, and 6995006, 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 disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for in vitro and in vivo transport of genes 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 cotransfecting the following plasmids into a cell line infected with a human helper virus (such as an adenovirus): the nucleic acid sequence of interest is flanked by two AAV inverted terminal repeats (ITR) Regional plasmids, and plasmids carrying AAV capsidization genes (rep and cap genes).
  • a human helper virus such as an adenovirus
  • the recombinant vector is capsidized to a virion (e.g., including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 And AAV16 AAV virions).
  • a virion e.g., including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 And AAV16 AAV virions.
  • a recombinant virion recombinant because it comprises a recombinant polynucleotide
  • Methods of 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 the expression of recombinant human type II mitochondrial dynein-like GTPase is not high and the therapeutic effect is poor.
  • the object of the present invention is to provide a recombinant human type II mitochondrial dynein-like GTPase optimized gene sequence.
  • Recombinant human type II mitochondrial dynein-like GTPase gene Its optimized CDS nucleotide sequence is shown in SEQ ID NO: 1. Its size is 2775bp. It starts at the codon ATG and encodes 924 amino acids. 1 to 288bp is OPA1.
  • the coding sequence of -MIS which encodes a peptide chain of 96 amino acids, whose function is to guide the GTP protein into the mitochondria and exert its physiological function; 289 to 2772bp, which encodes a peptide chain of 828 amino acids, is a GTP functional protein, and the last 3bp is Stop codon.
  • nucleotide sequence of the nucleic acid encoding human type II mitochondrial dynein-like GTPase according to the present invention is shown in SEQ ID NO.:1.
  • nucleotide sequence is greater than or equal to 95%, preferably greater than or equal to 98%, and more preferably greater than or equal to 99%, as shown in SEQ ID No.:1.
  • the nucleic acid encoding human type II mitochondrial dynein-like GTPase is also referred to as OPA1 optimized gene or OPA1 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 coding or non-coding.
  • the nucleotide sequence according to the present invention encodes the amino acid sequence shown in SEQ ID NO.:3.
  • OPA1-MIS signal peptide directs the protein into mitochondria. After being hydrolyzed by protease, the mature OPA1 isoform 2 protein enters mitochondria to play a role.
  • the nucleic acid sequence can be DNA, RNA, cDNA or PNA. Nucleic acid sequences can be genomic, recombinant, or synthetic. Nucleic acid sequences can be isolated or purified. The nucleic acid sequence may be single-stranded or double-stranded. Preferably, the nucleic acid sequence will encode a light-sensitive protein as described herein. Nucleic acid sequences can be derived by cloning, for example, using standard molecular cloning techniques including restriction digestion, ligation, and gel electrophoresis, such as described in Sambrook et al. Molecular Cloning: Laboratory, Cold Spring Laboratory Press). Nucleic acid sequences can be isolated, for example, using PCR technology.
  • Isolation means isolation of a nucleic acid sequence from any impurity and from other nucleic acid sequences and / or proteins that are naturally found to associate with the nucleic acid sequence in its source. Preferably, it will also be free of cellular material, culture medium or other chemicals from the purification / production process.
  • Nucleic acid sequences can be synthetic, such as by direct chemical synthesis. The nucleic acid sequence may be provided as a naked nucleic acid, or may be provided in combination with a protein or a lipid.
  • the full-length nucleotide sequence or a fragment thereof of the present invention can usually be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method.
  • primers can be designed according to published related nucleotide sequences, especially open reading frame sequences, 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 a primer.
  • Template amplified to obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments in the correct order.
  • a DNA sequence encoding a polypeptide (or a fragment thereof, or a derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into a variety of existing DNA molecules (or such as vectors) and cells known in the art.
  • the invention also relates to a vector comprising a polynucleotide of the invention, and to a host cell genetically engineered using the vector or polypeptide coding sequence of the invention.
  • the aforementioned polynucleotides, vectors or host cells 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 a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state.
  • nucleotide sequence is shown as SEQ ID NO.:1.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually cloned into a vector, and then transferred into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • synthetic methods can also be used to synthesize related sequences, especially when the fragment length is short.
  • long fragments can be obtained by synthesizing multiple small fragments first and then ligating them.
  • a method of applying PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.
  • the amplified DNA / RNA fragments can be isolated 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 that is genetically engineered using the vector or protein coding sequence of the present invention, and a method for utilizing the host cell to express the OPA1 protein by recombinant technology.
  • a host cell (such as a mammalian cell) expressing the OPA1 protein of the present invention can be obtained using the polynucleotide sequence of the present invention by conventional recombinant DNA technology. Generally, it includes the step of transducing a polynucleotide according to the first aspect of the present invention or a vector according to the third aspect of the present invention into a host cell.
  • an expression vector containing a coding DNA sequence of a polypeptide of the present invention and a suitable transcription / translation control signal. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology.
  • the DNA sequence can be operably 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 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 for eukaryotic cell culture Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a vector containing the appropriate DNA sequence and the appropriate promoter or control sequence described above can be used to transform an appropriate host cell to enable it to express a 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, NSO, COS7, or 293 cells.
  • 293T cells, photoreceptor cells (including cone cells and / or rod cells), other visual cells (such as biganglion cells), and neural cells are selected as host cells.
  • the host cell is selected from the group consisting of rod cells, cone cells, light-donating bipolar cells, light-releasing 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 prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the obtained transformants 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 may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • polypeptide in the above method may be expressed intracellularly, or on a cell membrane, or secreted extracellularly.
  • proteins can be isolated and purified by various separation methods using their 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 with a protein precipitant (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.
  • an optimized coding sequence for a recombinant human type II mitochondrial dynein-like GTPase which is particularly suitable for expression in mammalian cells.
  • the coding sequence is shown in SEQ ID NO.:1.
  • the "optimized OPA1 coding sequence” and “optimized OPA1 coding gene” both refer to a nucleotide sequence for encoding a recombinant human type II mitochondrial dynein-like GTPase, and the nucleotide sequence Encodes the amino acid sequence shown in SEQ ID NO.:3.
  • the wild DNA coding sequence (unoptimized DNA coding sequence) of the recombinant human type II mitochondrial dynein-like GTPase is shown in SEQ ID NO.:2.
  • the expression level of the unoptimized wild DNA coding sequence is very low.
  • sequence fragments that affect gene expression and protein localization. These sequence fragments include, but are not limited to, codon usage preferences, eliminate secondary structures (such as hairpin structures) that are not conducive to expression, change GC content, and CpG dinuclear Nucleotide content, secondary structure of mRNA, concealed splice site, early polyadenylation site, internal ribosome entry site and binding site, negative CpG island, RNA unstable region, repeat sequence (direct repeat, Inverted repeats, etc.) and restriction sites that may affect cloning.
  • SEQ ID NO.:1 Through analysis and experimental screening, a particularly optimized DNA coding sequence as shown in SEQ ID NO.:1 is finally obtained. This sequence is specially optimized, the transcription level is slightly increased, and the expression level is significantly increased.
  • the similarity between the specially optimized coding sequence shown in SEQ ID NO.:1 and the wild coding sequence shown in SEQ ID NO.:2 is 72.25% (2005/2775), as shown in Figure 1.
  • the present invention also provides a fusion nucleic acid comprising the nucleic acid sequence encoding a human type II mitochondrial dynein-like GTPase according to the first aspect of the present invention.
  • fusion nucleic acid refers to a nucleic acid formed by joining two or more nucleotide sequences from different sources, or two or more nucleosides from the same source but whose natural positions are not linked to each other. Nucleic acid formed by linking acid sequences.
  • the fusion nucleic acid further comprises a sequence selected from the group consisting of a UTR sequence, a promoter sequence, or a combination thereof.
  • the fusion nucleic acid is operably linked with a UTR sequence in the nucleic acid encoding human type II mitochondrial dynein-like GTPase.
  • the UTR sequence includes 3'-UTR and / or 5'-UTR.
  • the UTR sequence contains a ployA sequence with a stable structure.
  • the fusion nucleic acid has the structure of Formula I from the 5 ′ end to the 3 ′ end:
  • Each "-" is independently a bond or a nucleotide linking sequence
  • Z1 is a 5'-UTR sequence
  • Z2 is a nucleotide sequence according to the first aspect of the invention.
  • Z3 is a 3'-UTR sequence.
  • the invention also provides an expression vector for OPA1 protein, which contains the optimized OPA1 coding sequence of the invention.
  • sequence information By providing the sequence information, a skilled artisan can use available cloning techniques to generate a nucleic acid sequence or vector suitable for transduction into a cell.
  • a nucleic acid sequence encoding an OPA1 protein is provided as a vector, preferably an expression vector is provided.
  • an expression vector is provided.
  • it is provided as a gene therapy vector that is 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 mutant forms, retrovirus, lentivirus, herpes virus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV) , 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 replicating.
  • Viral vectors can often maintain an extrachromosomal state without integrating into the genome of target retinal cells.
  • a preferred viral vector for introducing a nucleic acid sequence encoding an OPA1 protein to a retinal target cell 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).
  • Viral vectors can be modified to delete any non-essential sequences.
  • the virus in AAV, can be modified to delete all or part of the IX gene, Ela, and / or Elb gene.
  • helper virus such as adenovirus
  • replication is very inefficient.
  • the replication gene and the 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 is required Provided by adenovirus or another plasmid. Similar modifications can 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 uptake of the viral vector 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 is preferably suitable for use in humans.
  • the vector will preferably contain one or more regulatory sequences to direct expression of the nucleic acid sequence in a retinal target cell. Regulatory sequences may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operably linked to a nucleic acid sequence.
  • the vector may also include a selectable marker, for example, to determine expression of the vector in a growth system (e.g., a bacterial cell) or in a retinal target cell.
  • “Operably linked” means that the nucleic acid sequences are functionally related to their operably linked sequences such that they are linked in a manner such that they affect each other's expression or function.
  • a nucleic acid sequence operably linked to a promoter will have an expression pattern that is affected by the promoter.
  • the promoter mediates expression of the nucleic acid sequence to which it is linked.
  • 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, such as for 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 glutamate receptor mGluR6 that is specific to optic nerve cells.
  • the preferred sources of the Grm6 gene are mouse and human.
  • 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.
  • CAG is then a fusion of the CMV early enhancer and the chicken ⁇ -actin promoter.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strongly 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 the 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 a constitutive promoter. Inducible promoters are also considered as part of the invention.
  • an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turning off expression when expression is undesirable.
  • inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.
  • OPA1 protein can be used for the expression of OPA1 protein 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 invention also provides a method for constructing an adeno-associated virus vector for recombination of a recombinant human type II mitochondrial dynein-like GTPase gene.
  • the method can quickly and easily construct a recombinant carrying a recombinant human type II mitochondrial dynein-like GTPase gene Adeno-associated virus vector and packaging to obtain complex defective adeno-associated virus vector.
  • the invention also provides a host cell for expressing OPA1 protein.
  • the host cell is a mammalian cell (preferably a human, more preferably a human optic nerve cell or a photoreceptor cell), and the expression amount of the OPA1 protein is increased.
  • the invention provides a formulation or composition comprising (a) a carrier according to the third aspect of the invention, and (b) a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical preparation is used for treating ocular 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 according to the present invention refers to a vector according to the present invention, such as a viral vector (including an adeno-associated virus vector).
  • the "active ingredients", formulations and / or compositions described herein can be used to treat ocular diseases.
  • safe and effective amount is meant an amount of the active ingredient 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 that are suitable for human use and must be of sufficient purity and sufficient Low toxicity.
  • “Compatibility” herein means that each component in the composition can blend with the active ingredient of the present invention and each other without significantly reducing the medicinal effect of the active ingredient.
  • the composition may be a liquid or a solid, such as a powder, a gel or a paste.
  • the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.
  • the carrier may be administered to the eye by subretinal or intravitreal administration.
  • the carrier is preferably provided as an injectable liquid.
  • the injectable liquid is provided as a capsule or syringe.
  • Examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and 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 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, etc.
  • the composition may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into a sterile injectable solution or dispersion.
  • Suitable aqueous and non-aqueous vehicles, 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 for preparing autosomal dominant optic atrophy (ADOA) Drug.
  • ADOA autosomal dominant optic atrophy
  • the optimized nucleic acid encoding human type II mitochondrial dynein-like GTPase has a higher expression level, which translates more OPA1 protein, and the optimized MIS sequence in OPA1 protein can accurately enter GTP functional proteins into the mitochondria Therefore, more GTPases are transfected into mitochondria.
  • An agent containing the nucleic acid of the present invention is injected into the vitreous cavity of a rabbit eye, the agent maintains viability in the vitreous cavity, and is transfected into optic nerve cells.
  • Optimizing the OPA1 nucleic acid encoding OPA1 protein that expresses more than the prior art can better treat autosomal dominant optic atrophy (ADOA).
  • ADOA autosomal dominant optic atrophy
  • the present invention mainly has the following advantages:
  • the recombinant human type II mitochondrial dynein-like GTPase (OPA1) coding gene sequence has been specifically optimized, including optimization of the MIS coding sequence and GTPase domain coding sequence in OPA1. Compared with OPA1's unoptimized DNA coding sequence, the expression was significantly increased, and more OPA1 proteins were transfected into mitochondria. The OPA1 protein expression of the optimized sequence was significantly increased and its biological activity was high.
  • the OPA1 coding gene (SEQ ID NO.:1) or fusion nucleic acid optimized by the present invention can treat autosomal dominant optic nerve atrophy (ADOA) very effectively and has good safety.
  • ADOA autosomal dominant optic nerve atrophy
  • the present invention provides an ADOA non-human mammalian model based on type II mitochondrial dynein-like GTPase gene inactivation, said model exhibiting pathological manifestations similar to autosomal dominant optic atrophy (ADOA), including the retina Decreased ATP content and changes in mitochondrial apoptosis can be used for experimental research, evaluation or screening of autosomal dominant optic atrophy (ADOA).
  • ADOA autosomal dominant optic atrophy
  • the rAAV2-hOPA1 recombinant adeno-associated virus provided by the present invention has a significant and efficient improvement effect on the pathological conditions of ADOA dominant genetic optic neuropathy point mutation model mice, and can provide continuous improvement effects, single administration for 3 months After being completely effective in reversing the effects of disease in model mice.
  • Example 1 Construction of recombinant adeno-associated virus vector of recombinant human type II mitochondrial dynein-like GTPase gene and method for virus packaging and purification thereof
  • the specially optimized recombinant human type II mitochondrial dynein-like GTPase gene (SEQ ID ID NO: 1) is added to two restriction sites of Kpn I and Sal I or the product amplified by PCR using the new gene design primers and
  • the pSNaV plasmid vector was digested with Kpn, I and Sal, respectively, and the digested product was recovered. T4DNA and Ligase were ligated overnight.
  • the ligation product was transformed into competent cells to obtain recombinant pSNaV / rAAV2 / 2-hOPA1 isoform 2 ( Figure 2).
  • the LB plate after 37 ° C culture showed blue and white spots, of which white was recombinant clone.
  • the PCR product was detected by electrophoresis, and the result is shown in FIG. 3, and a target band having a size of about 3000 bp was obtained.
  • the identification results showed that the clone contained the gene of interest.
  • 293T cells were seeded in a 225cm 2 cell culture flask with a seeding density of 3.0 ⁇ 10 7 cells / mL, the medium was DMEM + 10% bovine serum, and cultured at 37 ° C with 5% CO 2 Incubate overnight.
  • the SDS-PAGE gel was separated and laminated gel, and the gel concentration was 10%. Add 15 ⁇ g to each sample well. After electrophoresis, stain with Coomassie Brilliant Blue and decolorize with the corresponding decolorizing solution until a clear background band is displayed ( Figure 5).
  • the titer of rAAV2 / 2-hOPA1 isoform 2 was measured using a quantitative PCR method to detect the physical titer of rAAV2 / 2-hOPA1 isoform 2.
  • PCR reaction conditions pre-denaturation: 95 ° C for 10min; cycle: 95 ° C for 15sec, 60 ° C for 1min.
  • genomic titer was determined to be 1 ⁇ 10 12 vg / mL.
  • Example 2 Effect of rAAV2 / 2-hOPA1 recombinant adeno-associated virus on ADOA dominant genetic optic neuropathy 1. Rabbit eye vitreous cavity injection
  • Twenty-four rabbits were divided into three groups, which were divided into experimental group A, experimental group B and control group, and 50ul of 1 ⁇ 10 12 vg / mL rAAV2 / 2-optimized hOPA1 isoform 2 and rAAV2 / 2-original hOPA1 isoform 2 And rAAV-ZsGreen puncture the flat part of the ciliary body into the vitreous cavity at a distance of 3 mm from the limbus and inject the vitreous cavity.
  • the rabbits in the two groups were examined with slit lamp and intraocular pressure at 1, 3, 7, 30 days after surgery. All rabbits had no obvious abnormalities, no conjunctival hyperemia, secretions, no endophthalmitis, and no increase in intraocular pressure. Fundus photography one month after surgery showed.
  • the eyeballs of the experimental groups A and B and the control group were dissected out and paraffin sections were made. Paraffin sections were placed in a 65 ° C oven for 2h, dewaxed to water, and washed three times with PBS for 5min each time. The sections were placed in EDTA buffer solution for microwave repair. After the medium heat to boiling, the power was cut off. After natural cooling, PBS was washed 3 times for 5 min each. The sections were placed in a 3% hydrogen peroxide solution and incubated for 10 min at room temperature. Wash 3 times with PBS for 5 min each time, and block with 5% BSA for 20 min after drying.
  • the BSA solution was removed, and 50 ⁇ l of the diluted primary antibody was added to each section to cover the tissue, and overnight at 4 ° C. Wash three times in PBS for 5 min each. Remove the PBS solution, add 50 ⁇ l-100 ⁇ l of the fluorescent secondary antibody of the corresponding species to each section, and incubate at room temperature for 50min-1h in the dark. Wash 3 times in dark PBS for 5 min each. Remove the PBS solution. Add 50-100 ⁇ l of DAPI to each section to protect the nucleus from light for 5 min. Wash 3 times in PBS for 5 min each. The sections were dried a little and then mounted with anti-fluorescence quenched mounting tablets, and stored at 4 ° C in the dark to be photographed.
  • the retinal anti-hOPA1 immunofluorescence results are shown in Fig. 7.
  • the fluorescence intensity of the experimental group A was significantly higher than that of the experimental group B. There was a significant difference, which was more than doubled.
  • the expression of hOPA1 on the retina in experimental group A was significantly higher than that in control group and experimental group B.
  • primer design principles of fluorescent quantitative PCR primers were designed using primer 5:
  • Rabbit-actin-F CCTTCTACAACGAGCTGCGC (SEQ ID NO.:6)
  • Rabbit-actin-R TACAGGGACAGCACGGCC (SEQ ID NO.:7)
  • Optimized hOPA1-R GGGTGGTCACCTGGTGGGCC (SEQ ID NO.:11)
  • RNA from rabbit retinas was extracted using TRIZOL kit and cDNA template was synthesized by reverse transcription.
  • Real-time PCR was performed on a Real-time PCR Detection System instrument.
  • a 0.2 mL PCR reaction tube add 12.5 ⁇ L of SYBR Green Mix, 8 ⁇ L of ddH2O, 1 ⁇ L of each pair of primers, 2.5 ⁇ L of cDNA sample, and 25 ⁇ L of the total system.
  • Each sample was used to amplify both the target gene and the internal reference gene rabbit-actin.
  • Each gene was amplified in three replicates.
  • the reagents common in each PCR reaction tube can be added together and then aliquoted. After the sample is loaded, perform quantitative PCR.
  • a 2- ⁇ CT relative quantification method (Livak et al., 2001) was used to study the difference in gene expression. This method does not need to make a standard curve, and uses housekeeping rabbit-actin as an internal reference gene. The analysis software that comes with the instrument can automatically generate Express the value.
  • Fig. 10 The results are shown in Fig. 10.
  • the relative expression levels of hOPA1 mRNA in experimental group A and experimental group B were higher than the relative expression levels of hOPA1 gene in control group.
  • the relative expression level of mRNA in experimental group A was slightly higher than that in experimental group B. .
  • the retinas of rabbit eyeballs of different experimental groups were separated, and the corresponding volume of RIPA lysate was added according to 100 ⁇ L / 50 mg of tissue.
  • the homogenizer was homogenized and centrifuged to collect the supernatant. After the protein concentration was determined by the BCA method, the loading volume of the experimental group and the control group was calculated based on 50 ⁇ g of total protein, and SDS-PAGE gel electrophoresis and Western blot were performed. ECL was developed after antibody incubation.
  • Example 3 Long-term effect of rAAV2-hOPA1 recombinant adeno-associated virus on ADOA dominant genetic optic neuropathy
  • Wild-type C57BL / 6J mice were administered intravitreally. Mice were randomly divided into groups of 2 mice / cage, rAAV2 / 2-OPA1 concentration was 2E11vg / ml, and the volume was 1 ⁇ L. They were divided into 7-day group, 15-day group, 20-day group, 30-day group, 45-day group, 60-day group, and 90-day group after administration. After expiration, mice of different groups were sacrificed, eyeball samples were obtained, and relevant detection analysis was performed.
  • the primer sequences used are:
  • Primer F CTTTAGGTTATTTTGCTGTTGT (SEQ ID NO.:12)
  • Primer R TTGTAGTCACTTGGTGTGCC (SEQ ID NO .: 13)
  • the reaction conditions used were: pre-denaturation at 95 ° C. for 3 min, denaturation at 95 ° C. for 10 s, annealing at 60 ° C. for 15 s, and extension at 72 ° C. for 25 s. A total of 40 cycles of reaction procedures were used for amplification, and fluorescence signals were collected during the extension phase of each cycle. After the reaction, a melting curve analysis of 95 ° C to 60 ° C was performed.
  • the 2- ⁇ CT relative quantification method (Livak et al., 2001) was used to study the difference in gene expression. This method does not need to make a standard curve.
  • the housekeeping gene actin is used as the internal reference gene.
  • the analysis software provided by the instrument can automatically generate expression values. .
  • rAAV2-OPA1 virus was injected into the eyeballs of mice, RT-PCR detected that the target gene was higher than that at 7 days, 15 days, 20 days, 30 days, 45 days, 60 days, and 90 days.
  • the level before administration, and the expression level was highest around 20-30 days.
  • coding region uppercase
  • non-coding region bold
  • intron region lowercase
  • step (3) mating and breeding the chimeric mouse obtained in step (2) and a normal wild-type mouse, and screening the offspring to obtain OPA1 point mutation heterozygous mice;
  • OPA1Q (glutamine) 285Stop point mutation homozygous mouse is obtained by mating the heterozygous mice obtained in step (3) with each other, thereby obtaining an OPA1Q (glutamine) 285Stop point mutation mouse model.
  • a primer pair was synthesized, and a nucleic acid amplification product (length 530 bp) containing the Q285 site was obtained by PCR amplification.
  • Example 5 Improvement experiment of rAAV2-hOPA1 recombinant adeno-associated virus on ADOA dominant genetic optic neuropathy point mutation model mouse
  • OPA1 mutant mice / cages Take 2 OPA1 mutant mice / cages and divide them into 3 groups.
  • concentration of rAAV-OPA1 is 2E11vg / ml.
  • the administration volume is 1 ⁇ L and injected under the vitreous. Mice of different groups were sacrificed after 1 and 3 months, eyeball samples were obtained, OPA mutant group, 1 ⁇ L PBS was injected under the vitreous, 2 wild rats / cage were taken, and 1 ⁇ L PBS was injected under the vitreous as a control group. Detection and analysis of mitochondrial morphology observation.
  • Mobile phase A 15mM KH2PO4, 15mM KCL, adjusted to pH 6.0 with 0.1M KOH
  • Mobile phase B 85% of mobile phase A mixed with 15% of acetonitrile
  • mice eyeballs After the mouse eyeballs were taken, they were immersed in 2.5% glutaraldehyde, fixed overnight, and washed 3 times with 0.1 mol / L phosphate buffer for 15 min each time, fixed with osmic acid for 2 h, and washed 3 times with 0.1 mol / L phosphate buffer. After 15 minutes, dehydration with a gradient of ethanol (50%, 70%, 80%, 90%, 100%), then transition to pure acetone, and pure acetone was dehydrated twice. 15 minutes per step.
  • the present invention uses rabbit eyes as a model for intravitreal injection of rAAV2 / 2-hOPA1 isoform 2.
  • This experiment studies the injection dose, safety level, postoperative complications, and provides important references for future clinical trials.
  • the examination of the slit lamp and intraocular pressure in all rabbits showed no obvious abnormalities, no conjunctival congestion, secretions, no endophthalmitis, and no increase in intraocular pressure.
  • Fundus photography one month after surgery showed no significant complications or damage to the retinal blood vessels and optic nerve in all rabbits. Shows that this experiment is safe.
  • hOPA1 can be stably expressed on the retina of rabbits. Because ADOA lesions can cause apoptosis of retinal ganglion cells around the optic disc, retinal sections of the rAAV-ZsGreen group can detect stable fluorescent expression in the retina around the optic disc. hOPA1's fluorescent staining results can indicate that it can reach retinal ganglion cells, indicating that rAAV2 / 2-hOPA1 isoform2 can reach the diseased area in the patient's eye.

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Abstract

重组人Ⅱ型线粒体动力蛋白样GTP酶基因序列及其应用，其核苷酸序列如SEQ ID NO.:1所示。一种融合核酸，其包含该编码人Ⅱ型线粒体动力蛋白样GTP酶的核酸。一种重组表达载体，其包含该核酸或融合核酸。一种转化体，其在宿主中导入该核酸或融合核酸。一种基于Ⅱ型线粒体动力蛋白样GTP酶基因失活的非人哺乳动物的ADOA模型，利用编码人Ⅱ型线粒体动力蛋白样GTP酶的重组表达载体能有效改善ADOA的病理表现。编码人Ⅱ型线粒体动力蛋白样GTP酶的核酸表达量更高，因此可在线粒体中获得更多的人Ⅱ型线粒体动力蛋白样GTP酶，能较好地治疗ADOA等眼部疾病。

Description

重组人Ⅱ型线粒体动力蛋白样GTP酶基因序列及其应用 技术领域
本发明涉及生物制剂领域，尤其涉及重组人Ⅱ型线粒体动力蛋白样GTP酶基因序列及其应用。
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。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。但目前尚未出现任何类型的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。
因此，本领域亟需开发一种治疗效果好的人线粒体动力蛋白样GTP酶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治疗效果好的人线粒体动力蛋白样GTP酶的表达体系及制备方法。
本发明的目的是提供一种编码人线粒体动力蛋白样GTP酶的优化核酸序列、载体及制备方法。
本发明的第一方面，提供了一种所述核苷酸序列编码人Ⅱ型线粒体动力蛋白样GTP酶，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；和
(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％。
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。
在另一优选例中，所述核苷酸序列如SEQ ID NO.:1所示，其中
第1-288位为MIS的编码序列；
第289-2772位为GTP酶结构域、中枢动力蛋白结构域和GTPase效应结构域的编码序列；
第2773-2775位为终止密码子。
在另一优选例中，SEQ ID NO.:1的序列包括：MIS编码序列，如SEQ ID NO.:1中第1-288位所示；以及GTP酶结构域、中枢动力蛋白结构域和GTPase效应结构域的编码序列，如SEQ ID NO.:1中第289-2772位所示。
在另一优选例中，SEQ ID NO.:2的序列包括：MIS编码序列，如SEQ ID NO.:2中第1-288位所示；以及GTP酶结构域、中枢动力蛋白结构域和GTPase效应结构域的编码序列，如SEQ ID NO.:2中第289-2772位所示。本发明还提供单独编码所述MIS的核苷酸序列(SEQ ID No.:1或2中第1-288位)、以及单独编码GTP酶结构域、中枢动力蛋白结构域和GTPase效应结构域的核苷酸序列(SEQ ID No.:1或2中第289-2772或289-2775位)。
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：UTR序列、启动子序列、或其组合。
在另一优选例中，所述UTR序列包括3'-UTR和/或5'-UTR。
在另一优选例中，所述UTR序列中含有稳定结构的ployA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：
Z1-Z2-Z3  (I)
式中，
各“-”独立地为键或核苷酸连接序列；
Z1为无、或5'-UTR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5'-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，所述载体用于表达重组人Ⅱ型线粒体动力蛋白样GTP酶。
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：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)、(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，所述眼部疾病为视网膜病变。
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，较佳地为常染色体显性视神经萎缩(ADOA)。
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凋亡。
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 1-1×10 13个病毒/毫升，更佳地2×10 11-1×10 12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，较佳地治疗视网膜神经节细胞凋亡。
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，较佳地常染色体显性视神经萎缩(ADOA)。
在另一优选例中，所述药物制剂能显著提高眼球线粒体动力蛋白样GTP酶的表达和/或活性。
本发明的第七方面，提供了一种长期提高线粒体动力蛋白样GTP酶的表达和/或活性的方法，所述方法包括导入本发明第三方面所述的载体，和/或施用本发明第六方面所述的药物制剂。
在另一优选例中，所述方法能有效增加细胞产生的ATP含量，和/或抑制线粒体凋亡。
在另一优选例中，所述方法能持续上调GTP酶的表达和/或活性。
本发明的第八方面，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，较佳地为常染色体显性视神经萎缩(ADOA)。
所述治疗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。
所述治疗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。
本发明的第九方面，提供了一种重组人Ⅱ型线粒体动力蛋白样GTP酶的制备方法，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Ⅱ型线粒体动力蛋白样GTP酶。
本发明的第十方面，提供了一种ADOA显性遗传视神经病细胞模型，所述细胞中OPA1基因表达或活力下降。
在另一优选例中，所述细胞包括视网膜神经节细胞(Retinal ganglion cell，RGC)。
在另一优选例中，所述OPA1基因完全不表达或无活性。
本发明第十一方面，提供了一种ADOA显性遗传视神经病细胞模型的制备方法，该方法包括步骤：提供一种细胞，对所述细胞中的OPA1基因进行点突变，从而得到OPA1基因点突变的细胞；通过筛选获得点突变阳性单克隆细胞。
在另一优选例中，所述OPA1基因点突变指Q(谷氨酰胺)285Stop点突变。
在另一优选例中，所述细胞包括RGC细胞。
本发明的第十二方面，提供了一种ADOA显性遗传视神经病非人哺乳动物模型，所述动物模型中GRC细胞中的OPA1基因表达和/或活力降低。
在另一优选例中，所述OPA1基因存在点突变，具体地，存在Q(谷氨酰胺)285Stop点突变。
本发明的第十三方面，提供了一种ADOA显性遗传视神经病非人哺乳动物模型的制备方法，包括以下步骤：
(a)提供非人哺乳动物的细胞，对所述细胞中的OPA1基因进行点突变，从而得到OPA1基因点突变的细胞；
(b)利用步骤(a)中得到的OPA1基因点突变的细胞，制备具有OPA1基因点突变的动物模型。
在另一优选例中，所述的OPA1基因点突变是杂合型或纯合型。
在另一优选例中，将OPA1蛋白的第285位的谷氨酰胺位点突变为转录停止。
在另一优选例中，所述非人哺乳动物为啮齿动物或灵长目动物，较佳地包括小鼠、大鼠、兔、猴。
在另一优选例中，在步骤(b)中，包括步骤：
(b1)制备获得嵌合的OPA1基因点突变的动物模型，然后通过杂交，获得纯合型或杂合型OPA1基因点突变动物模型；
在另一优选例中，所述方法包括：
(1)利用CRISPR-CAS9技术，构建OPA1的第285位Q(谷氨酰胺)突变为终止(即Q285Stop)点突变的重组质粒，并用筛选标记的同源突变DNA序列替换，得到OPA1的Q285Stop点突变阳性单克隆胚胎干细胞；
(2)利用步骤(1)中得到的OPA1点突变的小鼠胚胎干细胞克隆株，制备得到嵌合小鼠；
(3)将步骤(2)中得到的嵌合小鼠和正常野生型小鼠交配繁育，在后代中筛选获得OPA1点突变的杂合子小鼠，即为OPA1的Q285Stop点突变小鼠模型。
在另一优选例中，将所述杂合子小鼠进行交配繁育，从而在其后代中筛选获得OPA1点突变的纯合子小鼠。
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显示了优化核苷酸序列与原人Ⅱ型线粒体动力蛋白样GTP酶基因序列的开放阅读框序列比较。两者的同源性为72.25％(2005/2775)，其中一致的用“|”表示，上行为优化开放阅读框核苷酸序列，下行为原人Ⅱ型线粒体动力蛋白样GTP酶基因序列(未优化的野生编码序列)。
图2显示了OPA1 isforms 2转录本的蛋白结构示意图。
图3显示了从重组克隆中筛选出目的条带为3000bp左右的正确克隆，M:蛋白marker；泳道1：正确的rAAV2/2-hOPA1 isoform 2重组克隆；泳道2：阳性对照；泳道3：阴性对照。
图4显示了重组腺相关病毒质粒pSNaV/rAAV2/2-hOPA1的结构示意图。
图5显示了SDS-PAGE电泳检测rAAV2/2-hOPA1 isoform 2纯度的考马斯亮蓝染色结果。其中，泳道1:蛋白marker；泳道2：rAAV2/2-hOPA1 isoform 2。
图6显示了兔眼玻切镜下眼底拍照，其中A为注射rAAV2/2-ZsGreen组(对照组)，B为注射rAAV2/2-优化hOPA1 isoform 2组(实验组A)。
图7显示了兔眼球切片免疫荧光检测，实验组A为注射rAAV2/2-优化hOPA1 isoform2组，实验B组为注射rAAV2/2-原hOPA1 isoform 2组。
图8显示了兔眼玻切镜下OCT拍照。其中A为注射rAAV2/2-ZsGreen组(对照组)，B为注射rAAV2/2-优化hOPA1 isoform 2组(实验组A)。
图9显示了显微镜下观察到的兔眼球HE切片内的视网膜。其中A为注射rAAV2/2-ZsGreen组(对照组)，B为注射rAAV2/2-优化hOPA1 isoform 2组(实验组A)。
图10显示了注射不同质粒的家兔眼球视网膜的hOPA1蛋白的实时荧光PCR相对表达量的检测结果，实验组A为注射rAAV2/2-优化hOPA1 isoform 2组，实验B组为注射 rAAV2/2-原hOPA1 isoform 2组，对照组为注射rAAV2/2-ZsGreen组。
图11显示了注射不同质粒的家兔眼球视网膜的hOPA1蛋白的western blot检测结果，实验组A为注射rAAV2/2-优化hOPA1 isoform 2组，实验B组为注射rAAV2/2-原hOPA1isoform 2组，对照组为注射rAAV2/2-ZsGreen组。
图12显示了ADOA显性遗传视神经病模型小鼠构建。
图13显示了ADOA显性遗传视神经病点突变模型小鼠基因型鉴定。
图14显示了高效液相HPLC检测给药后小鼠视网膜ATP含量变化。
图15显示了透射电镜TEM观察线粒体形貌。
具体实施方式
本发明人经过广泛而深入的研究，对重组人Ⅱ型线粒体动力蛋白样GTP酶(OPA1)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)细胞中高效转录和高效表达OPA1蛋白的核苷酸序列(SEQ ID NO.:1)，并构建了重组人Ⅱ型线粒体动力蛋白样GTP酶的重组表达载体。实验结果表明，相对于未优化的编码序列，经过特殊优化后的OPA1编码序列(SEQ ID NO.:1)转录效率略有提高，并且表达量显著提高了5倍以上，非常适合在哺乳动物(尤其是人)细胞内表达，能有效治疗ADOA等眼部疾病。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。
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。
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。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)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，其优化CDS核苷酸序列SEQ ID NO：1所示，其大小为2775bp，起始于密码子ATG，编码924个氨基酸，其中1到288bp是OPA1-MIS的编码序列，编码96个氨基酸的肽链，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(SEQ ID NO.:1)，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 NO.:1所示。在另一优选例中，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％。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 NO.:3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 2蛋白进入线粒体发挥作用。
Figure PCTCN2019102352-appb-000001
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 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
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 NO.:1所示。
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 NO.:3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.:2所示。所述未优化的野生DNA编码序列的表达量很低。
野生Homo sapiens:OPA1 isoform2CDS 2775bp
Figure PCTCN2019102352-appb-000002
Figure PCTCN2019102352-appb-000003
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 NO.:1所示的特别优化的DNA编码序列。此序列是经过特殊优化，转录水平略有提高，表达量显著提高。特别优化后的SEQ ID NO.:1所示的编码序列与SEQ ID NO.:2所示的野生编码序列相似度为72.25％(2005/2775)，如图1所示。
优化Homo sapiens:OPA1 isoform2CDS 2775bp
Figure PCTCN2019102352-appb-000004
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。
另一优选例中，所述融合核酸还包含选自下组的序列：UTR序列、启动子序列、或其组合。
优选地，所述融合核酸在所述的编码人Ⅱ型线粒体动力蛋白样GTP酶的核酸可操作性地连接有UTR序列。
在另一优选例中，所述UTR序列包括3'-UTR和/或5'-UTR。
在另一优选例中，所述UTR序列中含有稳定结构的ployA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：
Z1-Z2-Z3  (I)
式中，
各“-”独立地为键或核苷酸连接序列；
Z1为5'-UTR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。
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
Figure PCTCN2019102352-appb-000005
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)。
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Ⅱ型线粒体动力蛋白样GTP酶(OPA1)编码基因序列进行了特殊优化，包括对OPA1中的MIS编码序列和GTP酶结构域编码序列的优化。与OPA1的未优化的DNA编码序列相比，表达量显著提高，更多的OPA1蛋白转染到在线粒体中。优化后的序列OPA1蛋白表达量显著提高、生物活性高。
2.本发明优化的OPA1编码基因(SEQ ID NO.:1)或融合核酸能够非常有效地治疗常染色体显性视神经萎缩(ADOA)，并且安全性好。
3.本发明提供了一种基于Ⅱ型线粒体动力蛋白样GTP酶基因失活的ADOA非人哺乳动物模型，所述模型表现出类似于常染色体显性视神经萎缩(ADOA)的病理表现，包括视网膜ATP含量降低和线粒体凋亡变化，可以用于常染色体显性视神经萎缩(ADOA)的实验研究、评估或筛药用途。
4.本发明提供的rAAV2-hOPA1重组腺相关病毒对ADOA显性遗传视神经病点突变模型小鼠病理情况有明显而高效的改善效果，且能够提供持续的改善效果，单次给药3个月后能完全有效逆转模型小鼠中疾病的影响。
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。
实施例1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构建及其病毒包装纯化方法
1.含有人Ⅱ型线粒体动力蛋白样GTP酶基因重组腺相关病毒载体的构建
1)载体构建
将特殊优化后的重组人Ⅱ型线粒体动力蛋白样GTP酶基因(SEQ ID NO：1)加Kpn I 和Sal I两个酶切位点或者将以该新基因设计引物用PCR扩增的产物与pSNaV质粒载体，分别进行Kpn I和Sal I双酶切，回收酶切产物，T4DNA Ligase连接过夜，连接产物转化感受态细胞得到重组pSNaV/rAAV2/2-hOPA1 isoform 2(图2)。
2)重组子的筛选和鉴定
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。取1uL质粒作为模板，所述特异性引物为：
1F:5'-ATGTGGCGACTACGTCG-3'(SEQ ID NO.:4)；
1R:5'-TTATTTCTCCTGATGAAGAG-3'(SEQ ID NO.:5)；
PCR扩增程序
Figure PCTCN2019102352-appb-000006
对PCR产物电泳检测，结果如图3所示，得到一个大小约3000bp左右的目的条带。鉴定结果表明该克隆中含有目的基因。
3)菌液保存及PCR扩增及其片段测序
吸取1mL经过鉴定的菌液与灭菌后的的甘油1:3比例混匀,于-80℃保存，进行菌液测序，将测序得到的序列与重组人Ⅱ型线粒体动力蛋白样GTP酶基因比对分析，成功得到序列正确的重组腺相关病毒质粒pSNaV/rAAV2/2-hOPA1(图4)。
2.rAAV2/2-hOPA1 isoform 2重组腺相关病毒包被
1)转染前一天，将293T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。
2)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。具体步骤为：
(a)每个转染瓶取pAdHelper、pAAV-r2c5、pSNaV-hOPA1质粒按要求比例与DMEM+PlasmidTrans II(VGTC)(转染试剂)在1.5mL无菌Ep管中混匀，编号为A试剂，室温静止10～15min；
(b)将A试剂同30mL DMEM+10％牛血清混合均匀，编号B试剂；
(c)将B试剂均匀加入细胞培养瓶中，37℃含5％CO 2的培养箱中继续培养；
(d)转染16h后，换完全培养基(DMEM+10％牛血清)。
3)转染48h后，收取细胞。
4)将收取细胞用PBS重悬，并反复冻融3次。
3.rAAV2/2-hOPA1 isoform 2病毒的纯化与浓缩
采用氯仿处理-PEG/NaCl沉淀-氯仿抽提三步来分离、浓缩和纯化rAAV2/2-hOPA1病毒。总回收率＝终产物的病毒颗粒数/起始物的病毒颗粒数。
4.病毒纯度和滴度验证
灌制SDS-PAGE分离胶和积层胶,分离胶浓度为10％。分别按每个加样孔加样15μg。电泳完毕后用考马斯亮蓝染色，用相应的脱色液脱色直到显出低背景的、清晰的条带(图5)。
结果如图5所示，VP1/VP2/VP3＝1:1:10，条带清晰，比例正常，无可见杂带，纯度在99％以上。
rAAV2/2-hOPA1 isoform 2的滴度测定采用荧光定量PCR方法检测rAAV2/2-hOPA1isoform 2的物理滴度。
实验材料：SYBRⅡ(takara)；目的片段引物(20uM)；包装病毒用目的质粒(已知浓度)；待测病毒；PCR八联管(Bio-red)。
实验方法：模板1ul，SYBRⅡ7ul引物1 0.25ul，引物2 0.25ul，加nuclease-free水到14ul。
PCR反应条件：预变性：95℃10min；循环：95℃15sec，60℃1min。
最终确定其基因组滴度为1×10 12vg/mL。
实施例2：rAAV2/2-hOPA1重组腺相关病毒对ADOA显性遗传视神经病变的效果实验1.兔眼玻璃体腔注射
取24只兔子分为3组，分为实验组A、实验组B和对照组，分别吸取50ul 1×10 12vg/mL的rAAV2/2-优化hOPA1 isoform 2、rAAV2/2-原hOPA1 isoform 2和rAAV-ZsGreen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。
2.裂隙灯、眼压、眼底照相检查
两组兔子分别于术后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。
结果如图6所示，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。
3.视网膜的荧光照相
玻璃体腔注射30天后，ZsGreen组(对照组)视网膜的荧光照相显示，GFP成功在视网膜上表达，表明以rAAV为载体，携带GFP转染兔眼玻璃体内，证明rAAV2/2-hOPA1isoform 2重组基因可以在视网膜上表达。
4.hOPA1的免疫荧光检测
玻璃体腔注射30天后，剥离实验组A、B和对照组眼球，制成石蜡切片。石蜡切片置于65℃烘箱中烘片2h，脱蜡至水，用PBS冲洗三次，每次5min。切片置于EDTA缓冲液中微波修复，中火至沸后断电，间隔10min低火至沸。自然冷却后PBS洗3次，每次5min。切片放入3％过氧化氢溶液，室温下孵育10min。PBS洗3次，每次5min，甩干后5％BSA封闭20min。去除BSA液，每张切片加入50μl稀释的一抗覆盖组织，4℃过夜。PBS洗三次，每次5min。去除PBS液，每张切片加50μl-100μl相应种属的荧光二抗，避光室温下孵育50min-1h。避光PBS洗3次，每次5min。去除PBS液。每张切片加50-100μl DAPI避光染核5min。PBS洗3次，每次5min。切片稍干后用抗荧光淬灭封片剂封片，4℃避光保存待拍照。
视网膜抗hOPA1的免疫荧光结果如图7所示，实验组A的荧光强度显著高于实验组B的，存在显著差异，提高了一倍以上。表明实验组A视网膜上的hOPA1表达量明显高于对照组和实验组B。
5.OCT检测
结果显示实验组A和实验组B的视网膜神经纤维层厚度分别为67.55μm和66.00μm，无明显差别(P＞0.05，图8)。
6.HE检测
HE结果显示实验组和对照组的视网膜神经节细胞数量基本一样，且结构完整，表明rAAV-hOPA1重组基因是安全的，没有对视网膜造成损伤(图9)。
7.Real-Time PCR检测hOPA1的表达
首先用NCBI的保守结构域分析软件分析hOPA1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：
兔-actin-F：CCTTCTACAACGAGCTGCGC(SEQ ID NO.:6)
兔-actin-R：TACAGGGACAGCACGGCC(SEQ ID NO.:7)
原hOPA1-F：TTAGGTTATTTTGCTGTTGT(SEQ ID NO.:8)
原hOPA1-R：TTGTAGTCACTTGGTGTGCC(SEQ ID NO.:9)
优化hOPA1-F：CTGGGCTACTTCGCCGTGGT(SEQ ID NO.:10)
优化hOPA1-R：GGGTGGTCACCTGGTGGGCC(SEQ ID NO.:11)
1)提取RNA、反转录
利用TRIZOL试剂盒提取不同实验组兔子视网膜的总RNA并反转录合成cDNA模板。
2)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 mix 12.5μL、ddH2O 8μL，一对引物各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兔-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1s，94℃变性15s，55℃退火15s，72℃延伸45s，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94℃～55℃的融解曲线分析。
采用2-△△CT相对定量方法(Livak等，2001)研究基因表达量的差异，该方法无需制作标准曲线，以看家基因兔-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。
结果如图10所示，实验组A、实验组B的hOPA1基因mRNA的相对表达量均比对照组的hOPA1基因相对表达量高，实验组A的mRNA的水平相对表达水平比实验组B略高。
8.Western blot检测hOPA1蛋白的表达
分离不同实验组的家兔眼球的视网膜，按100μL/50mg组织加入对应体积的RIPA裂解液，匀浆器匀浆后离心收取上清。BCA法测定蛋白浓度后，按总蛋白50μg计算实验组和对照组上样体积，进行SDS-PAGE凝胶电泳和Western blot。抗体孵育后进行ECL显影。
结果如图11所示，蛋白质印迹的实验组A的hOPA1相对表达水平明(1.13)明显高于实验组B(0.19)和对照组(0.05)，有显著差异P＞0.05，表明，实验组A视网膜上hOPA1的表达水平明显提高，相对于实验组B和对照组分别提高了约5倍和22倍。
实施例3：rAAV2-hOPA1重组腺相关病毒对ADOA显性遗传视神经病变的长期效果实验
1.小鼠眼玻璃体腔注射
野生型C57BL/6J小鼠玻璃体腔给药，小鼠随机分组，2只/笼，给药rAAV2/2-OPA1浓度2E11vg/ml体积1μL，玻璃体下注射。分为给药后7天组、15天组、20天组、30天组、45天组、60天组、90天组。到期后处死不同组别的小鼠，获取眼球样本，进行相关的检测分析。
2.Real-Time PCR检测hOPA1的表达
如实施例2中所述的方法和步骤，进行总RNA的提取、RNA浓度测定及电泳鉴定、反转录和Real-Time PCR鉴定。
其中，所用引物序列为：
Primer F：CTTTAGGTTATTTTGCTGTTGT(SEQ ID NO.:12)
Primer R：TTGTAGTCACTTGGTGTGCC(SEQ ID NO.:13)
所用反应条件为：按照95℃预变性3min，95℃变性10s，60℃退火15s，72℃延伸25s，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95℃～60℃的融解曲线分析。
采用2-△△CT相对定量方法(Livak等，2001)研究基因表达量的差异，该方法无需制作标准曲线，以看家基因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。
结果如图11所示，使用rAAV2-OPA1病毒玻璃体腔注射小鼠眼球，RT-PCR检测到目的基因在7天、15天、20天、30天、45天、60天、90天均高于给药前水平，且第20-30天前后表达量最高。
实施例4：ADOA显性遗传视神经病模型小鼠的构建与鉴定
1.利用CRISPR/Cas9技术进行小鼠OPA1Q285Stop点突变，突变外显子为exon8(高度保守的GTPase区(外显子8—16))。在突变位点设计以NGG结尾的靶点，在CRISPR/Cas9作用下进行切割，体外构建约120bp的donor，外源donor以同源重组方式插入到切割位点。设计guideRNA，OPA1结构和设计策略如图12所示。
其中：编码区(大写)，非编码区(黑体)，内含子区(小写)；
Figure PCTCN2019102352-appb-000007
OPA1(Q285*)-sgRNA1：
Figure PCTCN2019102352-appb-000008
OPA1(Q285*)-sgRNA2：
Figure PCTCN2019102352-appb-000009
OPA1(Q285*)-120bp oligo:
Figure PCTCN2019102352-appb-000010
2.ADOA显性遗传视神经病模型小鼠的构建
(1)利用CRISPR-CAS9技术，构建OPA1Q285Stop点突变重组质粒，并用筛选标记的同源突变DNA序列替换，得到OPA1Q285Stop点突变阳性单克隆胚胎干细胞；
(2)利用步骤(1)中得到的OPA1点突变小鼠胚胎干细胞克隆株，制备得到嵌合小鼠；
(3)将步骤(2)中得到的嵌合小鼠和正常野生型小鼠交配繁育，在后代中筛选获得OPA1点突变的杂合子小鼠；
(4)通过将步骤(3)中得到的杂合子小鼠相互交配获得OPA1Q(谷氨酰胺)285Stop点突变的纯合子小鼠，从而得到OPA1Q(谷氨酰胺)285Stop点突变小鼠模型。
3.ADOA显性遗传视神经病点突变模型小鼠的鉴定
3.1基因组DNA提取
(1)消化：小鼠出生约一周内，剪取0.5cm小鼠脚趾，放入1.5ml EP管中，稍离心后加入500ul裂解液(配方：100mM Tris pH8.0，5mM EDTA pH8.0，0.5％SDS，NaCl 1.17g/100ml)、0.5ul蛋白酶K(浓度：20mg/ml，溶解在pH7.4,20mM Tris和1mM CaCl 2中，50％甘油缓冲液，-20℃保存)，混匀55℃水浴消化过夜；
(2)异丙醇沉淀抽提DNA：
1)从水浴锅中取出离心管，在室温静置10-15min，使样品温度降至室温，将离心管颠倒混匀。13000rpm，室温离心15min。
2)吸取400μl上清至另一个新的离心管中。加入等体积的异丙醇，立即温和地上下翻转，充分混匀，此时会出现白色絮状沉淀，室温下12000rpm离心10min，弃上清。
3)往离心管中加入700μl冰冷的75％乙醇漂洗，温和地上下翻转混匀。12000rpm，室温离心5min，将上清液全部吸除。
4)将离心管倒置在吸水纸上，以吸干乙醇。风干后用无菌ddH2O,50ul溶解DNA，55℃溶解2h(如不立即使用，-20℃保存)。
5)检测DNA的浓度，取100-200ng的DNA用做PCR模板。
3.2引物与PCR反应
合成一引物对，从而通过PCR扩增获得包含Q285位点的核酸扩增产物(长度为530bp)。
3.3鉴定结果
提取小鼠的总DNA，对PCR产物进行测序。
结果如图13所示，显示285位的碱基C变成T，点突变小鼠制备成功。
实施例5：rAAV2-hOPA1重组腺相关病毒对ADOA显性遗传视神经病点突变模型小鼠 的改善实验
1.方法
取OPA1突变鼠2只/笼，分为3组，rAAV-OPA1浓度为2E11vg/ml给药体积1μL，玻璃体下注射。1月和3月后处死不同组别的小鼠，获取眼球样本，OPA突变组，玻璃体下注射1μL PBS，取野生鼠2只/笼，玻璃体下注射1μL PBS作为对照组，进行ATP含量变化和线粒体形貌观察的检测分析。
2.高效液相HPLC检测给药后小鼠视网膜ATP含量变化
2.1取野生型和给药后OPA1突变鼠取视网膜，预冷PBS洗一遍，置于液氮中
2.2ATP提取
(1)液氮研磨
(2)加入1ml冰的酚-TE(含DTT)，加入200μl氯仿，150μl超纯水，震荡混匀，20s，4℃，10000g离心10min
(3)取200μl水相，0.22μm滤膜过滤，HPLC上机检测ATP
(4)BCA法测定蛋白浓度
2.3色谱分析方法
色谱柱：Sepax GP-C18,(4.6mm×250mm,5μm)
流动相A：15mM KH2PO4、15mM KCL，使用0.1M的KOH调PH为6.0
流动相B：85％的流动相A与15％的乙腈混合
上样体积：20μl
检测波长：254nm
梯度洗脱条件：
时间(min) 流动相A(％) 流动相B(％) 流速
0 100 0 0.54ml/min
0.28 97 3 0.54ml/min
9.72 91 9 0.54ml/min
13.89 0 100 0.54ml/min
19.44 0 100 0.54ml/min
33 100 0 0.54ml/min
40 100 0 0.54ml/min
结果如图14所示，HPLC检测发现对照组OPA1突变鼠中ATP含量降低，而使用rAAV2-OPA1病毒玻璃体腔注射小鼠眼球的实验组，给药后含量升高至正常水平，说明OPA1给药后可以恢复ATP含量。
由图14中可以看出，给药1个月和给药3个月，小鼠眼球ATP含量随时间而升高，直至给药后3个月OPA1突变鼠的眼球ATP含量与野生型小鼠眼球ATP含量相当，提示使用 rAAV2-OPA1病毒有效逆转了疾病的发生。
3.透射电镜TEM观察线粒体形貌
小鼠眼球取材后分别浸入2.5％的戊二醛中,固定过夜,0.1mol/L磷酸缓冲液清洗3次,每次15min,锇酸固定2h,0.1mol/L磷酸缓冲液清洗3次,每次15min,用乙醇(50％、70％、80％、90％、100％)梯度脱水，然后过度到纯丙酮，纯丙酮脱水2次。每步15分钟。812树脂：丙酮按3:1、3：2、3：3梯度渗透每步40分钟,然后纯包埋剂45℃包埋过夜，60℃聚合,切片,染色,透射电子显微镜观察。
结果如图15所示，TEM电镜观察到OPA1突变鼠中线粒体嵴消失，提示凋亡发生。而在给药1个月后观察到OPA1突变鼠中线粒体嵴的恢复，3个月有显著恢复，提示使用rAAV2-OPA1病毒有效逆转了疾病的发生。
讨论
随着基因治疗在眼部疾病治疗上的快速发展，治愈ADOA也将不是难事。因为人眼与兔眼的解剖和体积相似，本发明以兔子眼睛为模型进行rAAV2/2-hOPA1 isoform 2的玻璃体腔注射。本实验研究注射剂量，安全水平，术后并发症，为未来的临床试验提供重要的参考。所有兔子的裂隙灯，眼压的检查，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，所有兔子的视网膜血管和视神经均无明显并发症或损害。表明本实验是安全的。
免疫荧光、实时定量PCR和Western blot的结果可以证明hOPA1能稳定地表达于兔子的视网膜上。因为ADOA的病变会引起视盘周围的的视网膜神经节细胞凋亡，rAAV-ZsGreen组的视网膜切片能够检测到视盘周围的视网膜有稳定的荧光表达。hOPA1的荧光染色结果可以说明其能够到达视网膜神经节细胞中，表明rAAV2/2-hOPA1 isoform2能到达病人眼中病变的区域。眼底照相、OCT的结果显示单次玻璃体腔注射5×10 10vg/mL rAAV2/2-hOPA1 isoform 2是安全的，并且没有视网膜毒性，能被应用在未来的临床试验。
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 (20)

  1. 一种核苷酸序列，其特征在于，所述核苷酸序列编码人Ⅱ型线粒体动力蛋白样GTP酶，且所述核苷酸序列包括一种或多种选自下组的核苷酸序列：
    (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％；
    (c)所述核苷酸序列如SEQ ID NO.:2所示；和
    (d)所述核苷酸序列与SEQ ID NO.:2所示的核苷酸序列有≥95％相同性，优选地≥98％，更优选地≥99％。
  2. 如权利要求1所述的核苷酸序列，其特征在于，所述的核苷酸序列如SEQ ID No.:1所示。
  3. 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。
  4. 如权利要求3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：
    Z1-Z2-Z3  (I)
    式中，
    各“-”独立地为键或核苷酸连接序列；
    Z1为无、或5'-UTR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。
  5. 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3所述的融合核酸。
  6. 如权利要求4所述的载体，其特征在于，所述载体选自质粒或病毒载体，较佳地选自慢病毒载体、腺病毒载体、腺相关病毒载体或其组合。
  7. 如权利要求6所述的载体，其特征在于，所述载体为AAV载体，血清型选自AAV2、AAV5、AAV7、AAV8、或其组合。
  8. 如权利要求7所述的载体，其特征在于，所述载体为插入所述的核苷酸序列或所述的融合核酸的AAV载体。
  9. 如权利要求5所述的载体，其特征在于，所述载体选自DNA病毒载体或逆转录病毒载体。
  10. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求5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3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：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)、(视)神经 细胞、或其组合。
  12. 如权利要求5-9中任一所述的载体的用途，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. 一种药物制剂，其特征在于，所述的制剂含有(a)权利要求5-9中任一所述的载体，以及(b)药学上可接受的载体或赋形剂。
  14. 如权利要求13所述的药物制剂，其特征在于，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。
  15. 如权利要求13所述的药物制剂，其特征在于，药物制剂中，(a)载体的含量为1×10 9-1×10 16个病毒/毫升，较佳地1×10 11-1×10 13个病毒/毫升，更佳地2×10 11-1×10 12个病毒/毫升。
  16. 如权利要求13所述的药物制剂的用途，其特征在于，用于治疗眼部疾病，较佳地治疗视网膜神经节细胞凋亡。
  17. 如权利要求13所述的药物制剂的用途，其特征在于，用于治疗遗传性视神经病变，较佳地常染色体显性视神经萎缩(ADOA)。
  18. 一种长期提高线粒体动力蛋白样GTP酶的表达和/或活性的方法，所述方法：包括导入权利要求5-9所述的载体，和/或施用权利要求13-15所述的药物制剂。
  19. 一种治疗方法，其特征在于，所述方法包括将权利要求5-9中任一所述的载体施用于需要的对象。
  20. 一种重组人Ⅱ型线粒体动力蛋白样GTP酶的制备方法，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Ⅱ型线粒体动力蛋白样GTP酶。
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BR112021002566-9A BR112021002566A2 (pt) 2018-08-23 2019-08-23 sequência gênica da gtpase tipo dineína mitocondrial tipo ii humana recombinante e seus usos
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