WO2020010491A1 - Nucleic acid for encoding human nadh dehydrogenase subunit 4 protein and application thereof - Google Patents

Abstract

Provided are a nucleic acid for encoding a human NADH dehydrogenase subunit 4 protein, and an application thereof; the nucleotide sequence thereof is as shown in SEQ ID NO. 1. Also provided is a fusion nucleic acid, containing the nucleic acid for encoding human NADH dehydrogenase subunit 4 protein. Further provided is a recombinant expression vector, containing the described nucleic acid or fusion nucleic acid. Also provided is a transformant, which introduces the described nucleic acid or fusion nucleic acid into the host. The nucleic acid for encoding human NADH dehydrogenase subunit 4 protein and application thereof has a higher expression; therefore more of the human NADH dehydrogenase subunit 4 protein can be obtained in mitochondria, thus Leber hereditary optic neuropathy can be better treated.

Description

Nucleic acid encoding human NADH dehydrogenase subunit 4 protein and application thereof Technical field

The invention relates to the field of biological preparations, in particular to a nucleic acid encoding a human NADH dehydrogenase subunit 4 protein and an application thereof.

Background technique

Leber's hereditary optic neuroneopathy (LHON) is a degenerative visual disorder that usually manifests as bilateral loss of central vision. The average age of onset is in the middle of 20 years, and usually there is no pain within weeks to months, until the vision of both eyes deteriorates below 0.1, which seriously affects the quality of life of patients. LHON is caused by mutations in mitochondrial genes and is related to mutations in one of the three mitochondrial genes of the complex I subunit of the mitochondrial respiratory chain, NADH ubiquinone oxidoreductase. Studies have shown that the G3460A mutation that affects the ND1 gene, the T14484C mutation that affects the ND6 gene, and the G11778A mutation that affects the ND4 gene are considered to be the main causes of LHON, and each mutation has a significant risk of permanent vision loss. All of this has to do with focal degeneration of retinal ganglion cells.

Two major LHON mutations, G3460A and T14484C, resulted in 80% reduction in mitochondrial NADH dehydrogenase activity in patient platelets. However, mitochondria isolated from G11778A cells showed that the activity of complex I and most other components in the respiratory chain was close to normal. For Chinese LHON patients, 90% of patients with G11778A site mutations. The mutation at the 11778 site changes the arginine of human NADH dehydrogenase subunit 4 protein (ND4 protein) into histidine, leading to dysfunction, optic nerve damage, and Leber's hereditary optic neuropathy, which has a high incidence and prognosis. difference.

The main problem with LHON treatment arises from the obstacles in delivering DNA to organelles. The prior art CN 102634527 B discloses a recombinant human NADH dehydrogenase subunit 4 protein gene (ND4 gene) and a method for constructing an expression vector thereof. The 28-amino acid peptide chain of COX10 guides the ND4 protein into the mitochondria. in. CN 104450747A discloses the full length of a recombinant adeno-associated virus-NADH dehydrogenase subunit 4 (ND4) gene and a medicament for treating Leber hereditary optic neuropathy. The gene consists of a CAG promoter sequence, a ND4 coding sequence with a COX10 mitochondrial localization sequence, and a UTR. CN102634527B or CN104450747A containing CAG-Cox10-ND4 agent is injected into the vitreous cavity of the eye for the treatment of Leber's hereditary optic neuropathy. These agents can maintain viability in the vitreous cavity and be transfected into Optic nerve cells, the signal peptide on the N front of the protein directs the protein into the mitochondria, and the mature ND4 protein plays a role. However, this technique also has the disadvantages of low transfection efficiency and poor therapeutic effect.

Therefore, there is an urgent need in the art to develop an expression system and preparation method for human NADH dehydrogenase subunit 4 protein with low transfection efficiency and good therapeutic effect.

Summary of the invention

The purpose of the present invention is to provide an expression system and preparation method of human NADH dehydrogenase subunit 4 protein with low transfection efficiency and good therapeutic effect.

The purpose of the present invention is to provide an optimized nucleic acid sequence encoding a human NADH dehydrogenase subunit 4 protein, a vector and a preparation method thereof.

According to a first aspect of the present invention, a nucleotide sequence encoding a human NADH dehydrogenase subunit 4 (ND4) protein is provided, the nucleotide sequence is selected from the group consisting of:

(a) the nucleotide sequence is as shown in SEQ ID NO.:1; and

(b) The 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.

In another preferred example, the nucleotide sequence includes a DNA sequence, a cDNA sequence, or an mRNA sequence.

In another preferred example, the nucleotide sequence includes a single-stranded sequence and a double-stranded sequence.

In another preferred example, the nucleotide sequence includes a nucleotide sequence completely complementary to SEQ ID NO.:1.

According to a second aspect of the present invention, there is provided a fusion nucleic acid comprising the nucleotide sequence encoding the human NADH dehydrogenase subunit 4 protein according to the first aspect of the present invention.

In another preferred example, the fusion nucleic acid further comprises a sequence selected from the group consisting of a coding sequence of a mitochondrial targeting peptide, a UTR sequence, or a combination thereof.

In another preferred example, the coding sequence of the mitochondrial targeting peptide includes: a COX10 sequence and / or an OPA1 sequence.

In another preferred example, the coding sequence of COX10 has a sequence shown in SEQ ID NO.:2.

In another preferred example, the coding sequence of OPA1 has a sequence shown in SEQ ID NO.:3.

In another preferred example, the UTR sequence includes 3'-UTR and / or 5'-UTR, preferably 3'-UTR.

In another preferred example, the UTR sequence has a sequence as shown in SEQ ID NO .: 4 or 11.

In another preferred example, the fusion nucleic acid has the structure of Formula I from the 5 'end to the 3' end:

Z0-Z1-Z2-Z3 (I)

Where

Each "-" is independently a bond or a nucleotide linking sequence;

Z0 is none or 5'-UTR sequence;

Z1 is the coding sequence of the mitochondrial targeting peptide;

Z2 is a nucleotide sequence encoding a human NADH dehydrogenase subunit 4 protein according to the first aspect of the present invention; and Z3 is a 3'-UTR sequence.

In another preferred example, the Z1 is a coding sequence of COX10 or OPA1.

In another preferred example, the structure of the fusion nucleic acid from the 5'-3 'end is COX10-ND4-UTR.

In another preferred example, the sequence of the fusion nucleic acid is shown in SEQ ID NO .: 5;

Bits 1-84 are the COX10 coding sequence;

Positions 85-1464 are the nucleotide sequence encoding human NADH dehydrogenase subunit 4 protein;

Positions 1465-2889 are 3'-UTR sequences.

In another preferred example, the structure of the fusion nucleic acid from the 5'-3 'end is OPA1-ND4-UTR. Preferably, the fusion nucleic acid sequence is shown as SEQ ID NO.:10.

In another preferred example, the sequence is in a fusion nucleic acid shown in SEQ ID NO :: 10,

Bits 1-266 are the coding sequence of OPA1;

Positions 267-1646 are the nucleotide sequence encoding human NADH dehydrogenase subunit 4 protein;

Bits 1647-2271 are 3'-UTR sequences.

In another preferred example, the length of each nucleotide linking sequence is 1-30 nt, preferably 1-15 nt, and more preferably 3-6 nt.

In another preferred example, the nucleotide linking sequence is derived from a nucleotide adaptor sequence formed by restriction enzyme digestion.

According to a third aspect of the present invention, there is provided 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.

In another preferred example, the vector includes 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.

In another preferred example, the vector is selected from the group consisting of a plasmid and a viral vector.

In another preferred example, the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, or a combination thereof. Preferably, the vector is an AAV vector.

In another preferred example, the serotype of the AAV vector is selected from: AAV2, AAV5, AAV7, AAV8, or a combination thereof.

In another preferred example, the vector includes a DNA virus and a retrovirus vector.

In another preferred example, 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 .

In another preferred example, the vector is used for expressing recombinant human NADH dehydrogenase subunit 4 protein.

According to a fourth aspect of the present invention, there is provided 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.

In another preferred example, the host cell is a mammalian cell, and the mammal includes a human and a non-human mammal.

In another preferred example, the host cell is selected from the group consisting of HEK293 cells, photoreceptor cells (including cone cells and / or rod cells), other visual cells (such as biganglion cells), (optical) nerve cells, Or a combination.

In another preferred example, the host cell is selected from the group consisting of rod cells, cone cells, photophobic bipolar cells, photophobic bipolar cells, horizontal cells, ganglion cells, amacrine cells, or combination. Preferably, the host cell is a (retinal) ganglion cell.

According to a fifth aspect of the present invention, there is provided the use of the carrier according to the third aspect of the present invention, for preparing a preparation or a composition for restoring a subject's vision and / or treating retinal degeneration. Sexually transmitted diseases.

In another preferred example, the preparation or composition is used for treating ocular diseases.

In another preferred example, the preparation or composition is used to treat focal degeneration of retinal ganglion cells.

In another preferred example, the preparation or composition is used for treating hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).

According to a sixth aspect of the present invention, there is provided a pharmaceutical preparation containing (a) the carrier according to the third aspect of the present invention, and (b) a pharmaceutically acceptable carrier or excipient.

In another preferred example, the dosage form of the pharmaceutical preparation is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.

In another preferred example, the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, or a combination thereof. Preferably, the vector is an AAV vector.

In another preferred example, the content of the carrier in the pharmaceutical preparation is 1 × 10 ⁹ -1 × 10 ¹⁶ , preferably 1 × 10 ¹² -1 × 10 ¹³ viruses / ml.

In another preferred example, the pharmaceutical preparation is used to treat ocular diseases, preferably to treat focal degeneration of retinal ganglion cells.

In another preferred example, the pharmaceutical preparation is used for treating hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).

According to a seventh aspect of the present invention, there is provided a treatment method, which comprises applying the carrier according to the third aspect of the present invention to a subject in need.

In another preferred example, the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, or a combination thereof. Preferably, the vector is an AAV vector.

In another preferred example, the carrier is introduced into the eye of a desired subject.

In another preferred example, the required objects include humans and non-human mammals.

In another preferred example, the treatment method is a method for treating eye diseases.

In another preferred example, the eye disease is hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).

According to an eighth aspect of the present invention, a method for preparing a recombinant human NADH dehydrogenase subunit 4 protein is provided, comprising the steps of: culturing the host cell of claim 5 to obtain a recombinant human NADH dehydrogenase subunit 4 protein. .

It should be understood that, within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a comparison of the nucleotide sequence of an ND4 fusion nucleic acid with an optimized ND4 fusion nucleic acid. Among them, the "ND4 gene" line sequence is an unoptimized ND4 fusion nucleic acid sequence, and the "optimized ND4 gene" line sequence is an optimized ND4 fusion nucleic acid.

Figure 2 shows the results of PCR nucleic acid electrophoresis to verify ND4 (lane A) and optimized ND4 (lane B) gene clones.

Figure 3 shows the expression levels of rAAV2-optimized ND4 (black bar on the left) and rAAV2-ND4 (black bar on the right) at the gene level using q-actin as the internal reference gene (white bar).

Figure 4 shows the expression levels of rAAV2-optimized ND4 (black bar on the left) and rAAV2-ND4 (right black bar) on the protein level using β-actin as the internal reference protein (white bar).

Figure 5 shows the fundus photographs of rabbit eyes under glass microscope, where A is injected with rAAV2-ND4 virus and B is injected with rAAV2-optimized ND4 virus.

Figure 6 shows the fundus photograph results of the human eye vitreous cavity rAAV2-optimized ND4 virus injection experiment, where A is before treatment and B is after treatment.

detailed description

After extensive and intensive research, the present inventors optimized the coding sequence of the recombinant human NADH dehydrogenase subunit 4 protein gene, thereby obtaining a highly suitable transcription in mammalian (such as human) cells. The nucleotide sequence and fusion nucleic acid of ND4 protein were efficiently expressed, and a recombinant expression vector of recombinant human NADH dehydrogenase subunit 4 protein was constructed. After special optimization, the transcription efficiency and translation efficiency of the ND4 coding sequence (SEQ ID NO.:1) were significantly improved, and the expression of recombinant human NADH dehydrogenase subunit 4 protein was more than doubled. On this basis, the inventors have completed the present invention.

the term

In order that the present disclosure may be more easily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning set out below. Other definitions are set forth throughout the application.

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. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, 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 can be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of a 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.

As used herein, the terms "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, cattle, horses, dogs, cats, pigs, sheep, goats.

The terms "human NADH dehydrogenase subunit 4 protein", "ND4 (protein)", "polypeptide", "polypeptide of the invention" and "protein of the invention" are used interchangeably.

Adeno-associated virus

Adeno-associated virus (AAV), 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 adeno 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 (rAAV) 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.

In a preferred embodiment of the present invention, 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.

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. 4,797,368, 6,596,535 and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entireties). These patent publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced by the gene of interest, and these constructs are in vitro (into cultured cells) or in vivo (directly into organisms) ) Use of a gene of interest. Replication-deficient recombinant AAV can be prepared by co-transfecting 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). The AAV recombinants produced are then purified by standard techniques.

In some embodiments, 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). Accordingly, the disclosure includes a recombinant virion (recombinant because it comprises a recombinant polynucleotide) that contains any of the vectors described herein. Methods of generating such particles are known in the art and are described in US Patent No. 6,596,535.

Nucleic acid coding sequence

The technical problem to be solved by the present invention is to overcome the technical defects that the transfection efficiency of NADH dehydrogenase subunit 4 protein in the prior art is not high and the treatment effect is not good. The invention provides a targeted NADH dehydrogenase subunit 4 protein, and a preparation method and application thereof. It has been found through research that the optimized ND4 gene sequence (SEQ ID NO.:1) of the present invention makes the ND4 protein expression more efficient, and more ND4 proteins play a physiological role in the optic ganglion cells of patients.

The nucleotide sequence of the nucleic acid encoding human NADH dehydrogenase subunit 4 protein according to the present invention is shown in SEQ ID NO.:1. The nucleic acid encoding human NADH dehydrogenase subunit 4 protein has a full length of 1380 bp. In the present invention, the nucleic acid encoding the human NADH dehydrogenase subunit 4 protein is also referred to as an ND4 optimized gene or an ND4 optimized nucleic acid.

The polynucleotide of the present invention may be in the form of DNA or RNA. In another preferred example, the nucleotide is DNA. The form of DNA includes cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding.

The full-length nucleotide sequence of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. For the PCR amplification method, primers can be designed based on 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 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 stitch the amplified fragments together in the correct order. At present, 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.

As used herein, "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). For example, 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.

In a preferred embodiment of the present invention, the nucleotide sequence is as shown in SEQ ID NO.:1.

Once the relevant sequences are obtained, the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, synthetic methods can also be used to synthesize related sequences, especially when the fragment length is short. Generally, 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 expressing the ND4 protein using the host cell by recombinant technology.

A host cell (such as a mammalian cell) expressing the ND4 protein of the present invention can be obtained by using the polynucleotide sequence of the present invention through 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.

Methods well known to those skilled in the art can be used to construct 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.

In addition, 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.

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 so that it can 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. In a preferred embodiment of the present invention, HEK 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. In another preferred example, the host cell is selected from the group consisting of rod cells, cone cells, photophobic bipolar cells, photophobic 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. When 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. When the host is a eukaryote, 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 transformant can be cultured by a conventional method, and expresses the protein encoded by the gene of the present invention. Depending on the host cell used, 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.

The polypeptide in the above method may be expressed intracellularly, or on a cell membrane, or secreted extracellularly. If desired, proteins can be separated 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.

Sequence optimization

In the present invention, the mitochondrial coding sequence of COX10 is changed to a nucleic acid coding sequence (as shown in SEQ ID NO: 2) and the ND4 nucleotide sequence is optimized (as shown in SEQ ID NO: 1), which is referred to as the optimized ND4 gene for short in the present invention. Nucleic acid), this sequence is specially optimized, and the transcription efficiency and translation efficiency are significantly improved. The homology of the optimized COX10 + ND4 and unoptimized COX10 + ND4 sequences is 75.89%.

In the present invention, an optimized coding sequence of a recombinant human NADH dehydrogenase subunit 4 protein with high transcription efficiency and translation efficiency is provided, and the coding sequence is shown in SEQ ID NO.:1.

As used herein, the "optimized ND4 coding sequence" and "optimized ND4 coding gene" both refer to a nucleotide sequence for encoding a recombinant human NADH dehydrogenase subunit 4 protein, and the nucleotide sequence Encodes the amino acid sequence shown in SEQ ID NO.:1.

Fusion nucleic acid

The invention also provides a fusion nucleic acid, which comprises the nucleic acid encoding the human NADH dehydrogenase subunit 4 protein.

As used herein, a "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 protein encoded by the fusion nucleic acid of the present invention is called a fusion protein, which is an ND4-optimized protein in the present invention.

Preferably, the fusion nucleic acid is operably linked to the coding sequence and / or UTR sequence of the mitochondrial targeting peptide in the nucleic acid encoding the human NADH dehydrogenase subunit 4 protein. Preferably, when the nucleic acid encoding the human NADH dehydrogenase subunit 4 protein is linked to the coding sequence of a mitochondrial targeting peptide, the coding sequence of the mitochondrial targeting peptide is as shown in SEQ ID NO .: 2 The coding sequence of the mitochondrial targeting peptide of the COX10 gene (COX10 sequence for short), or the OPA1 sequence shown in SEQ ID NO :: 3; when the nucleic acid encoding the ND4 protein is linked to the UTR sequence, the UTR sequence is as follows SEQ ID NO.:4 or SEQ ID NO.:11.

In another preferred example, in the fusion nucleic acid, the coding sequence of COX10, the nucleic acid encoding human NADH dehydrogenase subunit 4 protein, and the UTR sequence are arranged in sequence from the 5 'end to the 3' end .

In another preferred example, the sequence of the fusion nucleic acid is shown in SEQ ID NO.:5. Specifically, the full length of the nucleotide sequence of the fusion nucleic acid is 2889bp, and the optimized COX10 sequence (a total of 84bp) is located from 1bp to 84bp; the optimized ND4 gene is located from 85bp to 1464bp, that is, the human NADH encoding The nucleic acid of the catalase subunit 4 protein (1380bp in total), the positions from 1465bp to 2889bp are UTR sequences (1425bp in total, also known as 3'UTR). The COX10 sequence guides the ND4 protein into the mitochondria and exerts its physiological functions; 3'UTR is a non-coding sequence designed behind the ND4 protein, and its role is to stabilize the coding sequence of the mitochondrial targeting peptide and the expression of ND4. Among them, the homology of optimized COX10 + ND4 and unoptimized COX10 + ND4 sequences was 75.89%.

In another preferred example, the sequence of the fusion nucleic acid is shown in SEQ ID NO.:10.

Expression vectors and host cells

The invention also provides an expression vector for ND4 protein, which contains the optimized ND4 coding sequence of the invention.

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.

Preferably, the nucleic acid sequence encoding the ND4 protein is provided as a vector, preferably an expression vector. Preferably, it is 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 mutant forms, retroviruses, lentivirus, herpes virus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV) , HIV, pox virus, and SV40. Preferably, 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 ND4 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. For example, in AAV, the virus can be modified to delete all or part of the IX gene, Ela, and / or Elb gene. For wild-type AAV, there is no helper virus such as adenovirus, and replication is very inefficient. For a recombinant adeno-associated virus, preferably, 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 also be made to lentiviral vectors.

Viral vectors have the ability to enter cells. However, non-viral vectors such as plasmids can be complexed with agents to facilitate uptake of the viral vector by target cells. Such agents include polycationic agents. Alternatively, 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. For example, 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 internal 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. For example, a suitable promoter may be selected from the group consisting of: L7, thy-1, restorer protein, calcium-binding protein, human CMV, GAD-67, chicken beta 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 early CMV enhancer and the chicken β-actin promoter.

An example of 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-1α (EF-1α). However, other 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. Further, the present invention should not be limited to the application of a constitutive promoter. Inducible promoters are also considered as part of the invention. The use of 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. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

Many expression vectors can be used to express ND4 protein in 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 host cell for expressing the ND4 protein. Preferably, the host cell is a mammalian cell (preferably a human, more preferably a human optic nerve cell or a photoreceptor cell), and the expression of the ND4 protein is increased.

Formulations and compositions

The invention provides a formulation or composition comprising (a) the carrier according to the third aspect of the invention, and (b) a pharmaceutically acceptable carrier or excipient.

In another preferred example, the pharmaceutical preparation is used for treating ocular diseases.

In another preferred example, the pharmaceutical preparation is used for treating hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).

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. A "safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. "Pharmaceutically acceptable carrier or excipient" means: one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must be of sufficient purity and sufficient Low toxicity. "Compatibility" here 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, gel or paste. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.

In the present invention, the carrier may be administered to the eye by subretinal or intravitreal administration. In either mode of administration, the carrier is preferably provided as an injectable liquid. Preferably, the injectable liquid is provided as a capsule or syringe.

)、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。 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 (such as Tween

), Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, 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 ND4-encoding nucleic acid or fusion nucleic acid provided by the present invention can produce ND4 protein or ND4 fusion protein in vitro or in vivo, and the fusion protein or the preparation containing the fusion protein can be used to prepare a medicine for treating Leber's hereditary optic neuropathy.

The optimized nucleic acid encoding human NADH dehydrogenase subunit 4 protein has a higher expression level, which translates more ND4 fusion proteins, and the COX10 sequence can accurately locate the ND4 fusion protein on the inner mitochondrial membrane, so there is more Many ND4 proteins are transfected into mitochondria. The agent containing COX10-optimized ND4 fusion nucleic acid was injected into the vitreous cavity of rabbit eyes, and the agent maintained viability in the vitreous cavity and was transfected into optic nerve cells. The optimized ND4 nucleic acid encodes more ND4 protein than the existing technology, and its transfection efficiency is higher, which can better treat Leber hereditary optic neuropathy.

Compared with the prior art, the present invention mainly has the following advantages:

1. The gene sequence encoding the recombinant human NADH dehydrogenase subunit 4 protein (ND4) of the present invention is specially optimized. Compared with ND4's unoptimized DNA coding sequence, transcription efficiency and translation efficiency are significantly improved. The expression of the optimized sequence ND4 protein was significantly increased, and its biological activity was high.

2. The optimized COX10 sequence or OPA1 sequence of the present invention can accurately locate the ND4 fusion protein on the inner mitochondrial inner membrane, so more ND4 proteins are transfected into the mitochondria.

3. The optimized ND4 coding gene (SEQ ID No .: 1) or fusion nucleic acid of the present invention can treat Leber's hereditary optic neuropathy very effectively and has good safety.

The present invention will be further described below in combination with specific implementation. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions Conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

experiment equipment

Gel imaging system: Gene Genius; Taq DNA polymerase: Biotech Bioengineering (Shanghai) Co., Ltd .; Marker: Biotech Bioengineering (Shanghai) Co., Ltd .; 6 × DNA Loading Dye: Biotech Bioengineering (Shanghai) Co., Ltd. ; PCR product purification and recovery kit: Biotech Bioengineering (Shanghai) Co., Ltd .; KpnI / SalI enzyme: Biotech Bioengineering (Shanghai) Co., Ltd .; Lipofectamine 2000 kit: American Invitrogen company.

Example 1 Construction of plasmid and preparation of recombinant adeno-associated virus

1.1 Plasmid preparation: After obtaining the human ND4 nucleotide sequence (National Biotechnology Information Center reference sequence: yp_003024035.1), the present invention changes the mitochondrial coding sequence of COX10 to a nucleic acid coding sequence (as shown in SEQ ID NO: 2 ) And perform ND4 nucleotide sequence optimization (as shown in SEQ ID NO: 1, referred to as the optimized ND4 gene / nucleic acid in the present invention), this sequence is specially optimized, the transcription efficiency and translation efficiency are significantly improved, and the optimized COX10 + The homology of ND4 and unoptimized COX10 + ND4 sequences is 75.89%. And the UTR sequence is connected to the 3 ′ end of the optimized ND4 gene (as shown in SEQ ID NO: 4), and the sequence of its fusion gene (or fusion nucleic acid) is shown as SEQ ID ID NO.:5. Synthetic from Biotechnology Co., Ltd. The full-length gene was amplified by PCR (Figure 2), and a sticky end was formed on the fusion gene by EcoRI / SalI digestion, and the fusion gene was embedded in an adeno-associated virus vector pSNaV, ie, pSNaV / rAAV2 / 2-Optimized ND4 (hereinafter referred to as pAAV2-Optimized ND4). The steps for screening and identifying recombinants are the same as CN 102634527B, and they are briefly described as follows: Take the LB plate cultured at 37 ° C, blue spots and white spots appear, and white is the recombinant clone. Pick white colonies and add them to LB liquid medium containing 100 mg / L of Amp and incubate at 37 ° C and 200 rpm for 8 h. After culturing, take the bacterial solution and extract the plasmid. Refer to Biomiga's instructions for plasmid extraction and use EcoRI / SalI to digest and identify. Control pSNaV / rAAV2 / 2-ND4 (hereinafter abbreviated as pAAV2-ND4) is prepared by referring to CN 102634527.

1.2 Cell transfection: One day before transfection, HEK293 cells were seeded in a 225cm ² cell culture flask with a seeding density of 3.0 × 10 ⁷ cells / mL, and the culture medium was DMEM + 10% bovine serum, which was placed at 37 ° C and containing 5% CO ₂ incubator overnight. The medium was changed on the day of transfection, and the culture was continued in fresh DMEM medium containing 10% bovine serum. When the cells grow to 80-90%, discard the culture medium and transfect pAAV2-ND4 and pAAV2-optimized ND4 with PlasmidTrans II (VGTC) transfection kit (for specific transfection steps, see CN 102634527 B Example 1). 48h after transfection, cells were harvested.

1.3 Collection, concentration and purification of recombinant adeno-associated virus:

1.3.1 Virus collection: 1) Prepare dry ice ethanol bath (or liquid nitrogen) and 37 ° C water bath; 2) collect the toxic cells together with the culture medium into a 15 ml centrifuge tube; 3) centrifuge at 1000 rpm / min 3 minutes, the cells and supernatant were separated, the supernatant was stored separately, and the cells were resuspended in 1 ml of PBS; 4) the cell suspension was repeatedly transferred in a dry ice ethanol bath and a 37 ° C water bath, and freeze-thaw four times, freeze and thaw each 10 Minutes, with slight shock after each melt.

1.3.2 Virus concentration: 1) Remove cell debris by centrifugation at 10,000g, transfer the centrifuged supernatant to a new centrifuge tube; 2) filter with a 0.45 μm filter to remove impurities; 3) add 1/2 volume of 1M NaCl, 10% PEG8000 solution, mix evenly at 4 ° C overnight; 4) Centrifuge at 12,000 rpm for 2 h, discard the supernatant, and dissolve the virus pellet with an appropriate amount of PBS solution. After complete dissolution, filter through a 0.22 μm filter to remove bacteria; 5) Add Benzonase nuclease Residual plasmid DNA was removed by digestion (final concentration of 50 U / ml). Close the tube cap and invert several times to mix thoroughly. Incubate at 37 ° C for 30 minutes; 6) Filter through a 0.45 μm filter, and take the filtrate as the concentrated rAAV2 virus.

1.3.3 Virus purification: 1) Add solid CsCl to the virus concentrate until the density is 1.41 g / ml (refractive index is 1.372); 2) Add the sample to the ultracentrifuge tube and use the pre-made 1.41 g / Fill the remaining space of the centrifuge tube with ml CsCl solution; 3) Centrifuge at 175,000g for 24 hours to form a density gradient. Samples of different densities were collected in sequence, and samples were taken for titer determination. Collect the components enriched with rAAV2 particles; 4) Repeat the above process once. The virus was loaded into a 100 kDa dialysis bag and dialyzed and desalted overnight at 4 ° C.

So far, concentrated and purified recombinant adeno-associated viruses rAAV2-ND4 and rAAV2-optimized ND4 were obtained.

As known to those skilled in the art, in addition to COX10, OPA1 (sequence is shown in SEQ ID No .: 3) can also be fused with the optimized ND4 gene of the present invention. The coding sequence OPA1 of the mitochondrial targeting peptide can optimize the expression of ND4 The protein is introduced into the inner mitochondrial membrane, thereby achieving mitochondrial targeted expression of the protein.

Example 2 Experiment of Injecting rAAV2 into the Vitreous Cavity of Rabbit Eyes

Twelve rabbits were divided into two groups, and the virus solution rAAV2-ND4 and rAAV2-optimized ND4 (1 × 10 ¹⁰ vg / 0.05mL) were punctured into the vitreous cavity at a distance of 3 mm from the limbal limbus into the vitreous cavity. After injection in the vitreous cavity, slit lamp and fundus photography were performed. After 30 days of injection, each group was tested by RT-PCR and immunoblot.

Example 3 RT-PCR detection of ND4 expression

RNA and reverse transcription of rabbit optic nerve cells transfected with rAAV2-ND4 and rAAV2-optimized-ND4 were extracted, and total RNA was extracted using the TRIZOL kit and cDNA templates were synthesized by reverse transcription. NCBI's conservative domain analysis software was used to analyze the conserved structure of ND4 to ensure that the amplified fragments of the designed primers were located in non-conserved regions. Then, based on the primer design principles of fluorescent quantitative PCR, primers were designed using primer 5:

β-actin-S: CGAGATCGTGCGGGACAT (SEQ ID NO.:6);

β-actin-A: CAGGAAGGAGGGCTGGAAC (SEQ ID NO .: 7);

ND4-S: CTGCCTACGACAAACAGAC (SEQ ID NO.:8);

ND4-A: AGTGCGTTCGTAGTTTGAG (SEQ ID NO .: 9);

Reaction system and procedure of real-time PCR:

Real-time PCR was performed on a Real-time PCR Detection System instrument. In a 0.2 mL PCR reaction tube, 12.5 μL of SYBR Green mix, ₈ μL of ddH ₂ O, ₁ μL of each primer, 2.5 μL of cDNA sample, and 25 μL of the total system were added. Each sample was used to amplify both the target gene and the internal reference gene β-actin. Each gene was amplified in three replicates. In order to reduce the error during the actual loading, the reagents common to each PCR reaction tube can be added together and then aliquoted. After the sample is loaded, perform quantitative PCR.

According to the reaction procedure of pre-denaturation at 95 ° C for 1 s, denaturation at 94 ° C for 15 s, annealing at 55 ° C for 15 s, and extension at 72 ° C for 45 s, a total of 40 cycles were performed, and fluorescence signals were collected during the extension phase of each cycle. After the reaction, a melting curve analysis of 94 ° C to 55 ° C was performed.

Relative quantitative methods were used to study the differences in gene expression, with β-actin as the internal reference gene.

The results are shown in Figure 3. At the mRNA level, the relative expression levels of mRNA in the unoptimized rAAV2-ND4 group and the optimized rAAV2-optimized ND4 group were 0.42 ± 0.23 and 0.57 ± 0.62, respectively. There was a significant difference between the two groups ( p <0.05, Figure 3). This result unexpectedly shows that the optimized ND4 coding nucleotide sequence (positions 85-1464 of SEQ ID No .: 5) and the corresponding fusion nucleic acid (position 1 of SEQ ID NO .: 5) of the present invention -2889), unexpectedly increased the transcription efficiency, so that the rAAV2-optimized ND4 group gene expression was significantly higher than the rAAV2-ND4 group, an increase of about 36%. The results indicate that in terms of transcription efficiency, the transcription efficiency of the rAAV2-optimized ND4 group is significantly higher.

Example 4 Detection of ND4 Expression by Western Blot

Mitochondrial ND4 protein of rabbit neurons transfected with rAAV2-ND4 and rAAV2-optimized-ND4 was extracted separately, and then subjected to 10% polyacrylamide gel electrophoresis, and the spots were transferred to a polyvinylidene fluoride membrane (Bio-Rad, Her-cules, CA, USA), used for immunoassay, using β-actin as the internal reference gene, observation and analysis of the bands on the film with an automatic image analysis instrument (Li-Cor; Lincoln, NE, USA), and the integrated optical density of each protein band Integrate with normalization method to get the corresponding optical density value of the same sample. Statistical analysis was performed using SPSS 19.0 statistical software for statistical analysis.

The results are shown in Figure 4. Analysis of mitochondrial ND4 protein expression The average expression value of rAAV2-ND4 group was 0.32 ± 0.11, while the average expression value of rAAV2-optimized ND4 group was 0.68 ± 0.20, and the increase of rAAV2-optimized ND4 group was about 112%. There was a significant difference between the two groups Sexual differences (p <0.01, Figure 4). The protein expression level of the rAAV2-optimized ND4 group was significantly higher than that of the non-optimized group, which indicates that, in terms of translation efficiency, the optimized ND4 coding nucleotide sequence of the present invention (SEQ ID No. 85-1464 of No. 5: 5464 Position) and the corresponding fusion nucleic acid (SEQ ID No .: 1-2889) are more efficient in translation.

Example 5 Rabbit intraocular pressure and fundus photography

The rabbits of the two groups were examined by 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. The fundus photographs for one month after the operation are shown in FIG. 5, where FIG. 5A is the fundus photograph result of the injection of rAAV2-ND4, and FIG. 5B is the fundus photograph result of the injection of rAAV2-optimized ND4. As can be seen from the figure, there were no obvious complications or damage to the retinal blood vessels and optic nerve in all rabbits. It shows that the normal standard vitreous cavity injection is safe without obvious inflammation or other complications.

Example 6 Clinical Trial

Nine clinical trials were performed from 2011 to 2012. Patients were injected with 1 × 10 ¹⁰ vg / 0.05mL rAAV2-ND4 as a control group in monocular vitreous cavity. Twenty clinical trials were performed between 2017 and January 2018. Patients were injected 1 × 10 ¹⁰ vg / 0.05mL rAAV2-optimized ND4 was used as the experimental group to observe the clinical efficacy of the two groups. Statistical analysis was performed using SPSS 19.0 statistical software.

The clinical efficacy comparison between the two groups is shown in Table 1. The fastest improvement of visual acuity in the experimental group was 1 month, which was significantly faster than the control group in 3 months. There was a significant difference between the two groups (p <0.01). 1.0, significantly higher than 0.8 in the control group, there was a significant difference between the two groups (p <0.01); the average visual acuity recovery of the experimental group was 0.582 ± 0.086, which was significantly higher than 0.344 ± 0.062 in the control group. p <0.01). Among them, the fundus photographs of the rAAV2-optimized ND4 group before and after treatment are shown in Figure 6. Generally, no complications occurred in the control group and the experimental group, and the safety was very good.

Table 1 Comparison of LHON between two gene therapies

Example 7

Replace the sequence such as the COX10 sequence (positions 1-84) in SEQ ID NO.:5 with the OPA1 mitochondrial targeting peptide sequence (SEQ ID NO.:3), and replace the positions 1465-2889 with SEQ ID ID NO :: The UTR sequence shown in FIG. 11 yields a fusion nucleic acid with the structure OPA1-ND4-UTR. The sequence is shown in SEQ ID NO.:10.

The experimental method is the same as that in Examples 1-6, and the fusion nucleic acid shown in SEQ ID NO .: 5 is replaced with the fusion nucleic acid shown in SEQ ID NO .: 10 in this example. As a result, compared with the unoptimized ND4 coding sequence, the optimized ND4 coding nucleotide sequence (positions 267-1646 of SEQ ID NO .: 10) and the corresponding fusion nucleic acid (SEQ ID ID NO .: The ND4 transcription efficiency and translation efficiency of ND4 were significantly increased, and the expression was significantly higher. It can effectively treat Leber's hereditary optic neuropathy and has good safety.

All documents mentioned in the present invention are incorporated by reference in this application, as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

A nucleotide sequence encoding a human NADH dehydrogenase subunit 4 (ND4) protein, wherein the nucleotide sequence is selected from the following group: (a) the nucleotide sequence is as shown in SEQ ID NO.:1; and (b) The 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. A fusion nucleic acid, characterized in that the fusion nucleic acid comprises the nucleotide sequence encoding the human NADH dehydrogenase subunit 4 protein according to claim 1. The fusion nucleic acid according to claim 2, wherein the fusion nucleic acid has a structure of formula I from the 5 'end to the 3' end: Z0-Z1-Z2-Z3 (I) Where Each "-" is independently a bond or a nucleotide linking sequence; Z0 is none or 5'-UTR sequence; Z1 is the coding sequence of the mitochondrial targeting peptide; Z2 is a nucleotide sequence encoding a human NADH dehydrogenase subunit 4 protein according to claim 1; and Z3 is a 3'-UTR sequence. A vector, wherein the vector contains the nucleotide sequence according to claim 1 or the fusion nucleic acid according to claim 2. A host cell, characterized in that the host cell contains the vector according to claim 4, or the chromosome has an exogenous nucleotide sequence according to claim 1 or a fusion according to claim 2 Nucleic acid. The host cell according to claim 5, wherein the host cell is selected from the group consisting of HEK293 cells, photoreceptor cells (including cone cells and / or rod cells), and other visual cells (such as biganglocytes). , (Optical) nerve cells, or a combination thereof. The use of the carrier according to claim 4, characterized in that it is used for preparing a preparation or composition for restoring vision and / or treating degenerative retinal diseases in a subject. A pharmaceutical preparation, characterized in that the preparation contains (a) the carrier according to claim 4, and (b) a pharmaceutically acceptable carrier or excipient. A treatment method, characterized in that the method comprises applying the carrier according to claim 4 to a subject in need. A method for preparing a recombinant human NADH dehydrogenase subunit 4 protein, comprising the steps of: culturing the host cell according to claim 5 to obtain a recombinant human NADH dehydrogenase subunit 4 protein.

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