CN111518813B

CN111518813B - Coding sequence of rhodopsin, construction of expression vector and application thereof

Info

Publication number: CN111518813B
Application number: CN201910108800.8A
Authority: CN
Inventors: 李斌
Original assignee: Wuhan Niufusi Biological Technology Co ltd
Current assignee: Wuhan Niufusi Biological Technology Co ltd
Priority date: 2019-02-03
Filing date: 2019-02-03
Publication date: 2023-04-28
Anticipated expiration: 2039-02-03
Also published as: CN111518813A

Abstract

The invention provides a coding sequence of rhodopsin, an expression vector construction and application thereof. Specifically, the invention carries out targeted special optimization design on RHO gene coding sequence, thereby obtaining a nucleotide sequence which is particularly suitable for efficiently expressing RHO protein in mammalian (such as human) cells (such as photoreceptor cells and optic nerve cells), and constructing a recombinant AAV which expresses normal human RHO protein. Compared with the non-optimized coding sequence, the expression quantity of the RHO coding sequence (SEQ ID NO: 1) after special optimization is obviously improved, is very suitable for intracellular expression of mammals (especially human), and can effectively treat the retinitis pigmentosa.

Description

Coding sequence of rhodopsin, construction of expression vector and application thereof

Technical Field

The invention relates to the field of biological agents, in particular to a coding sequence of rhodopsin, construction of an expression vector thereof and application thereof.

Background

Retinitis pigmentosa (retinitis pigmentosa, RP) is the most common group of hereditary blinding fundus diseases in which retinal photoreceptor and pigment epithelial cell degeneration leads to progressive visual field defects. Patients often present with night blindness, progressive visual field reduction and vision loss, eventually leading to reduced or even complete loss of vision function due to apoptosis of the retinal photoreceptor cells. RP has high genetic heterogeneity and clinical heterogeneity, and has a plurality of genetic modes such as emission, autosomal dominant, autosomal recessive, X-linked inheritance, double-gene inheritance and the like. At present, the incidence rate of the disease is 1/3500, and at least 100 tens of thousands of people suffer from the disease worldwide, wherein the incidence rate of the disease is about 1/4 of the incidence rate of Chinese people, and the incidence rate of the disease increases year by year. 40% -50% of patients are sporadic or single shot with no family history of disease, known as sporadic retinitis pigmentosa (sporadic retinitis pigmentosa, SRP). X-linked inheritance accounts for about 6-20% of all RP patients. Autosomal recessive inheritance accounts for 5% -15%. Autosomal dominant inherited retinitis pigmentosa (autosomal dominant retinitis pigmentosa, ADRP) accounts for 15% -25%. Rhodopsin (RHO) gene mutations account for 16% -25% of autosomal dominant inherited retinal pigment degeneration (ADRP) in europe and north america, around 7% in china and japan. Mutation of the base of the RHO gene may cause amino acid substitution, termination, or deletion of the RHO protein to cause abnormal functioning of the RHO protein, thereby causing visual dysfunction.

Therefore, there is a need in the art to develop a gene therapy method and therapeutic drug capable of effectively treating retinitis pigmentosa.

Disclosure of Invention

The invention aims to provide a gene therapy method and a therapeutic drug capable of effectively treating retinitis pigmentosa.

The invention also aims at providing a coding sequence for coding rhodopsin, a vector and a preparation method thereof.

In a first aspect of the invention, there is provided a nucleotide sequence encoding rhodopsin, and selected from the group consisting of:

(a) The nucleotide sequence is shown as SEQ ID NO. 1;

(b) The nucleotide sequence has more than or equal to 95 percent identity, preferably more than or equal to 98 percent, more preferably more than or equal to 99 percent with the nucleotide sequence shown in SEQ ID No. 1; and

(c) A nucleotide sequence complementary to the nucleotide sequence of (a) or (b).

In another preferred embodiment, the nucleotide sequence comprises a DNA sequence, a cDNA sequence, or an mRNA sequence.

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

In another preferred embodiment, the nucleotide sequence comprises a nucleotide sequence that is fully complementary to SEQ ID NO. 1.

In a second aspect of the invention there is provided a fusion nucleic acid comprising a nucleotide sequence encoding rhodopsin according to the first aspect of the invention.

In another preferred embodiment, the fusion nucleic acid further comprises a UTR sequence.

In another preferred embodiment, the UTR sequence comprises a 3'UTR and/or a 5' UTR.

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

Z0-Z1-Z2 (I)

in the method, in the process of the invention,

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

z0 is none, or a 5' UTR sequence;

z1 is a nucleotide sequence according to the first aspect of the invention; and

z2 is the none, or 3' UTR sequence.

In another preferred embodiment, the fusion nucleic acid has a structure of 5'UTR-RHO-3' UTR from the 5'-3' end.

In another preferred embodiment, each nucleotide linking sequence has a length of 1-30nt, preferably 1-15nt, more preferably 3-6nt.

In another preferred embodiment, the nucleotide connecting sequence is derived from a nucleotide linker sequence formed by restriction enzyme cleavage.

In a third aspect of the invention there is provided a vector comprising a nucleotide sequence according to the first aspect of the invention or a fusion nucleic acid according to the second aspect of the invention.

In another preferred embodiment, the vector comprises one or more promoters operably linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, selectable marker, nucleic acid restriction site, and/or homologous recombination site.

In another preferred embodiment, the carrier is selected from the group consisting of: plasmid and viral vector.

In another preferred embodiment, the carrier is selected from the group consisting of: lentiviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV), or combinations thereof.

In another preferred embodiment, the vector is in the form of a viral particle.

In another preferred embodiment, the vector is an adeno-associated viral AAV vector.

In another preferred embodiment, the serotype of the AAV vector is selected from the group consisting of: AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or a combination thereof. Preferably, the serotype of the AAV vector is AAV8 or AAV9.

In another preferred embodiment, the vector is an AAV8 vector or an AAV9 vector.

In another preferred embodiment, the vector is a capsid transferred AAV vector.

In another preferred embodiment, the vector comprises an AAV2 genome and an AAV8 capsid protein (AAV 2/8), or an AAV2 genome and an AAV9 capsid protein (AAV 2/9).

In another preferred embodiment, the vector is a recombinant adeno-associated viral vector rAAV2/8 or rAAV2/9.

In another preferred embodiment, the vector comprises a DNA viral vector, a retroviral vector.

In another preferred embodiment, the vector is an AAV vector comprising or inserted with a nucleotide sequence according to the first aspect of the invention or a fusion nucleic acid according to the second aspect of the invention; preferably rAAV2/8 or rAAV2/9 vectors.

In another preferred embodiment, the backbone of the vector is the adeno-associated viral vector plasmid pSNaV.

In another preferred embodiment, the vector is used to express rhodopsin.

In a fourth aspect of the invention there is provided a host cell comprising a vector according to the third aspect of the invention, or a chromosome thereof, into which has been integrated an exogenous nucleotide sequence according to the first aspect of the invention or a fusion nucleic acid according to the second aspect of the invention.

In another preferred embodiment, the host cell is a mammalian cell, including human and non-human mammals.

In another preferred embodiment, the host cell is selected from the group consisting of: HEK293 cells, photoreceptor cells (including cone cells and/or rod cells), other vision cells (e.g., binodal cells), (optic) nerve cells, or combinations thereof.

In another preferred embodiment, the host cell is selected from the group consisting of: rod cells, cone cells, light-donating bipolar cells, light-withdrawing bipolar cells, horizontal cells, ganglion cells, non-long process cells, or combinations thereof. Preferably, the host cell is a (retinal) ganglion cell or a photoreceptor cell.

In a fifth aspect of the invention there is provided the use of a carrier according to the third aspect of the invention for the preparation of a formulation or composition for restoring vision in a subject and/or for treating or preventing an ocular disorder.

In another preferred embodiment, the ocular disease is Retinitis Pigmentosa (RP).

In another preferred embodiment, the formulation or composition is for use in the treatment or prevention of retinal pigment degeneration, such as autosomal dominant inherited retinal pigment degeneration (ADRP).

In another preferred embodiment, the formulation or composition is used to reduce photoreceptor cell or optic nerve cell death in a subject suffering from or at risk of developing retinitis pigmentosa.

In a sixth aspect of the invention there is provided a pharmaceutical formulation comprising (a) a carrier according to the third aspect of the invention, and (b) a pharmaceutically acceptable carrier or excipient.

In another preferred embodiment, the dosage form of the pharmaceutical formulation is selected from the group consisting of: lyophilized formulations, liquid formulations, or combinations thereof.

In another preferred embodiment, the carrier is selected from the group consisting of: lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, or combinations thereof. Preferably, the vector is an AAV vector; more preferably AAV2/8 or AAV2/9 vectors.

In another preferred embodiment, the carrier is present in the pharmaceutical formulation in an amount of 1X 10 ⁹ -1×10 ¹⁶ Individual viruses/ml, preferably 1X 10 ¹² -1×10 ¹³ Individual viruses/milli-virusLifting.

In another preferred embodiment, the pharmaceutical formulation is for use in the treatment or prevention of an ocular disease, preferably in the treatment or prevention of Retinitis Pigmentosa (RP).

In a seventh aspect of the invention there is provided a method of treating or preventing a disease, the method comprising administering to a subject in need thereof a vector according to the third aspect of the invention.

In another preferred embodiment, the method is a method of treating or preventing an ocular disease.

In another preferred embodiment, the method is a method of reducing photoreceptor cell or optic nerve cell death in a patient suffering from or at risk of developing retinitis pigmentosa.

In another preferred embodiment, the carrier is introduced into the eye of a subject in need thereof.

In another preferred embodiment, the subject in need thereof includes humans and non-human mammals.

In another preferred embodiment, the method comprises administering the vector of the third aspect of the invention to the eye of a subject in need thereof by subretinal, direct retinal injection or intravitreal injection.

In another preferred embodiment, the method may cause photoreceptor cell or optic nerve cell death due to retinitis pigmentosa to be substantially prevented for the lifetime of the subject in need thereof.

In another preferred embodiment, the method is such that visual function is substantially restored or maintained in the treated eye.

In an eighth aspect of the present invention, there is provided a method for preparing recombinant rhodopsin, comprising the steps of: culturing the host cell of the fourth aspect of the invention, thereby obtaining recombinant rhodopsin.

In a ninth aspect of the invention there is provided a viral vector production system comprising a set of polynucleotides encoding components required for the production of said viral vector, wherein the viral vector genome comprises a nucleotide sequence according to the first aspect of the invention.

In a tenth aspect of the present invention, there is provided a DNA construct for use in the viral vector production system according to the ninth aspect of the present invention, comprising the nucleotide sequence according to the first aspect of the present invention.

In an eleventh aspect of the invention there is provided a viral vector producing cell comprising a nucleotide sequence according to the first aspect of the invention, or a viral vector production system according to the ninth aspect of the invention, or a DNA construct according to the tenth aspect of the invention.

In a twelfth aspect of the invention there is provided a method of producing a viral vector comprising introducing a nucleotide sequence according to the first aspect of the invention into a cell and culturing the cell under conditions suitable for production of the viral vector.

In another preferred embodiment, the cell is a HEK293 or HEK293T cell.

In a thirteenth aspect of the invention there is provided a vector comprising a nucleic acid molecule encoding a RHO protein.

In another preferred embodiment, the RHO protein has the amino acid sequence set forth in SEQ ID NO. 3.

In another preferred embodiment, the nucleic acid molecule encoding a RHO protein has a nucleotide sequence as set forth in SEQ ID No. 1, 2, 6 or 7.

In another preferred embodiment, the vector is an adeno-associated viral vector, preferably an adeno-associated viral vector AAV2/8 or AAV2/9.

In a fourteenth aspect of the invention there is provided the use of a vector according to the thirteenth aspect of the invention for the preparation of a formulation or composition for restoring vision in a subject and/or for treating or preventing an ocular disorder.

In another preferred embodiment, the ocular disease comprises retinitis pigmentosa.

In a fifteenth aspect of the present invention there is provided a pharmaceutical formulation comprising (a) a carrier according to the thirteenth aspect of the present invention, and (b) a pharmaceutically acceptable carrier or excipient.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the results of an alignment of the optimized RHO nucleotide sequence with the original human rhodopsin gene sequence. The optimized RHO nucleotide sequence had a homology of 88.31% (922/1044) to the original rhodopsin gene open reading frame sequence. Wherein the upstream sequence is an optimized open reading frame nucleotide sequence and the downstream sequence is a protorhodopsin gene sequence (wild sequence).

FIG. 2 shows the cloning results of the PCR nucleic acid electrophoresis verification of the wild RHO gene (lane 1) and the optimized RHO gene (lane 2).

FIG. 3 shows the structure of the recombinant plasmid pSNaV/rAAV-optimized hRHO.

FIG. 4 shows a protein electrophoresis pattern of rAAV-RHO virus. Lane 1, protein marker; lane 2: rAAV-optimized hRHO.

Fig. 5 shows fundus photographing results of different groups of rabbit eyes.

FIG. 6 shows bar graphs of RHO immunofluorescence measurements of different groups of rabbit eyes.

Fig. 7 shows OCT detection results for different groups of rabbit eyes. Fig. 7A shows OCT results of group a, fig. 7B shows OCT results of group B, fig. 7C shows OCT results of group M, and fig. 7D shows OCT results of group O.

FIG. 8 shows the mRNA detection results of RHO gene of different groups of rabbit eyes.

FIG. 9 shows RHO protein detection results for different groups of rabbit eyes.

Figure 10 shows the weight change of mice of different groups.

FIG. 11 shows the relative expression levels of mRNA of the RHO gene in muscle of mice of different groups.

Detailed Description

Through extensive and intensive research, the inventor performs targeted optimization design on Rhodopsin (RHO) gene coding sequences, so that a nucleotide sequence which is particularly suitable for high-efficiency transcription and high-efficiency expression of human RHO protein in mammalian (such as human) cells is obtained, and a recombinant expression vector of rhodopsin is constructed. Experimental results show that compared with an unoptimized coding sequence, the expression quantity of the RHO coding sequence (SEQ ID NO: 1) after special optimization is obviously improved. Furthermore, the applicant has unexpectedly found that for the specific optimized RHO coding sequences of the present application AAV2/8 and AAV2/9 are better able to express RHO proteins as vectors and more effective in treating RP than AAV vectors of other serotypes. On this basis, the inventors completed the present invention.

Terminology

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

The term "about" may refer to a value or composition that is within an acceptable error of 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 measured. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of a reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a well known method to those skilled in the art.

As used herein, the terms "subject," "subject in need thereof" refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cows, horses, dogs, cats, pigs, sheep, goats.

Adeno-associated virus

Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus dependovirus of the family picoviridae, and is the simplest class of structurally single-stranded DNA-deficient viruses currently found, requiring helper virus (typically adenovirus) to participate in replication. It encodes cap and rep genes in inverted repeats (ITRs) at both ends. ITRs are decisive for viral replication and packaging. The cap gene encodes viral capsid proteins and the rep gene is involved in viral replication and integration. AAV can infect a variety of cells.

Recombinant adeno-associated virus (rAAV) is derived from non-pathogenic wild adeno-associated virus, and is regarded as one of the most promising gene transfer vectors due to the characteristics of good safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, long time for expressing exogenous genes in vivo, etc., and is widely applied to gene therapy and vaccine research worldwide. Through more than 10 years of research, the biological properties of recombinant adeno-associated viruses have been well understood, and in particular, many data have been accumulated on their utility in various cell, tissue and in vivo experiments. In medical research, rAAV is used in research (including in vivo, in vitro experiments) for gene therapy of various diseases; meanwhile, the gene transfer vector is used as a characteristic gene transfer vector and is also widely used in aspects of gene function research, disease model construction, gene knockout mouse preparation and the like.

In a preferred embodiment of the invention, the vector is a recombinant AAV vector. AAV is a relatively small DNA virus that can integrate 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 appear to be involved in human pathology. AAV genomes have been cloned, sequenced and characterized. AAV contains approximately 4700 bases and contains an Inverted Terminal Repeat (ITR) region of about 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two important regions with encapsidation functions: the left part of the genome comprising the rep gene involved in viral replication and viral gene expression; and the right part of the genome comprising the cap gene encoding the viral capsid protein.

AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying the vectors can be found, for example, in U.S. Pat. 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 genes has been described (see, e.g., international patent application publication Nos. WO91/18088 and WO93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535 and 5,139,941, and European patent No.0488528, each of which is incorporated herein by reference in its entirety). These patent publications describe various AAV-derived constructs in which rep and/or cap genes are deleted and replaced by genes of interest, and the use of these constructs to transport genes of interest in vitro (into cultured cells) or in vivo (directly into organisms). Replication-defective recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (e.g., adenovirus): plasmids containing the nucleic acid sequence of interest flanked by two AAV Inverted Terminal Repeat (ITR) regions, and plasmids carrying AAV encapsidation genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.

In some embodiments, the recombinant vector is encapsidated into a virion (e.g., an AAV virion including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV 16). Thus, the present disclosure includes recombinant viral particles (recombinant as they comprise recombinant polynucleotides) comprising any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. patent No.6,596,535. The capsid proteins of AAV of different serotypes recognize different cell surface receptors, so that the infection efficiency of different tissue cells is greatly different, and certain organ targeting specificity is shown. AAV of different serotypes may also have different results in terms of expression efficiency for different protein coding sequences of interest.

Retinitis Pigmentosa (RP)

Retinitis pigmentosa (retinitis pigmentosa, RP) is the most common group of hereditary blinding fundus diseases in which retinal photoreceptor and pigment epithelial cell degeneration leads to progressive visual field defects. Patients often present with night blindness, progressive visual field reduction and vision loss, eventually leading to reduced or even complete loss of vision function due to apoptosis of the retinal photoreceptor cells. RP has high genetic heterogeneity and clinical heterogeneity, and has a plurality of genetic modes such as emission, autosomal dominant, autosomal recessive, X-linked inheritance, double-gene inheritance and the like. At present, the incidence rate of the disease is 1/3500, and at least 100 tens of thousands of people suffer from the disease worldwide, wherein the incidence rate of the disease is about 1/4 of the incidence rate of Chinese people, and the incidence rate of the disease increases year by year. 40% -50% of patients are sporadic or single shot with no family history of disease, known as sporadic retinitis pigmentosa (sporadic retinitis pigmentosa, SRP). X-linked inheritance accounts for about 6-20% of all RP patients. Autosomal recessive inheritance accounts for 5% -15%. Autosomal dominant inherited retinitis pigmentosa (autosomal dominant retinitis pigmentosa, ADRP) accounts for 15% -25%. Rhodopsin (RHO) gene mutations account for 16% -25% of autosomal dominant inherited retinal pigment degeneration (ADRP) in europe and north america, around 7% in china and japan. With the development of molecular genetics, the discussion of the molecular pathogenesis of the disease has been advanced in breakthrough. Rhodopsin (RHO) gene, which encodes a protein that is exclusively expressed in rod photoreceptor cells and consistent with rod degeneration occurring early in RP onset, becomes the first candidate for RP molecular defect studies.

RHO

As used herein, the terms "rhodopsin", "RHO protein", "polypeptide", "human RHO protein" and "hRHO protein" have the same meaning and are used interchangeably herein.

Since Dryja et al first discovered that there are RHO gene mutations in 1990, more than 100 pathogenic mutations have been reported so far, most of which are unintentional or misintended mutations. More than 90% are single base point mutations, a few are microdeletions, nonsense mutations, and insertion mutations. The RHO gene is located on chromosome 3q21-q24, contains 4 introns and 5 exons, codes RHO protein containing 348 amino acids, is a photosensitive protein playing an important role in the process of converting optical signals into visual signals in human eyes, and is mainly distributed on membrane discs of outer segments of visual rods, and is a main substance for producing scotopic vision of visual rod cells and is also a main component part of the membrane discs. The RHO protein with photosensitivity is one member of a G protein coupled receptor family, and consists of two parts, namely, opsin and 11-cis-retinal, wherein the secondary structure of the opsin contains 7 transmembrane alpha helices and consists of 348 amino acids, the 11-cis-retinal of the RHO is positioned on lysine of a seventh alpha helix of the opsin, the N end of the 11-cis-retinal is positioned outside a membrane disc, the C end of the 11-cis-retinal is positioned in the membrane disc, and after the 11-cis-retinal is irradiated by light, the 11-cis-retinal is isomerized into all-trans-retinal, and the structure of the protein is rapidly changed to enable the rhodopsin to be in an excited state, so that photosensitive transduction cascade reaction is caused. With all-trans retinal separated from the opsin, RHO becomes a drag-assistant opsin without photosensitivity, which can be restored when it is combined with 11-cis retinal again. Mutation of the base of the RHO gene may cause amino acid substitution, termination, or deletion of the RHO protein to cause abnormal functioning of the RHO protein, thereby causing visual dysfunction.

Mutation of one allele of the RHO gene results in abnormal protein structure, i.e. abnormal opsin production which leads to altered glycosylation; the protein is retained in the endoplasmic reticulum of the rod cells and cannot be transported to the outer disc membrane of the rod cells, or the variant opsin cannot be folded due to abnormal structure, cannot be integrated into the disc membrane or causes unstable disc membrane structure to cause degeneration of the rod cells, resulting in RP. There is currently no FDA or european drug administration approved treatment for the treatment of such autosomal dominant inherited retinal pigment degeneration. In recent years, gene therapy RP has been greatly advanced. A variety of animal models of RP have been established, including retinal degenerated (retinal degeneration, rd) mice, chronic retinal degenerated (retinal degeneration slow, rds) mice, and the like, which help in the pathogenesis and therapeutic study of RP. The protein coded by the gene related to RP is mainly related to light level enzyme linked reaction or rhodopsin light metabolism, photoreceptor structural protein and photoreceptor cell transcription factor, and the research shows that the pathological change of RP takes apoptosis as a common path. Gene therapy is to assemble therapeutic genes into a certain vector to guide genes defective in human cells so as to prevent the development of diseases. The gene vector comprises a viral vector and a non-viral vector, wherein the viral vector mainly comprises an adeno-associated viral vector, a lentiviral vector, an adenovirus vector, a recombinant adeno-associated viral vector and the like. The AAV vector is selected as a viral vector, and the AAV is not integrated into a human genome, but is used as a circulating vector, so that the risk of causing other genetic diseases is reduced, only a small immune response is caused, and long-term transgene expression in various retina cells can be realized.

Nucleic acid coding sequences

The invention aims to overcome the technical defects of low RHO expression efficiency and poor treatment effect in the prior art. The present invention provides an optimized RHO gene sequence. The optimized RHO coding sequence is shown as SEQ ID NO:1, the size of which is 1044bp. The research shows that the optimized RHO gene sequence (SEQ ID NO: 1) of the invention has higher RHO protein expression efficiency and more RHO proteins play a physiological role in the optic nerve cells or photoreceptor cells of patients.

The nucleotide sequence of the nucleic acid for encoding rhodopsin is shown as SEQ ID NO. 1. In another preferred embodiment, the nucleotide sequence has an identity of 95% or more, preferably 98% or more, more preferably 99% or more to the nucleotide sequence set forth in SEQ ID No. 1. In the present invention, the optimized rhodopsin-encoding nucleic acid is also referred to as a RHO-optimized coding sequence, a RHO-optimized gene, or a RHO-optimized nucleic acid or an optimized hhho.

The polynucleotides of the invention may be in the form of DNA or RNA. In another preferred embodiment, the nucleotide is DNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The nucleotide sequence disclosed by the invention encodes an amino acid sequence shown as SEQ ID NO. 3.

The amino acid sequence of the RHO protein is shown as SEQ ID NO. 3.

MNGTEGPNFYVPFSNATGVVRSPFEYPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVLGGFTSTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLAGWSRYIPEGLQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIIIFFCYGQLVFTVKEAAAQQQESATTQKAEKEVTRMVIIMVIAFLICWVPYASVAFYIFTHQGSNFGPIFMTIPAFFAKSAAIYNPVIYIMMNKQFRNCMLTTICCGKNPLGDDEASATVSKTETSQVAPA(SEQ ID NO.:3)

The nucleic acid sequence may be DNA, RNA, cDNA or PNA. The nucleic acid sequence may be genomic, recombinant or synthetic. The nucleic acid sequence may be isolated or purified. The nucleic acid sequence may be single-stranded or double-stranded. Preferably, the nucleic acid sequence will encode a RHO protein as described herein. The nucleic acid sequences may be derived by cloning, for example using standard molecular cloning techniques including restriction, ligation, gel electrophoresis, as described, for example, in Sambrook et al Molecular Cloning: A laboratory manual, cold Spring Harbour Laboratory Press). The nucleic acid sequence may be isolated, for example, using PCR techniques. Isolation means isolating a nucleic acid sequence from any impurities and from other nucleic acid sequences and/or proteins that are naturally found associated with the nucleic acid sequence in their source. Preferably, it will also be free of cellular material, culture medium or other chemicals from the purification/production process. The nucleic acid sequence may be synthetic, for example produced by direct chemical synthesis. The nucleic acid sequence may be provided as naked nucleic acid, or may be provided complexed with a protein or lipid.

The full-length nucleotide sequence or a fragment thereof of the present invention can be usually 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 the disclosed nucleotide sequences, particularly open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order. At present, it is entirely possible to obtain DNA sequences encoding the polypeptides of the invention (or fragments or derivatives thereof) by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered with the vectors or polypeptide coding sequences of the invention. The polynucleotide, vector or host cell described above may be isolated.

As used herein, "isolated" refers to a substance that is separated from its original environment (i.e., the natural environment if it is a natural substance). If the polynucleotides and polypeptides in the native state in living cells are not isolated or purified, the same polynucleotides or polypeptides are isolated or purified if they are separated from other substances that are present in the native state.

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

Once the relevant sequences are obtained, recombinant methods 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 isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining the genes of the present invention. Primers for PCR can be appropriately selected according to 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 invention also relates to vectors comprising the polynucleotides of the invention, host cells genetically engineered with the vectors or protein coding sequences of the invention, and methods of expressing RHO proteins using the host cells via recombinant techniques.

Host cells (e.g., mammalian cells) expressing the RHO proteins of the invention can be obtained by conventional recombinant DNA techniques using the polynucleotide sequences of the invention. Generally comprising the steps of: transduction of a polynucleotide according to the first aspect of the invention or a vector according to the third aspect of the invention into a host cell.

Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequences for the polypeptides of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the polypeptides.

The host cell may be a prokaryotic cell, or a lower eukaryotic cell, or a higher eukaryotic cell, such as a mammalian cell (including human and non-human mammals). Representative examples are: CHO, NS0, COS7, or 293 cells. In a preferred embodiment of the invention HEK cells, photoreceptor cells (including cone cells and/or rod cells), other vision cells (e.g. binodal cells), neural cells are selected as host cells. In another preferred embodiment, the host cell is selected from the group consisting of: rod cells, cone cells, light-donating bipolar cells, light-withdrawing bipolar cells, horizontal cells, ganglion cells, non-long process cells, or combinations thereof.

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, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformant obtained can be cultured by a conventional method to express the protein encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

Sequence optimization

In the present invention, a specially optimized coding sequence for rhodopsin with significantly improved expression efficiency (including transcription efficiency and/or translation efficiency) is provided, as shown in SEQ ID NO. 1.

As used herein, the terms "optimized RHO coding sequence", "optimized RHO coding gene", "hRHO optimizing gene", "optimized hhho gene", "optimized RHO nucleic acid" are used interchangeably and refer to nucleotide sequences encoding rhodopsin after specific optimization of the present invention, which nucleotide sequences encode the amino acid sequence shown in SEQ ID No.: 3. The optimized RHO coding sequences of this invention are particularly suitable for expression in mammalian cells.

In the invention, the wild DNA coding sequence (non-optimized DNA coding sequence) of the RHO is shown as SEQ ID NO. 2, and the expression level of the non-optimized wild DNA coding sequence is very low. The specific nucleic acid sequence of the RHO wild coding sequence is shown as SEQ ID NO. 2.

ATGAATGGCACAGAAGGCCCTAACTTCTACGTGCCCTTCTCCAATGCGACGGGTGTGGTACGCAGCCCCTTCGAGTACCCACAGTACTACCTGGCTGAGCCATGGCAGTTCTCCATGCTGGCCGCCTACATGTTTCTGCTGATCGTGCTGGGCTTCCCCATCAACTTCCTCACGCTCTACGTCACCGTCCAGCACAAGAAGCTGCGCACGCCTCTCAACTACATCCTGCTCAACCTAGCCGTGGCTGACCTCTTCATGGTCCTAGGTGGCTTCACCAGCACCCTCTACACCTCTCTGCATGGATACTTCGTCTTCGGGCCCACAGGATGCAATTTGGAGGGCTTCTTTGCCACCCTGGGCGGTGAAATTGCCCTGTGGTCCTTGGTGGTCCTGGCCATCGAGCGGTACGTGGTGGTGTGTAAGCCCATGAGCAACTTCCGCTTCGGGGAGAACCATGCCATCATGGGCGTTGCCTTCACCTGGGTCATGGCGCTGGCCTGCGCCGCACCCCCACTCGCCGGCTGGTCCAGGTACATCCCCGAGGGCCTGCAGTGCTCGTGTGGAATCGACTACTACACGCTCAAGCCGGAGGTCAACAACGAGTCTTTTGTCATCTACATGTTCGTGGTCCACTTCACCATCCCCATGATTATCATCTTTTTCTGCTATGGGCAGCTCGTCTTCACCGTCAAGGAGGCCGCTGCCCAGCAGCAGGAGTCAGCCACCACACAGAAGGCAGAGAAGGAGGTCACCCGCATGGTCATCATCATGGTCATCGCTTTCCTGATCTGCTGGGTGCCCTACGCCAGCGTGGCATTCTACATCTTCACCCACCAGGGCTCCAACTTCGGTCCCATCTTCATGACCATCCCAGCGTTCTTTGCCAAGAGCGCCGCCATCTACAACCCTGTCATCTATATCATGATGAACAAGCAGTTCCGGAACTGCATGCTCACCACCATCTGCTGCGGCAAGAACCCACTGGGTGACGATGAGGCCTCTGCTACCGTGTCCAAGACGGAGACGAGCCAGGTGGCCCCGGCC(SEQ ID NO.:2)

The present invention optimizes sequence fragments that affect gene expression, including, but not limited to, codon usage bias, elimination of secondary structures that are detrimental to expression (e.g., hairpin structures), changes in GC content, cpG dinucleotide content, secondary structures of mRNA, cryptic splice sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA instability regions, repetitive sequences (direct repeat, inverted repeat, etc.), and restriction sites that may affect cloning. A number of optimized RHO coding sequences were finally obtained, including the nucleotide sequences shown as SEQ ID NO. 1, 6, 7, etc.

Wherein the similarity of the wild RHO sequence to the RHO optimized sequence shown in SEQ ID NO. 6 is 77.97% (814/1044).

ATGAATGGTACTGAAGGCCCTAACTTTTATGTTCCCTTCTCTAATGCTACCGGAGTCGTACGTTCCCCATTTGAGTACCCGCAATATTACTTAGCCGAACCTTGGCAGTTCTCAATGTTGGCAGCGTATATGTTTCTTCTCATTGTGCTAGGGTTCCCCATCAACTTTCTGACATTATACGTTACGGTCCAACATAAAAAGTTGCGCACTCCACTTAATTATATACTCCTAAACCTGGCTGTAGCCGATTTATTCATGGTGTTGGGTGGCTTTACCTCGACACTTTACACGAGTCTCCACGGATATTTCGTTTTTGGGCCGACTGGTTGTAATCTAGAGGGCTTCTTTGCAACCCTGGGAGGGGAAATTGCGTTATGGAGCTTGGTCGTACTTGCTATCGAGCGATACGTGGTTGTCTGCAAACCTATGTCTAACTTCCGGTTTGGTGAAAATCATGCCATAATGGGCGTAGCATTCACATGGGTGATGGCGCTCGCTTGTGCCGCACCCCCACTAGCGGGATGGTCCAGATATATTCCGGAGGGGCTGCAGTGCTCATGTGGTATCGACTACTATACGTTAAAGCCTGAAGTTAACAATGAGTCGTTTGTCATATACATGTTCGTAGTGCACTTTACTATTCCCATGATCATAATTTTCTTTTGCTATGGCCAATTGGTTTTCACCGTCAAAGAAGCTGCCGCACAGCAACAGGAGAGTGCGACAACGCAAAAGGCTGAAAAAGAGGTAACTAGGATGGTGATCATAATGGTTATTGCCTTTCTTATCTGTTGGGTCCCATACGCAAGCGTAGCGTTCTATATATTTACCCATCAGGGATCTAACTTCGGGCCGATTTTTATGACAATCCCTGCTTTCTTTGCCAAGTCCGCAGCGATATACAATCCCGTGATTTATATCATGATGAACAAACAATTCCGTAATTGCATGCTCACGACTATATGTTGCGGTAAGAACCCACTAGGCGATGACGAAGCTTCAGCCACCGTTTCGAAAACAGAGACGAGTCAGGTCGCACCGGCG(SEQ ID NO.:6)

Wherein the wild RHO sequence has 81.42% (850/1044) similarity to the RHO optimized sequence shown as SEQ ID NO. 7.

ATGAACGGCACCGAGGGCCCCAACTTCTACGTGCCCTTCAGCAACGCCACCGGCGTGGTGCGCAGCCCCTTCGAGTACCCCCAGTACTACCTGGCCGAGCCCTGGCAGTTCAGCATGCTGGCCGCCTACATGTTCCTGCTGATCGTGCTGGGCTTCCCCATCAACTTCCTGACCCTGTACGTGACCGTGCAGCACAAGAAGCTGCGCACCCCCCTGAACTACATCCTGCTGAACCTGGCCGTGGCCGACCTGTTCATGGTGCTGGGCGGCTTCACCAGCACCCTGTACACCAGCCTGCACGGCTACTTCGTTTTTGGGCCGACTGGTTGTAATCTAGAGGGCTTCTTTGCAACCCTGGGAGGGGAAATTGCGTTATGGAGCTTGGTCGTACTTGCTATCGAGCGATACGTGGTTGTCTGCAAACCTATGTCTAACTTCCGGTTTGGTGAAAATCATGCCATAATGGGCGTAGCATTCACATGGGTGATGGCGCTCGCTTGTGCCGCACCCCCACTAGCGGGATGGTCCAGATATATTCCGGAGGGGCTGCAGTGCTCATGTGGTATCGACTACTATACGTTAAAGCCTGAAGTTAACAATGAGTCGTTTGTCATATACATGTTCGTAGTGCACTTTACTATTCCCATGATCATAATTTTCTTTTGCTATGGCCAATTGGTTTTCACCGTCAAAGAAGCTGCCGCACAGCAACAGGAGAGTGCGACAACGCAAAAGGCTGAAAAAGAGGTAACTAGGATGGTGATCATAATGGTTATTGCCTTTCTTATCTGTTGGGTCCCATACGCAAGCGTAGCGTTCTATATATTTACCCATCAGGGATCTAACTTCGGGCCGATTTTTATGACAATCCCTGCTTTCTTTGCCAAGTCCGCAGCGATATACAATCCCGTGATTTATATCATGATGAACAAACAATTCCGTAATTGCATGCTCACGACTATATGTTGCGGTAAGAACCCACTAGGCGATGACGAAGCTTCAGCCACCGTTTCGAAAACAGAGACGAGTCAGGTCGCACCGGCG(SEQ ID NO.:7)

Through a large number of analyses and experimental screening, a particularly optimized DNA coding sequence shown as SEQ ID NO. 1, with the size of 1044bp, starting from the codon ATG and consisting of 5 exons, codes for 348 amino acids, is finally obtained, and is a G-protein coupled receptor which plays a role in the visual photoelectric conversion process. The sequence is specially optimized, so that the transcription level and the translation level are improved, and the RHO expression quantity is obviously improved. The similarity of the coding sequence shown in SEQ ID No. 1 with the wild-type coding sequence shown in SEQ ID No. 2 is 88.31% (922/1044).

ATGAACGGCACCGAGGGCCCCAACTTCTACGTGCCCTTCAGCAACGCCACCGGCGTGGTGCGCAGCCCCTTCGAGTACCCCCAGTACTACCTGGCCGAGCCCTGGCAGTTCAGCATGCTGGCCGCCTACATGTTCCTGCTGATCGTGCTGGGCTTCCCCATCAACTTCCTGACCCTGTACGTGACCGTGCAGCACAAGAAGCTGCGCACCCCCCTGAACTACATCCTGCTGAACCTGGCCGTGGCCGACCTGTTCATGGTGCTGGGCGGCTTCACCAGCACCCTGTACACCAGCCTGCACGGCTACTTCGTGTTCGGCCCCACCGGCTGCAACCTGGAGGGCTTCTTCGCCACCCTGGGCGGCGAGATCGCCCTGTGGAGCCTGGTGGTGCTGGCCATCGAGCGCTACGTGGTGGTGTGCAAGCCCATGAGCAACTTCCGCTTCGGCGAGAACCACGCCATCATGGGCGTGGCCTTCACCTGGGTGATGGCCCTGGCCTGCGCCGCCCCCCCCCTGGCCGGCTGGAGCCGCTACATCCCCGAGGGCCTGCAGTGCAGCTGCGGCATCGACTACTACACCCTGAAGCCCGAGGTGAACAACGAGAGCTTCGTGATCTACATGTTCGTGGTGCACTTCACCATCCCCATGATCATCATCTTCTTCTGCTACGGCCAGCTGGTGTTCACCGTGAAGGAGGCCGCCGCCCAGCAGCAGGAGAGCGCCACCACCCAGAAGGCCGAGAAGGAGGTGACCCGCATGGTGATCATCATGGTGATCGCCTTCCTGATCTGCTGGGTGCCCTACGCCAGCGTGGCCTTCTACATCTTCACCCACCAGGGCAGCAACTTCGGCCCCATCTTCATGACCATCCCCGCCTTCTTCGCCAAGAGCGCCGCCATCTACAACCCCGTGATCTACATCATGATGAACAAGCAGTTCCGCAACTGCATGCTGACCACCATCTGCTGCGGCAAGAACCCCCTGGGCGACGACGAGGCCAGCGCCACCGTGAGCAAGACCGAGACCAGCCAGGTGGCCCCCGCC(SEQ ID NO.:1)

Expression vectors and host cells

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

By providing sequence information, the skilled artisan can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transduction into cells.

Preferably, the nucleic acid sequence encoding the RHO protein is provided as a vector, preferably an expression vector. Preferably, it may be provided as a gene therapy vector preferably suitable for transduction and expression in retinal target cells. The vector may be viral or non-viral (e.g., a plasmid). Viral vectors include those derived from adenovirus, adeno-associated virus (AAV), including mutated forms, retrovirus, lentivirus, herpes virus, vaccinia virus, MMLV, gaLV, simian Immunodeficiency Virus (SIV), HIV, poxvirus, and SV40. Preferably, the viral vector is replication defective (replication defective), although it is contemplated that it may be replication deficient (replication deficient), replication-competent or conditionally replication-competent. Viral vectors can generally remain extrachromosomal without integrating into the genome of the target retinal cell. A preferred viral vector for introducing a nucleic acid sequence encoding RHO protein into a retinal target cell is an AAV vector, such as a self-complementing adeno-associated virus (scAAV). Selective targeting can be achieved using specific AAV serotypes (AAV serotype 2 through AAV serotype 12) or modified versions of any of these serotypes, including AAV 4YF and AAV 7m8 vectors.

The viral vector may be modified to delete any non-essential sequences. For example, in AAV, the virus may be modified to delete all or part of the IX gene, ela and/or Elb gene. Replication is very inefficient for wild-type AAV in the absence of helper viruses such as adenovirus. For recombinant adeno-associated viruses, preferably, the replication gene and capsid gene are provided in trans (in the pRep/Cap plasmid) and only the 2ITR of the AAV genome is retained and packaged into the virion, while the desired adenovirus gene is provided by the 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 may be complexed with agents to facilitate uptake of the viral vector by the target cell. Such agents include polycationic agents. Alternatively, a delivery system such as a liposome-based delivery system may 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 comprise one or more regulatory sequences to direct expression of the nucleic acid sequence in the retinal target cells. 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., bacterial cells) or in a retinal target cell.

"operably linked" means that the nucleic acid sequences are functionally related to the sequences to which they are operably linked such that they are linked in such a way that they affect the expression or function of each other. 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. Promoters may be constitutive or may be inducible. Promoters may direct ubiquitous expression in inner retinal cells, or neuronal specific expression. In the latter case, the promoter may direct cell type specific expression, for example, to 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, calbindin, human CMV, GAD-67, chicken actin, hSyn, grm6 enhancer SV40 fusion proteins. Targeting can be achieved using cell-specific promoters, for example, 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 encoding the metabotropic glutamate receptor mGluR6 specific for optic nerve cells. Preferred sources of the Grm6 gene are mice and humans. Ubiquitous expression can be achieved using promoters of pan-neurons, examples of which are known and available in the art. One such example is CAG. CAG promoter is a fusion of CMV early enhancer and chicken actin promoter.

One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the ebustan-balr (Epstein-Barr) virus immediate early promoter, the ruses 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 invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or switching off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

Many expression vectors can be used for expression of the RHO protein in mammalian cells (preferably human, more preferably human optic nerve cells or photoreceptor cells). The present invention preferably uses adeno-associated virus as an expression vector, preferably AAV2/8 or AAV2/9 vectors.

The invention also provides a host cell for expressing the RHO protein. Preferably, the host cell is a mammalian cell (preferably a human, more preferably an optical nerve cell or a photoreceptor cell) that increases the expression level of the RHO protein.

Formulations and compositions

The present 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.

In another preferred embodiment, the pharmaceutical formulation is for use in the treatment of an ocular disease.

In another preferred embodiment, the pharmaceutical formulation is for use in the treatment of retinitis pigmentosa.

The "active ingredient" in the pharmaceutical composition of the present invention refers to the vector (vector) of the present invention, for example, a viral vector (including adeno-associated viral vectors). The "active ingredients", formulations and/or compositions described herein may be used to treat ocular disorders. "safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition or symptom without causing serious side effects. "pharmaceutically acceptable carrier or excipient" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatibility" as used herein means that the components of the composition are capable of blending with and between the active ingredients of the present invention without significantly reducing the efficacy of the active ingredients.

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

Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, and the like), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, and the like), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, and the like), emulsifiers (e.g.

) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.

The compositions may comprise a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

The nucleic acid for encoding RHO provided by the invention can be used for producing RHO protein in vitro or in vivo and can be applied to preparing medicines for treating retinal pigment degeneration.

The optimized nucleic acid encoding rhodopsin is expressed more efficiently, thereby translating more RHO protein. Compared with the prior art, the optimized RHO nucleic acid codes and expresses more RHO proteins, has higher transfection efficiency, and can better treat the retinitis pigmentosa. The optic nerve cells are infected, and RHO protein is expressed in the nerve cells to play a role.

Therapeutic method

The present invention provides a method of providing photoreceptor or optic nerve cell function to a cell, the method comprising introducing into the eye a vector comprising an optimized sequence encoding RHO. The method can comprise administering the nucleic acid vector subretinally or intravitreally to an inner retinal cell of the eye.

The present invention provides a nucleic acid vector for use in a method of treating retinal degeneration by providing photoreceptor or optic nerve cell function to a cell, the nucleic acid vector comprising an optimized sequence encoding RHO. The compositions of the invention may be administered alone or in combination (e.g., formulated in the same pharmaceutical composition) with other therapeutic agents.

The invention also provides a method of expanding photoreceptor or optic nerve cell function in the retina, particularly after degeneration of the rods and/or cones, comprising introducing into the vitreous cavity of the eye a nucleic acid vector comprising an optimized sequence encoding RHO. The method may comprise administering the nucleic acid vector to an inner retinal cell, subretinal or intravitreally of the eye. The present invention provides nucleic acid vectors for use in treating retinal degeneration by enlarging photoreceptor or optic nerve cell function in the retina, the nucleic acid vectors comprising an optimized sequence encoding RHO.

The invention also provides a method of restoring vision to a subject, the method comprising introducing into the eye a vector comprising an optimized sequence encoding RHO. The method may comprise subretinal or intravitreal administration of the nucleic acid vector to inner retinal cells of the eye. The present invention provides a nucleic acid vector for use in restoring vision to a subject, the nucleic acid vector comprising an optimized sequence encoding RHO.

The invention also provides a method of treating a retinal disease in a subject, the method comprising introducing into the eye a vector comprising an optimized sequence encoding RHO. The method may comprise subretinal or intravitreal administration of the nucleic acid vector to inner retinal cells of the eye. The disease may be retinal dystrophy, including rod dystrophy, rod cone dystrophy, cone rod dystrophy, cone dystrophy, and macular dystrophy; other forms of retinal or macular degeneration, ischemic conditions, retinal pigment degeneration, leber congenital amaurosis, uveitis, and any other disease caused by a loss of photoreceptor or optic nerve cell capacity.

As used herein, providing a cell with photoreceptor or optic nerve cell function means that a cell that has not previously had photoreceptor or optic nerve cell capacity or whose photoreceptor or optic nerve cell capacity has been completely or partially degenerated, becomes sensitized upon expression therein of a foreign nucleic acid sequence encoding RHO. Such cells may be referred to herein as transformed cells because they contain non-native nucleic acids therein. Preferably, the transformed retinal cells exhibit some or all of the photoreceptor cell capacity of natural photoreceptor cells. Preferably, the transformed retinal cells exhibit some or all of the ganglion cell capabilities of the native ganglion cells. Preferably, the transformed cells exhibit at least the same or substantially the same photoreceptor capacity of natural retinal photoreceptor cells. Preferably, the transformed cells exhibit a higher photoreceptor capacity than the diseased or degenerating native retinal photoreceptor cells. Thus, transformed cells will preferably have increased photoreceptor cells or optic nerve cells compared to untreated degenerated or diseased cells from the same source, maintained under the same conditions. Transformed cells are distinguishable from natural cells by the presence of exogenous nucleic acid therein.

As used herein, expanding photoreceptor or optic nerve cell function means increasing photoreceptor or optic nerve cell function of the retina by increasing function in photoreceptor or optic nerve cells such as rods or cones and/or by providing photoreceptor or optic nerve cell function to cells. Thus, the retina will have an increased ability to receive light signals and transmit such signals, which may be any amount, as compared to a retina that is not treated with the methods as described herein.

As used herein, restoring vision in a subject means that the subject exhibits improved vision compared to prior to treatment, e.g., using a vision test as described herein. Recovery includes any degree of improvement, including complete recovery of vision to perfect or near perfect vision.

As used herein, treating a disease means administering a nucleic acid or vector as described herein to ameliorate or alleviate one or more symptoms of a disease selected from the group consisting of retinal dystrophy, including rod dystrophy, rod cone dystrophy, cone rod dystrophy, cone dystrophy, and macular dystrophy; another form of retinal or macular degeneration, retinitis pigmentosa, ischemic conditions, leber congenital amaurosis, uveitis, and any other disease caused by a loss of photoreceptor or optic nerve cell capacity. Improvement or alleviation may result in improvement of peripheral or central vision, and/or daytime or nighttime vision.

The method of the invention comprises introducing a nucleic acid sequence encoding a RHO protein into the vitreous cavity of an eye. Preferably, the method comprises contacting the cell with a vector (preferably a virus, more preferably an adeno-associated virus) comprising a nucleic acid sequence encoding a RHO protein. Preferably, the cells are retinal cells, preferably cone cells, rod cells, donor bipolar cells, withdrawal bipolar cells, level cells, ganglion cells and/or amacrine cells.

When the nucleic acid sequence and the one or more enzymes are provided in multiple (two or more) doses, the doses may be separated by a suitable time interval, for example 30 seconds to several hours or 1 or more days.

Each dose may comprise an effective amount of a nucleic acid sequence or viral vector. An effective dose of the nucleic acid sequence or viral vector may be 1X 10 per treatment regimen ⁹ -1×10 ¹⁶ The range of viruses.

The present invention is based on targeting optimized nucleic acid sequences encoding RHO to retinal cells to compensate for degeneration of photoreceptor cells or optic nerve cells in the retina. The cell to which the nucleic acid sequence is targeted is a cell of the retina, which is living and capable of expressing the foreign nucleic acid sequence. Retinal cells are cells of the retina, which are nerve or neuronal cells and are capable of becoming excited and transmitting electrical signals. Preferably, the target retinal cells will be able to generate an electrical signal and initiate a signaling cascade, resulting in the transmission of the signal to the optic nerve. Preferably, the target retinal cell is a cell of the inner retina. The target cells may be rods or cones, and/or may be non-photoreceptor cells (i.e., retinal cells that are not responsive to light in their native form). The target retinal cells may include one or more cell types selected from the group consisting of: rod cells, cone cells, donor bipolar cells, withdrawal bipolar cells, level cells, ganglion cells, miller cells, and/or non-long process cells.

Thus, when the target retinal cell is a retinal-targeted donor bipolar, a remover bipolar, a level cell, a ganglion cell, and/or an amacrine cell, expression of a nucleic acid encoding a RHO may be referred to as ectopic expression. Thus, the present invention includes within its scope methods for ectopic expression of a nucleic acid sequence encoding RHO in non-photoreceptor cells. Such ectopic expression has the effect of providing the cell with photoreceptor or optic nerve cell function through expression of heterologous RHO proteins therein. This serves to increase the photosensitivity of the retina where degeneration is observed.

The horizontal cells are inner retinal cells, and participate in signal processing and feedback to photoreceptor cells; bipolar cells are inner retinal cells and communicate between rods/cones and amacrine and/or ganglion cells; no long process cells are found in the inner retina and allow communication between photoreceptor cell pathways and ganglion cells; ganglion cells are the innermost retinal cells that transmit signals from the photoreceptor cells to the optic nerve.

Reference herein to a cell includes the progeny of the cell. Preferably, the modification of the cells according to the invention also takes place in the subsequent generations of the transformed host cell. The progeny cells may not be identical to the original targeted cells, but preferably will also exhibit expression of unnatural RHO.

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

1. the coding gene sequence of rhodopsin (hRHO) of the inventor is subjected to targeted special optimization, and the gene sequence is different from the prior art. The expression efficiency is significantly improved compared to the non-optimized DNA coding sequence of RHO. The expression level of the optimized sequence RHO protein is obviously improved, and the biological activity is high.

2. The rAAV2/8 and rAAV2/9 of the invention have the advantages of best expression effect of hRHO gene, strong penetrating power, high infection efficiency and capability of expressing in various tissue layers.

3. The optimized RHO coding gene (SEQ ID NO: 1) or recombinant expression vector can be used for very effectively treating eye diseases (such as retinitis pigmentosa), has good safety, and can not generate obvious inflammatory reaction or other complications.

The invention is further described below in conjunction with the specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1 sequence optimization

In this example, the inventors have optimized the coding sequence based on the amino acid sequence of the RHO protein (SEQ ID NO.: 3) and the natural coding sequence (SEQ ID NO.: 2). In particular, the present invention optimizes sequence fragments that affect gene expression, including but not limited to: codon usage bias, elimination of secondary structures that are detrimental to expression (e.g., hairpin structures), changes in GC content, cpG dinucleotide content, secondary structures of mRNA, cryptic splice sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA instability regions, repeat sequences (direct repeat, inverted repeat, etc.), and restriction sites that may affect cloning.

In this example, tens of optimized RHO coding sequences were designed and analyzed and experimentally screened. As a result, compared with the natural RHO coding sequence (SEQ ID NO: 2), the specific optimized DNA coding sequence shown as SEQ ID NO: 1 has the advantage that the RHO protein expression efficiency is remarkably improved. The RHO protein expression efficiency of the nucleotide sequences shown in SEQ ID NO. 6 and SEQ ID NO. 7 is not remarkably improved, and the improvement range is less than 10%.

EXAMPLE 2 construction of recombinant adeno-associated Virus vector of recombinant human rhodopsin Gene and purification of Virus packaging thereof

1. Construction of recombinant adeno-associated virus vector containing human rhodopsin gene

1) Vector construction

The natural coding sequence (SEQ ID NO: 2) and the optimized human rhodopsin gene of example 1 (SEQ ID NO: 1) were digested with two cleavage sites of KpnI and SalI or the product amplified by PCR with the new gene design primer was digested with pSNaV plasmid vector, the digested product was recovered, T4DNA Ligase was ligated overnight, and competent cells were transformed with the ligation product to obtain recombinant pSNaV/-wild hRHO and pSNaV/-optimized hRHO.

2) Screening and identification of recombinants

LB plates were incubated at 37℃and blue and white spots appeared, white being a recombinant clone. White colonies were picked and added to LB liquid medium containing 100mg/L of Amp, and cultured at 37℃for 8 hours at 200 rpm. After culturing, the bacterial liquid is taken, plasmids are extracted, and the plasmid extraction step is referred to the Biomiga instruction. Taking 1uL plasmid as a template, wherein the specific primers are as follows:

1F:5’-ACTTCTACGTGCCCTTCTCCAATG-3’(SEQ ID NO.:4)；

1R:5’-GTCTTGGACACGGTAGCAGAGGC-3’(SEQ ID NO.:5)；

PCR amplification procedure

The PCR products were electrophoretically detected (FIG. 2) to obtain a target band of about 1000bp in size.

3) Bacterial liquid preservation and PCR amplification and fragment sequencing thereof

And (3) sucking 1mL of the identified bacterial liquid and sterilized glycerol in a ratio of 1:3, uniformly mixing, preserving at-80 ℃, sequencing the bacterial liquid, comparing and analyzing the sequence obtained by sequencing with the optimized human rhodopsin gene, and successfully obtaining the recombinant adeno-associated virus plasmid skeleton pSNaV/rAAV-optimized hRHO with correct sequence (figure 3).

2. rAAV-hRHO recombinant adeno-associated virus coating

1) The day before transfection, 293T cells were seeded in 225cm2 cell culture flasks at a density of 3.0X107 cells/mL in DMEM+10% bovine serum and incubated overnight at 37℃in an incubator containing 5% CO 2.

2) The day of transfection was changed and culture was continued with fresh DMEM medium containing 10% bovine serum. When the cells grow to 80-90%, the medium is discarded and transfection is performed with a plasmid II (VGTC) transfection kit. The method comprises the following specific steps:

(a) Mixing pAdHelper, pAAV-r2c5 and pSNaV-hRHO plasmids with DMEM+PlasmidTrans II (VGTC) (transfection reagent) in a 1.5mL sterile Ep tube according to a required proportion, and standing at room temperature for 10-15 min, wherein the number of the transfection reagent is A;

(b) Uniformly mixing the reagent A with 30mL of DMEM+10% bovine serum, and numbering the reagent B;

(c) Uniformly adding the reagent B into a cell culture flask, and continuously culturing in an incubator containing 5% CO2 at 37 ℃;

(d) 16h after transfection, the complete medium (DMEM+10% bovine serum) was changed.

3) Cells were harvested 48h after transfection.

4) The harvested cells were resuspended in PBS and freeze-thawed 3 times repeatedly.

3. Purification and concentration of rAAV-hRHO virus

The rAAV-hRHO virus is separated, concentrated and purified by three steps of chloroform treatment, PEG/NaCl precipitation and chloroform extraction. Total recovery = number of virions of end product/number of virions of starting material.

4. Virus purity and titer validation

SDS-PAGE separating gel and laminating gel are packed, and the concentration of the separating gel is 10%. 15. Mu.g was applied to each well. After electrophoresis, the gel was stained with coomassie blue and decolorized with a corresponding decolorization solution until a low background, clear band was developed (fig. 4).

The results show that VP1/VP2/VP 3=1:1:10, the bands are clear, the proportion is normal, no visible impurity band exists, and the purity is more than 99%.

Titer determination of rAAV-hRHO physical titer of rAAV-RHO was detected by fluorescent quantitative PCR method.

SYBR II (takara); fragment of interest primer (20 uM); packaging the plasmid of interest (known concentration) for the virus; test virusThe method comprises the steps of carrying out a first treatment on the surface of the PCR octant (Bio-red). The experimental method comprises the following steps: template 1ul, SYBR II 7ul primer 1.25 ul, primer 2.25 ul, and nucleotide-free water were added to 14ul. PCR reaction conditions: pre-denaturation: 95 ℃ for 10min; and (3) circulation: 15sec at 95℃and 1min at 60 ℃. The genome titre was determined to be 1X 10 ¹² vg/mL。

Example 3 experiment of the Effect of rAAV-hRHO recombinant adeno-associated Virus on retinitis pigmentosa

1. Rabbit eye vitreous cavity injection

51 rabbits were divided into 3 groups, an experimental group A-P and a control group, and 50ul of 1X 10 were respectively aspirated ¹² The vg/mL adeno-associated virus penetrated the pars plana 3mm beyond the limbus and entered the vitreous cavity for intravitreal injection.

Wherein, the recombinant adeno-associated virus vector rAAV/optimized-hRHO carries an optimized RHO coding sequence; recombinant adeno-associated viral vector rAAV/wild-hhho carrying wild (non-optimized) RHO coding sequence.

2. Slit lamp, intraocular pressure and fundus photographic examination

Group 9 rabbits were subjected to slit lamp and ocular pressure examination at 1, 3, 7, 30 days post-surgery, respectively. All rabbits had no obvious abnormality, conjunctival congestion, secretion, endophthalmitis and rise of intraocular pressure. Fundus photography one month post-surgery showed no significant complications or damage to retinal blood vessels and optic nerves in all rabbits (fig. 5). Indicating that normal standard intravitreal injections do not produce significant inflammatory reactions or other complications.

3. Fluorescence photography of retina

After 30 days of intravitreal injection, fluorescence photography of EGFP group retinas is expected to show successful expression of EGFP on retinas, indicating normal expression on retinas by transfection of rabbit eye vitruses with rAAV as a vector, carrying EGFP.

4. Immunofluorescence detection of hRHO

After 30 days of intravitreal injection, the eyeballs of the experimental and control groups were peeled off to prepare paraffin sections. The paraffin sections were dried in an oven at 65℃for 2h, dewaxed to water, washed three times with PBS for 5min each. Placing the slices in EDTA buffer solution for microwave restoration, powering off after medium fire to boiling, and keeping the temperature for 10min until low fire to boiling. After natural cooling, PBS is washed for 3 times, each time for 5min. The sections were placed in 3% hydrogen peroxide solution and incubated at room temperature for 10min. Washed 3 times with PBS for 5min each, and blocked with 5% BSA for 20min after spin-drying. The BSA solution was removed and 50. Mu.l of diluted primary antibody was added to cover the tissue per slice at 4℃overnight. The cells were washed three times with PBS for 5min each. The PBS solution is removed, 50 mu l-100 mu l of the fluorescent secondary antibodies of the corresponding species are added to each slice, and the slices are incubated for 50min-1h at room temperature in the absence of light. Light-shielding PBS is used for washing for 3 times, each time for 5min. The PBS was removed. 50-100 μl DAPI was added to each slice to dye nuclei in the dark for 5min. PBS was washed 3 times, each for 5min. And (5) after the slices are slightly dried, sealing the slices by using an anti-fluorescence quenching sealing tablet, and preserving the slices at 4 ℃ in a dark place for photographing.

The immunofluorescence results of the retina against hRHO are shown in FIG. 6. It can be seen that the relative intensity of immunofluorescence of each rAAV 2-optimized hRHO group is significantly higher than that of the corresponding rAAV 2-wild hRHO group (non-optimized group). The relative intensity of immunofluorescence of group A (rAAV 2/2-optimized hRHO) is 0.81, and the relative intensity of immunofluorescence of group B (rAAV 2/2-wild hRHO) is 0.58, which is improved by about 40%. The relative intensity of immunofluorescence of M group (rAAV 2/8-optimized hRHO) is 1.38, and the relative intensity of immunofluorescence of N group (rAAV 2/8-wild hRHO) is 0.37, which is improved by about 2.73 times. The relative intensity of immunofluorescence of group O (rAAV 2/9-optimized hRHO) is 1.89, and the relative intensity of immunofluorescence of group P (rAAV 2/9-wild hRHO) is 0.56, which is improved by about 2.38 times. This shows that the protein expression of the coding nucleotide sequence of the specially optimised hRHO of the invention (SEQ ID NO: 1) is higher than that of the non-optimised coding sequence SEQ ID NO: 2.

As can also be seen from FIG. 6, the expression of hRHO gene on retina of O group and M group is obviously improved compared with other groups, and the expression effect of hRHO gene carried by AAV8 and AAV9 is better.

5. OCT detection

The OCT results for each group are shown in fig. 7, showing no significant differences in retinal nerve fibers.

6. Real-Time PCR detection of hRHO expression

Firstly, analyzing the conserved structure of hRHO by using NCBI conserved domain analysis software to ensure that the amplified fragment of the designed primer is positioned in a non-conserved region; then, primer design is carried out by using primer premier 5 according to the primer design principle of fluorescence quantitative PCR:

rabbit-actin-F: CCTTCTACAACGAGCTGCGC (SEQ ID NO.: 8)

rabbit-actin-R: TACAGGGACAGCACGGCC (SEQ ID NO.: 9)

Original hRHO-F: CTTCACCCACCAGGGCTCCAACT (SEQ ID NO.: 10)

Original hRHO-R: AGTGGGTTCTTGCCGCAGCAGAT (SEQ ID NO.: 11)

Optimizing hRHO-F: CTTCACCCACCAGGGCAGCAACT (SEQ ID NO.: 12)

Optimizing hRHO-R: ACCTGGCTGGTCTCGGTCTTGCT (SEQ ID NO.: 13)

1) Extraction of RNA and reverse transcription

Total RNA of retinas of different experimental groups of rabbits was extracted using TRIZOL kit and reverse transcribed to synthesize cDNA templates.

2) Reaction system and reaction program for fluorescent quantitative PCR

Fluorescent quantitative PCR was performed on a Real-time PCR Detection System instrument. Into a 0.2mL PCR reaction tube was added SYBR Green mix 12.5. Mu. L, ddH2O 8. Mu.L, 1. Mu.L each of a pair of primers, 2.5. Mu.L each of cDNA sample, and 25. Mu.L total. Each sample was used to amplify both the target gene and the reference gene rabbit-actin, and the amplification of each gene was repeated three times. In the actual sample application, the reagents common to each PCR reaction tube can be added together and then split-packed in order to reduce errors. And (5) after the sample addition is finished, performing fluorescent quantitative PCR.

Amplification was performed according to a reaction program of 95℃for 1s,94℃for 15s,55℃for 15s,72℃for 45s, 40 cycles total, and fluorescent signals were collected at the extension stage of each cycle. After the reaction is finished, melting curve analysis is carried out at 94-55 ℃.

The difference of gene expression quantity is researched by adopting a 2-delta CT relative quantitative method (Livak et al 2001), a standard curve is not required to be manufactured in the method, and the housekeeping gene rabbit-actin is taken as an internal reference gene, so that the expression value can be automatically generated by analysis software of an instrument.

As a result, as shown in FIG. 8, the relative expression amount of mRNA was higher for the rAAV 2-optimized hRHO group than for the corresponding rAAV 2-wild hRHO group for AAV2/2, AAV2/3, AAV2/4, AAV2/5, and AAV 2/6. In particular to AAV2/4, AAV2/5 and AAV2/6, and the relative expression quantity of mRNA of the rAAV 2-optimized hRHO group is obviously improved. This result surprisingly shows that the optimized coding nucleotide sequence of hRHO of the invention (SEQ ID NO: 1) surprisingly increases transcription efficiency, resulting in a significant increase in expression of the rAAV 2-optimized hRHO group gene level over the rAAV 2-wild hRHO group. The results indicate that the transcription efficiency of the rAAV 2-optimized hho group is significantly higher in terms of transcription efficiency.

Furthermore, it can be seen that mRNA expression levels of AAV2/2, AAV2/8, AAV2/9 (groups A, O and M) were significantly higher than those of AAV of the other serotypes. The expression of hRHO genes carried by AAV2/2, AAV8 and AAV9 is better as proved by obviously improving the expression of hRHO genes on retina of A group, O group and M group compared with other groups.

7. Western blot detection of hRHO protein expression

Retinas of the eyeballs of rabbits of different experimental groups were isolated, RIPA lysate was added in a corresponding volume to 100. Mu.L/50 mg of tissue, homogenized by a homogenizer, and the supernatant was collected by centrifugation. After the protein concentration is measured by ultraviolet spectrophotometry at 280nm, the loading volumes of the experimental group and the control group are calculated according to 50 mug of total protein, and SDS-PAGE gel electrophoresis and Western blot are carried out. ECL development was performed after antibody incubation.

The results are shown in FIG. 9. It can be seen that the expression at the protein level of each rAAV 2-optimized hRHO group is significantly improved over the corresponding rAAV 2-wild hRHO group (non-optimized group). The optimal rAAV 2/9-optimized hRHO group is improved by 3.3 times, the rAAV 2/8-optimized hRHO group is improved by 1.8 times, and the rAAV 2/2-optimized hRHO group is improved by 1 time. This shows that the optimized coding nucleotide sequence of hRHO of the invention (SEQ ID NO.: 1) is more efficient in translation.

In addition, the expression of hRHO gene protein carried by AAV8 and AAV9 is best compared with other groups, and the optimized sequence has better expression effect than the original sequence hRHO gene protein.

With the rapid development of gene therapy in the treatment of ocular diseases, healing retinal pigment degeneration would not be a problem. Because the human eyes and rabbit eyes are similar in anatomy and volume, the invention uses the rabbit eyes as a model to carry out the intravitreal injection of rAAV-hRHO. The experiment researches injection dosage, safety level and postoperative complications, and provides important references for future clinical tests. All rabbits were tested for slit lamp, eye pressure, conjunctival congestion, secretion, endophthalmitis, and elevation of eye pressure. Fundus photography one month post-surgery showed that all rabbits had no obvious complications or damage to retinal blood vessels and optic nerves. Indicating that the experiment was safe.

Immunofluorescence, real-time quantitative PCR and Western blot results prove that hRHO can be stably expressed on the retina of the rabbit. Since the lesions cause apoptosis of retinal ganglion cells around the optic disc, retinal sections of the rAAV-EGFP group can detect stable fluorescent expression of the retina around the optic disc. The fluorescence staining of hRHO indicates that it can reach retinal ganglion cells, indicating that rAAV2-hRHO can reach diseased areas in the patient's eye. The results of fundus photography and OCT show that a single intravitreal injection of adeno-associated virus carrying the RHO gene is safe and has no retinal toxicity and can be applied to future clinical trials.

Example 4

20 mice were divided into 5 groups and into control groups according to the above table (vitrectomy 1X 10) ¹² vg rAAV-EGFP [ Guangzhou Pi Biotechnology Co., ltd]) Experimental group A, B, C, D vitrectomy of the 4 recombinant human RHO Gene recombinant adeno-associated Virus (rAAV 2/2-optimized hRHO, rAAV2/2-hRHO and rAAV 2/9-optimized hRHO and rAAV2/9-hRHO, 50. Mu.l, 1X 10) ¹² vg/mL), and is injected by puncturing the inner thigh of the mouse. The mice were observed for body weight changes over a month and rat thigh tissue mRNA was extracted. QPCR detects the hRHO gene and compares the relative expression levels of the hRHO gene in A, B group with control group.

As can be seen from fig. 10, the A, B, C, D group mice gain weight over time, and injection of AAV2/2 and AAV2/9 was safe.

As can be seen from FIG. 11, the relative expression levels of the group A, B rAAV 2/2-optimized hRHO and the group C, D rAAV 2/9-optimized hRHO and the group rAAV 2/9-wild hRHO are improved by about 40 times, and the relative expression levels of the group C, D rAAV 2/9-optimized hRHO and the group rAAV 2/9-wild hRHO are improved by about 100 times.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Wohan New gos biotechnology Co., ltd

<120> coding sequence of rhodopsin, construction of expression vector and application thereof

<130> P2019-0018

<160> 13

<170> SIPOSequenceListing 1.0

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<213> Artificial sequence (artificial sequence)

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atgaacggca ccgagggccc caacttctac gtgcccttca gcaacgccac cggcgtggtg 60

cgcagcccct tcgagtaccc ccagtactac ctggccgagc cctggcagtt cagcatgctg 120

gccgcctaca tgttcctgct gatcgtgctg ggcttcccca tcaacttcct gaccctgtac 180

gtgaccgtgc agcacaagaa gctgcgcacc cccctgaact acatcctgct gaacctggcc 240

gtggccgacc tgttcatggt gctgggcggc ttcaccagca ccctgtacac cagcctgcac 300

ggctacttcg tgttcggccc caccggctgc aacctggagg gcttcttcgc caccctgggc 360

ggcgagatcg ccctgtggag cctggtggtg ctggccatcg agcgctacgt ggtggtgtgc 420

aagcccatga gcaacttccg cttcggcgag aaccacgcca tcatgggcgt ggccttcacc 480

tgggtgatgg ccctggcctg cgccgccccc cccctggccg gctggagccg ctacatcccc 540

gagggcctgc agtgcagctg cggcatcgac tactacaccc tgaagcccga ggtgaacaac 600

gagagcttcg tgatctacat gttcgtggtg cacttcacca tccccatgat catcatcttc 660

ttctgctacg gccagctggt gttcaccgtg aaggaggccg ccgcccagca gcaggagagc 720

gccaccaccc agaaggccga gaaggaggtg acccgcatgg tgatcatcat ggtgatcgcc 780

ttcctgatct gctgggtgcc ctacgccagc gtggccttct acatcttcac ccaccagggc 840

agcaacttcg gccccatctt catgaccatc cccgccttct tcgccaagag cgccgccatc 900

tacaaccccg tgatctacat catgatgaac aagcagttcc gcaactgcat gctgaccacc 960

atctgctgcg gcaagaaccc cctgggcgac gacgaggcca gcgccaccgt gagcaagacc 1020

gagaccagcc aggtggcccc cgcc 1044

<210> 2

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<213> Homo sapiens (Homo sapiens)

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atgaatggca cagaaggccc taacttctac gtgcccttct ccaatgcgac gggtgtggta 60

cgcagcccct tcgagtaccc acagtactac ctggctgagc catggcagtt ctccatgctg 120

gccgcctaca tgtttctgct gatcgtgctg ggcttcccca tcaacttcct cacgctctac 180

gtcaccgtcc agcacaagaa gctgcgcacg cctctcaact acatcctgct caacctagcc 240

gtggctgacc tcttcatggt cctaggtggc ttcaccagca ccctctacac ctctctgcat 300

ggatacttcg tcttcgggcc cacaggatgc aatttggagg gcttctttgc caccctgggc 360

ggtgaaattg ccctgtggtc cttggtggtc ctggccatcg agcggtacgt ggtggtgtgt 420

aagcccatga gcaacttccg cttcggggag aaccatgcca tcatgggcgt tgccttcacc 480

tgggtcatgg cgctggcctg cgccgcaccc ccactcgccg gctggtccag gtacatcccc 540

gagggcctgc agtgctcgtg tggaatcgac tactacacgc tcaagccgga ggtcaacaac 600

gagtcttttg tcatctacat gttcgtggtc cacttcacca tccccatgat tatcatcttt 660

ttctgctatg ggcagctcgt cttcaccgtc aaggaggccg ctgcccagca gcaggagtca 720

gccaccacac agaaggcaga gaaggaggtc acccgcatgg tcatcatcat ggtcatcgct 780

ttcctgatct gctgggtgcc ctacgccagc gtggcattct acatcttcac ccaccagggc 840

tccaacttcg gtcccatctt catgaccatc ccagcgttct ttgccaagag cgccgccatc 900

tacaaccctg tcatctatat catgatgaac aagcagttcc ggaactgcat gctcaccacc 960

atctgctgcg gcaagaaccc actgggtgac gatgaggcct ctgctaccgt gtccaagacg 1020

gagacgagcc aggtggcccc ggcc 1044

<210> 3

<211> 348

<212> PRT

<213> Homo sapiens (Homo sapiens)

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Met Asn Gly Thr Glu Gly Pro Asn Phe Tyr Val Pro Phe Ser Asn Ala

1 5 10 15

Thr Gly Val Val Arg Ser Pro Phe Glu Tyr Pro Gln Tyr Tyr Leu Ala

20 25 30

Glu Pro Trp Gln Phe Ser Met Leu Ala Ala Tyr Met Phe Leu Leu Ile

35 40 45

Val Leu Gly Phe Pro Ile Asn Phe Leu Thr Leu Tyr Val Thr Val Gln

50 55 60

His Lys Lys Leu Arg Thr Pro Leu Asn Tyr Ile Leu Leu Asn Leu Ala

65 70 75 80

Val Ala Asp Leu Phe Met Val Leu Gly Gly Phe Thr Ser Thr Leu Tyr

85 90 95

Thr Ser Leu His Gly Tyr Phe Val Phe Gly Pro Thr Gly Cys Asn Leu

100 105 110

Glu Gly Phe Phe Ala Thr Leu Gly Gly Glu Ile Ala Leu Trp Ser Leu

115 120 125

Val Val Leu Ala Ile Glu Arg Tyr Val Val Val Cys Lys Pro Met Ser

130 135 140

Asn Phe Arg Phe Gly Glu Asn His Ala Ile Met Gly Val Ala Phe Thr

145 150 155 160

Trp Val Met Ala Leu Ala Cys Ala Ala Pro Pro Leu Ala Gly Trp Ser

165 170 175

Arg Tyr Ile Pro Glu Gly Leu Gln Cys Ser Cys Gly Ile Asp Tyr Tyr

180 185 190

Thr Leu Lys Pro Glu Val Asn Asn Glu Ser Phe Val Ile Tyr Met Phe

195 200 205

Val Val His Phe Thr Ile Pro Met Ile Ile Ile Phe Phe Cys Tyr Gly

210 215 220

Gln Leu Val Phe Thr Val Lys Glu Ala Ala Ala Gln Gln Gln Glu Ser

225 230 235 240

Ala Thr Thr Gln Lys Ala Glu Lys Glu Val Thr Arg Met Val Ile Ile

245 250 255

Met Val Ile Ala Phe Leu Ile Cys Trp Val Pro Tyr Ala Ser Val Ala

260 265 270

Phe Tyr Ile Phe Thr His Gln Gly Ser Asn Phe Gly Pro Ile Phe Met

275 280 285

Thr Ile Pro Ala Phe Phe Ala Lys Ser Ala Ala Ile Tyr Asn Pro Val

290 295 300

Ile Tyr Ile Met Met Asn Lys Gln Phe Arg Asn Cys Met Leu Thr Thr

305 310 315 320

Ile Cys Cys Gly Lys Asn Pro Leu Gly Asp Asp Glu Ala Ser Ala Thr

325 330 335

Val Ser Lys Thr Glu Thr Ser Gln Val Ala Pro Ala

340 345

<210> 4

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<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

acttctacgt gcccttctcc aatg 24

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

gtcttggaca cggtagcaga ggc 23

<210> 6

<211> 1044

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 6

atgaatggta ctgaaggccc taacttttat gttcccttct ctaatgctac cggagtcgta 60

cgttccccat ttgagtaccc gcaatattac ttagccgaac cttggcagtt ctcaatgttg 120

gcagcgtata tgtttcttct cattgtgcta gggttcccca tcaactttct gacattatac 180

gttacggtcc aacataaaaa gttgcgcact ccacttaatt atatactcct aaacctggct 240

gtagccgatt tattcatggt gttgggtggc tttacctcga cactttacac gagtctccac 300

ggatatttcg tttttgggcc gactggttgt aatctagagg gcttctttgc aaccctggga 360

ggggaaattg cgttatggag cttggtcgta cttgctatcg agcgatacgt ggttgtctgc 420

aaacctatgt ctaacttccg gtttggtgaa aatcatgcca taatgggcgt agcattcaca 480

tgggtgatgg cgctcgcttg tgccgcaccc ccactagcgg gatggtccag atatattccg 540

gaggggctgc agtgctcatg tggtatcgac tactatacgt taaagcctga agttaacaat 600

gagtcgtttg tcatatacat gttcgtagtg cactttacta ttcccatgat cataattttc 660

ttttgctatg gccaattggt tttcaccgtc aaagaagctg ccgcacagca acaggagagt 720

gcgacaacgc aaaaggctga aaaagaggta actaggatgg tgatcataat ggttattgcc 780

tttcttatct gttgggtccc atacgcaagc gtagcgttct atatatttac ccatcaggga 840

tctaacttcg ggccgatttt tatgacaatc cctgctttct ttgccaagtc cgcagcgata 900

tacaatcccg tgatttatat catgatgaac aaacaattcc gtaattgcat gctcacgact 960

atatgttgcg gtaagaaccc actaggcgat gacgaagctt cagccaccgt ttcgaaaaca 1020

gagacgagtc aggtcgcacc ggcg 1044

<210> 7

<211> 1044

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

atgaacggca ccgagggccc caacttctac gtgcccttca gcaacgccac cggcgtggtg 60

cgcagcccct tcgagtaccc ccagtactac ctggccgagc cctggcagtt cagcatgctg 120

gccgcctaca tgttcctgct gatcgtgctg ggcttcccca tcaacttcct gaccctgtac 180

gtgaccgtgc agcacaagaa gctgcgcacc cccctgaact acatcctgct gaacctggcc 240

gtggccgacc tgttcatggt gctgggcggc ttcaccagca ccctgtacac cagcctgcac 300

ggctacttcg tttttgggcc gactggttgt aatctagagg gcttctttgc aaccctggga 360

ggggaaattg cgttatggag cttggtcgta cttgctatcg agcgatacgt ggttgtctgc 420

aaacctatgt ctaacttccg gtttggtgaa aatcatgcca taatgggcgt agcattcaca 480

tgggtgatgg cgctcgcttg tgccgcaccc ccactagcgg gatggtccag atatattccg 540

gaggggctgc agtgctcatg tggtatcgac tactatacgt taaagcctga agttaacaat 600

gagtcgtttg tcatatacat gttcgtagtg cactttacta ttcccatgat cataattttc 660

ttttgctatg gccaattggt tttcaccgtc aaagaagctg ccgcacagca acaggagagt 720

gcgacaacgc aaaaggctga aaaagaggta actaggatgg tgatcataat ggttattgcc 780

tttcttatct gttgggtccc atacgcaagc gtagcgttct atatatttac ccatcaggga 840

tctaacttcg ggccgatttt tatgacaatc cctgctttct ttgccaagtc cgcagcgata 900

tacaatcccg tgatttatat catgatgaac aaacaattcc gtaattgcat gctcacgact 960

atatgttgcg gtaagaaccc actaggcgat gacgaagctt cagccaccgt ttcgaaaaca 1020

gagacgagtc aggtcgcacc ggcg 1044

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

ccttctacaa cgagctgcgc 20

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

tacagggaca gcacggcc 18

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 10

cttcacccac cagggctcca act 23

<210> 11

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 11

agtgggttct tgccgcagca gat 23

<210> 12

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 12

cttcacccac cagggcagca act 23

<210> 13

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 13

acctggctgg tctcggtctt gct 23

Claims

1. A nucleotide sequence encoding rhodopsin, wherein the nucleotide sequence is selected from the group consisting of:

(a) The nucleotide sequence is shown as SEQ ID NO. 1; or (b)

(b) A nucleotide sequence complementary to the nucleotide sequence of (a).

2. A fusion nucleic acid comprising a structure of formula I from the 5 'end to the 3' end:

Z0-Z1-Z2(I)

in the method, in the process of the invention,

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

z0 is none, or a 5' UTR sequence;

z1 is the nucleotide sequence of claim 1; and

z2 is the none, or 3' UTR sequence.

3. A vector comprising the nucleotide sequence of claim 1 or the fusion nucleic acid of claim 2.

4. The vector of claim 3, wherein the vector is an adeno-associated viral vector.

5. The vector of claim 3, wherein the vector is an adeno-associated viral vector AAV2/8 or AAV2/9.

6. A host cell comprising the vector of claim 3, or a chromosome thereof having incorporated therein an exogenous nucleotide sequence of claim 1 or the fusion nucleic acid of claim 2.

7. Use of the vector of claim 3 for the preparation of a formulation or composition for restoring vision in a subject and/or for treating or preventing an ocular disorder, which ocular disorder is retinitis pigmentosa.

8. A pharmaceutical formulation comprising (a) the carrier of claim 3, and (b) a pharmaceutically acceptable carrier or excipient.