CN111909935B - Expression vector of recombinant human frizzled protein receptor 4 (FZD 4) and application thereof - Google Patents

Abstract

The present application relates to a recombinant nucleic acid comprising a nucleotide sequence encoding human frizzled receptor 4 (FZD 4).

Description

Expression Vector and Application of Recombinant Human Frizzled Receptor 4 (FZD4)

technical field

This application relates to the field of biomedicine, in particular to an expression vector of recombinant human Frizzled receptor 4 (FZD4) and its application.

Background technique

Familial exudative vitreoretinopathy (FEVR) is a severe hereditary vitreoretinopathy. It is one of the causes of retinal detachment and blindness in adolescents. It is typically characterized by incomplete peripheral retinal vascularization and brush-like distal capillaries. Patients with incomplete peripheral retinal vascularization may not have any ocular symptoms. When abnormal blood vessels lead to further development of retinal hypoxia, it can cause various degrees of lesions and visual impairment. FEVR is inherited in a variety of ways, and the identified causative genes of FEVR exist in the Wnt and Norrin-β-catenin signaling pathways that are highly conserved in evolution and play an important role in the development of ocular structures and neovascularization. Currently, there are no studies on FEVR gene therapy. Therefore, there is an urgent need in the art to develop a therapeutically effective recombinant human Frizzled receptor 4 (FZD4) expression system and its application.

Contents of the invention

The present application provides a recombinant nucleic acid, which contains a nucleotide sequence encoding human Frizzled receptor 4 (FZD4), and the nucleotide sequence includes at least one sequence selected from the following group:

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

(b) The nucleotide sequence has ≥99% identity with the nucleotide sequence shown in SEQ ID NO.:1;

(c) The nucleotide sequence has ≥98% identity with the nucleotide sequence shown in SEQ ID NO.:1;

(d) the nucleotide sequence has ≥95% identity with the nucleotide sequence shown in SEQ ID NO.:1; and

(e) The nucleotide sequence has ≥80% identity, preferably ≥85% identity, more preferably ≥90% identity with the nucleotide sequence shown in SEQ ID NO.:1.

In certain embodiments, the nucleotide sequence includes at least one sequence selected from the group consisting of:

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

(b) The nucleotide sequence has ≥99% identity with the nucleotide sequence shown in SEQ ID NO.:1;

(c) the nucleotide sequence has ≥98% identity with the nucleotide sequence shown in SEQ ID NO.:1; and

(d) The nucleotide sequence has ≥95% identity with the nucleotide sequence shown in SEQ ID NO.:1.

In some embodiments, the protein sequence encoded by the recombinant nucleic acid is shown in SEQ ID NO.:3.

The application also provides a fusion nucleic acid, which has a structure of formula I from the 5' end to the 3' end:

Z1-Z2-Z3(I)

In the formula,

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

Z1 is none, or 5'-UTR sequence;

Z2 is a nucleotide sequence as described in the application; and

Z3 is the 3'-UTR sequence.

The present application also provides a vector containing the recombinant nucleic acid or the fusion nucleic acid described in the present application.

In certain embodiments, the vector is selected from a plasmid or a viral vector.

In certain embodiments, the vector is selected from lentiviral vectors, adenoviral vectors, adeno-associated viral vectors or combinations thereof.

In certain embodiments, the vector is an AAV vector.

In certain embodiments, the serotype of the vector is selected from AAV2, AAV5, AAV7, AAV8, or combinations thereof.

In certain embodiments, the vector is selected from a DNA viral vector or a retroviral vector.

The present application also provides a use of the carrier as described in the present application, which is used for preparing a preparation or composition, and the preparation or composition is used for restoring vision of a subject and/or treating eye diseases.

The present application also provides a host cell, which contains the vector described in the present application, or the exogenous recombinant nucleic acid as described in the present application or the fusion nucleic acid as described in the present application is integrated in its chromosome.

In certain embodiments, the host cell is selected from 293T cells, photoreceptor cells, other visual cells, (optic) nerve cells, or combinations thereof.

In certain embodiments, the photoreceptor cells are cone cells and/or rod cells, and the other visual cells are bisected cells.

The application also provides a pharmaceutical preparation, which contains:

(a) the carrier described in this application, and

(b) Pharmaceutically acceptable pharmaceutical carriers and/or excipients.

In certain embodiments, the dosage form of the pharmaceutical preparation is selected from the group consisting of lyophilized preparations, liquid preparations and combinations thereof.

In some embodiments, the content of the carrier in the pharmaceutical preparation is 1×10 ⁹ -1×10 ¹⁶ viruses/ml, preferably 1×10 ¹¹ -1×10 ¹³ viruses/ml, more preferably 2× 10 ¹¹ -1×10 ¹² viruses/ml.

The present application also provides the use of the pharmaceutical preparation as described in the present application, which is used for treating eye diseases, preferably retinopathy.

In certain embodiments, the retinopathy is an inherited retinopathy, preferably familial exudative retinopathy FEVR.

In some embodiments, the carrier or pharmaceutical preparation can significantly increase the expression and/or activity of Frizzled receptor 4 (FZD4).

In some embodiments, the carrier or the pharmaceutical preparation can effectively increase the expression and/or activity of Frizzled receptor 4 (FZD4) for up to 3 months, preferably up to 6 months.

The present application also provides a method for preparing recombinant human Frizzled receptor 4 (FZD4), which includes the step of: cultivating the host cell described in the present application to obtain recombinant human Frizzled receptor 4 (FZD4).

The recombinant nucleic acid, fusion nucleic acid, vector, host cell and/or pharmaceutical preparation of the present application have one or more of the following effects: can improve the transcription and/or expression of Frizzled receptor 4 (FZD4), can be used to restore the visual acuity and/or treat eye disease, can treat hereditary retinopathy and/or can treat familial exudative retinopathy FEVR.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Fig. 1 shows the open reading frame sequence comparison between the applicant's frizzled receptor 4 (FZD4) nucleotide sequence after sequence optimization and the wild type human frizzled receptor 4 nucleotide sequence. Wherein, the upper row represents the optimized nucleotide sequence of the present application, and the lower row represents the wild-type nucleotide sequence, and "|" indicates that the corresponding sites of the two are consistent; the homology of the two is 77.5%.

Figure 2 shows a schematic diagram of the protein structure of the Frizzled receptor 4 transcript. Among them, domain A encodes the Frizzled domain (CRD_FZ domain) of Frizzled receptor 4, and domain B encodes the G protein-coupled receptor family 2 domain (7transmembrane receptor domain, 7tm_2domain) of Frizzled receptor 4.

Figure 3 shows the electrophoresis detection of the PCR product, and the correct clone with a target band of about 1614bp was screened from the recombinant clones. Among them, lane M is the protein marker; lane 1 is the correct recombinant adeno-associated virus expression vector AAV-MCS-rAAV2/2-rhFZD4.

Fig. 4 is a schematic diagram showing the structure of the recombinant adeno-associated virus expression vector AAV-MCS-rAAV2/2-rhFZD4 of the present application.

Figure 5 shows the results of Coomassie Brilliant Blue staining for SDS-PAGE electrophoresis to detect the purity of the recombinant adeno-associated virus rAAV2/2-rhFZD4 of the present application. Among them, lane 1 is the protein marker; lane 2 is the recombinant adeno-associated virus rAAV2/2-rhFZD4 of the present application.

Figure 6 shows the detection results of the relative expression level of human Frizzled receptor 4 in the retina of the rabbit eye by fluorescent quantitative PCR. Among them, the experimental group A is the group injected with rAAV2/2-rhFZD4 of the present application, the experimental group B is the group injected with wild-type rAAV2/2-hFZD4, and the control group is the group injected with rAAV2/2-ZsGreen.

Figure 7 shows the quantitative analysis results of Western blot detection of human Frizzled receptor 4 in the retina of rabbit eyeballs. Experimental group A is the group injected with rAAV2/2-rhFZD4 of this application, and experimental group B is injected with wild-type rAAV2/2 -hFZD4 group, and the control group was rAAV2/2-ZsGreen injection group.

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the term "about" generally refers to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, "about 100" includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).

In this application, the term "comprising" or "comprising (comprising)" generally refers to open, semi-closed and closed. In other words, the term also includes "consisting essentially of", or "consisting of".

In this application, sequence "identity" or "identity" is usually measured along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 80%, or 90%, 95%, or 100%) are determined by comparing two aligned sequences and determining the number of positions where the same residue occurs. Typically, this is expressed as a percentage. The measurement of sequence "identity" or "identity" of nucleotide sequences is well known to those skilled in the art. For example, "identity" can refer to the relatedness of two sequences based on a nucleotide-by-nucleotide comparison over a specified comparison window or segment. Thus, identity is defined as the degree of identity, correspondence or equivalence between the same strand (sense or antisense strand) of two DNA segments. "Percent sequence identity" is calculated by comparing two optimally aligned sequences over a specified region; determining the number of positions where the same base or amino acid occurs in the two sequences to obtain a matching position the number of such locations; divide the number of such locations by the total number of locations in the segment being compared and multiply the quotient by 100. The optimal alignment of sequences can be performed by the algorithm of Smith & Waterman, Appl. Math. 2: 482 (1981), the algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), Pearson & Lipman, Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988) method and computer program (for example Clustal Macaw Pileup (Higgins et al., CABIOS.5L151-153 (1989)), FASTDB (Intelligenetics), BLAST (National Center for Biomedical Information; Altschul et al., Nucleic Acids Research 25:3389-3402 (1997)), PILEUP (Genetics Computer Group, Madison, Wis) or GAP, BESTFIT, FASTA and TFASTA (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, Madison, Wis ) to carry out. (See US Patent 5,912,120.)

In this application, the terms "subject", "subject in need" generally refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, rabbits, rats, mice, horses, dogs, cats, pigs, sheep, goats.

In this application, the terms "(recombinant) human Frizzled receptor 4", "FZD4 (protein)", "hFZD4 (protein)", "polypeptide", "polypeptide of the present application" and "protein of the present application" generally have the same are used interchangeably in this paper. FZD4 is a member of the Frizzled gene family. Its encoded protein contains 537 amino acids and has a seven-time transmembrane helical structure, forming 3 intracellular loops and 3 extracellular loops; the PDZ domain located at the C-terminal of the cell is intracellular Receptors provide action sites, and the highly conserved cysteine-rich region (CRD) at the extracellular N-terminal is the receptor of Wnt signaling pathway on the cell surface. For example, the Frizzled receptor 4 can be the amino acid sequence shown in SEQ ID NO.:3.

In this application, the term "familial exudative retinopathy" or "FEVR" generally refers to a severe inherited vitreoretinal disease. It is one of the causes of retinal detachment and blindness in adolescents.

In this application, the term "encoding" generally refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, DNA or mRNA, that the sequence can serve as a template for the synthesis of a nucleotide sequence in a biological process. Other polymers and macromolecules of nucleotide sequences (such as rRNA, tRNA and mRNA) or defined amino acid sequences and the resulting biological properties. Thus, a gene, cDNA, or RNA can encode a protein when transcription and translation of mRNA corresponding to the gene in a cell or other biological system results in the production of the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence, usually given in the Sequence Listing) and the non-coding strand (used as a template for transcription of a gene or cDNA) can both be referred to as the coding protein, or part of the gene or cDNA. other products. For example, the gene encoding Frizzled receptor 4 of the present application can be used to synthesize corresponding mRNA and/or corresponding polypeptide in biological processes.

In the present application, the term "vector" generally refers to an expression vector containing the recombinant nucleic acid or fusion nucleic acid DNA sequence of the present application and appropriate transcription/translation control signals. The method of constructing the vector may include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and so on. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Expression vectors may also include ribosomal binding sites for translation initiation, regulatory sequences, and transcription terminators. 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 the nucleic acid sequence. Vectors may also include selectable markers, for example, to determine expression of the vector in growth systems such as bacterial cells or in retinal target cells. For example, a vector comprising the above-mentioned appropriate DNA sequence and an appropriate promoter or regulatory sequence can be used to transform an appropriate host cell so that it can express the polypeptide. For example, a plasmid vector and/or a viral vector comprising a sequence encoding Frizzled receptor 4.

In this application, the term "viral vector" generally means that a gene therapy vector suitable for transduction and expression in target cells is provided. For example the target cell may be a retinal target cell. 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. For example, viral vectors can often remain extrachromosomal without integrating into the genome of target retinal cells, or can integrate into the genome of target retinal cells. The viral vector used to introduce the nucleic acid sequence encoding FZD4 protein into target retinal cells may be an AAV vector, such as self-complementary adeno-associated virus (scAAV). Selective targeting can be achieved using specific AAV serotypes (AAV serotype 2 to AAV serotype 12) or modified versions of any of these serotypes, including AAV 4YF and AAV 7m8 vectors.

In this application, the term "adeno-associated virus (adeno-associated virus)" or "AAV" is also commonly referred to as adeno-associated virus, which belongs to the family Parvoviridae and is the most simple single-stranded DNA defect found so far. Type viruses require a helper virus (usually an adenovirus) to replicate. It encodes cap and rep genes in two terminal inverted repeats (ITRs). The terminal inverted repeat sequence plays a decisive role in the replication and packaging of the virus. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. Adeno-associated virus can infect a variety of cells. For example, adeno-associated viruses can act as recombinant adeno-associated virus vectors, integrating in a stable and site-specific manner into the genome of the cells they infect. Adeno-associated virus vectors can be prepared by standard methods in the art, and any serotype of adeno-associated virus is suitable. Replication-deficient recombinant adeno-associated viruses can be prepared by co-transfecting into cell lines infected with a human helper virus (eg, adenovirus) a plasmid containing a nucleic acid sequence of interest flanked by two adeno-associated virus inverted terminal repeats sequence (ITR) region, and plasmids carrying the adeno-associated virus capsidation genes (rep and cap genes). The resulting adeno-associated virus recombinants are then purified by standard techniques. For example, recombinant adeno-associated viral vectors can be encapsidated into virions (such as, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16 in AAV virions). For example, the recombinant adeno-associated virus vector comprising the sequence encoding Frizzled receptor 4 of the present application.

In this application, the term "serotype" generally refers to the detection of epitopes on the capsid surface of the adeno-associated virus by serological methods, and the typing of the adeno-associated virus. Adeno-associated virus has a variety of common serotypes and more than 100 virus variants. In this application, the AAV capsid, ITR and other selected AAV components are selected from any AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVInc80, any Variants of AAVs known or mentioned or AAVs yet to be discovered, or variants or mixtures thereof.

In this application, the term "host cell" may generally refer to prokaryotic cells, or lower eukaryotic cells, or higher eukaryotic cells, such as mammalian cells (including human and non-human mammals). Representative examples may include the following groups: animal cells of CHO, NSO, COS7, or 293 cells, and the like. For example, 293T cells, photoreceptor cells (including cone cells and/or rod cells), other visual cells (such as bisected cells), and nerve cells are selected as host cells. For example, the host cell is selected from the group consisting of rod cells, cone cells, light-giving bipolar cells, light-withdrawing bipolar cells, horizontal cells, ganglion cells, amacrine 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 prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

In this application, the term "culturing" can usually mean culturing host cells by conventional methods to express the protein encoded by the gene of this application. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

In this application, the term "obtaining" the Frizzled receptor 4 can usually be obtained by conventional methods to obtain recombinant human Frizzled receptor 4 expressed by the host cell. Frizzled receptor 4 can be expressed intracellularly, on the cell membrane, or secreted extracellularly. Proteins can be isolated and purified by various separation methods by taking advantage of their physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

In this application, the term "recombinant nucleic acid" may refer to the optimized gene sequence of recombinant human Frizzled receptor FZD4. Recombinant human Frizzled receptor FZD4 gene, its optimized nucleotide sequence is shown in SEQ ID NO: 1, its size is 1614bp, starting from the codon ATG, encoding 537 amino acids, of which the 40th-483rd position can be coiled The coding sequence of the Frizzled domain; 654-1509 may be the coding sequence of G protein-coupled receptor family 2 (7tm_2domain). The recombinant nucleic acid of the present application may be in the form of DNA or RNA. For example, the recombinant nucleic acid is DNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA, DNA can be single-stranded or double-stranded, and DNA can be coding or non-coding.

In this application, the term "fusion nucleic acid" may generally refer to a nucleic acid formed by linking nucleotide sequences from two or more different sources, or two or more nucleotide sequences from the same source but not linked to each other in their natural positions. A nucleic acid formed by linking two or more nucleotide sequences. For example, the nucleic acid encoding human Frizzled receptor 4 (FZD4) may be operably linked to a UTR sequence. For example, the nucleic acid encoding human Frizzled receptor 4 (FZD4) may be operably linked to a promoter sequence. For example, the nucleic acid encoding human Frizzled receptor 4 (FZD4) may be operably linked to a polyA sequence containing a stable structure.

In this application, the term "nucleotide linking sequence" generally refers to a nucleic acid formed by linking two or more nucleotide sequences. For example, a nucleotide linking sequence can be a linker. For example, a linker comprises an oligonucleotide of length selected from the group consisting of 1-30 nucleotides, 1-15 nucleotides, and 3-6 nucleotides. For example, a linker comprises a nucleotide linker sequence formed by cleavage with restriction enzymes EcoRI and/or SalI.

In the present application, the term "operably linked" may mean that the nucleic acid sequences are functionally related to the sequences to which they are operably linked such 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.

In the present application, the term "expression" may mean that the recombinant nucleic acid, fusion nucleic acid, or vector of the present application transcribes the target RNA and/or translates the target protein in the host cell. For example, the protein of interest may be Frizzled receptor 4. The recombinant nucleic acid or fusion nucleic acid encoding Frizzled receptor 4 provided by this application can produce Frizzled receptor 4 or Frizzled receptor 4 fusion protein in vitro or in vivo, and the fusion protein or the preparation containing the fusion protein can be used In the preparation of FEVR for the treatment of familial exudative retinopathy. The expression level of the nucleic acid encoding Frizzled receptor 4 optimized in the present application is higher, so that more Frizzled receptor 4 can be translated, and the optimized Frizzled receptor 4 can be expressed in human host cells more easily. The medicament containing the recombinant nucleic acid, fusion nucleic acid and carrier of the present application is injected into the vitreous cavity of the rabbit eye, and the medicament can maintain activity in the vitreous cavity and be transfected to retinal cells. The nucleic acid of the present application can encode Frizzled receptor 4 expressed more than the prior art, and can treat familial exudative retinopathy (FEVR).

In this application, the term "treatment" generally refers to an intervention that seeks to alter the natural course of the subject being treated, and may be performed prophylactically or during the course of clinical pathology. Desirable effects include, but are not limited to, preventing the occurrence or recurrence of disease, relieving symptoms, containing, reducing or inhibiting any direct or indirect pathological consequences of disease, ameliorating or relieving the disease state, and causing remission or improving prognosis. For example, treating an eye disease can be restoring vision in a subject, can be increasing or maintaining retinal nerve fiber layer thickness in a subject, can be increasing or maintaining the number of retinal ganglion cells in a subject.

In the present application, the term "hereditary retinopathy" generally refers to a pathological condition in which retinal abnormalities are caused by genetic factors. For example, the mode of inheritance may be autosomal dominant inheritance, autosomal recessive inheritance or X-linked inheritance. For example, the hereditary retinopathy may be familial exudative vitreoretinopathy (FEVR). For example, retinal abnormalities or retinopathy may include, but are not limited to, retinal folds, macular ectopia, and retinal detachment and/or concomitant ocular secondary lesions.

In this application, the term "retrovirus" generally refers to an integrating virus that infects dividing cells. For example, the retroviral vector of the present application can be constructed from the following retroviruses: HIV, MoMuLV (Moloney Murine Leukemia Virus), MSV (Moloney Murine Sarcoma Virus), HaSV (Hawey Sarcoma Virus), SNV ( Spleen necrosis virus), RSV (Rous sarcoma virus), Friend virus, murine stem cell virus (MSCV), lentivirus, or the disclosed defective retroviral vectors.

In this application, the term "DNA viral vector" generally refers to any DNA virus known to those skilled in the art. For example, the DNA viral vector infects mammalian cells. For example, examples of mammals may include laboratory animals (such as dogs, cats, rats, mice, and rabbits), farm animals (such as cows, horses, and sheep), and primates (such as monkeys and humans). For example, a DNA virus can be a single-stranded or double-stranded DNA virus. For example, the DNA virus can be Epstein-Barr virus (EBV). For example, the DNA virus can be Kaposi's sarcoma-associated herpes virus, also known as herpesvirus 8 (KSHV). For example, the DNA virus can be cytomegalovirus (HCMV).

In this application, the term "integration" generally means, for example, that retroviruses, following infection of cells, produce a double-stranded DNA molecule from two RNA molecules carried in the virion by a molecular process Covalently integrated into the host cell genome, the genes of the virus are expressed with the help of cellular and/or viral factors.

In this application, the term "plasmid" generally refers to a self-contained molecule of double-stranded DNA, which readily accepts additional (foreign) DNA and is readily introduced into a suitable host cell. A number of vectors, including plasmid and fungal vectors, have been described that can replicate and/or express in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples may include pKK plasmid (Clonetech), pUC plasmid, pET plasmid (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmid (Invitrogen, San Diego, Calif.) or pMAL plasmid (New England Biolabs, Beverly, Mass.) and a number of suitable host cells, using methods disclosed or referenced herein or known to those skilled in the relevant art.

In this application, the term "pharmaceutically acceptable carrier" or "excipient" means: one or more compatible solid or liquid filler or gel substances, which It is suitable for human use and must be of sufficient purity and low enough toxicity. Compatibility here means that each component in the composition can be blended with the active ingredient of the present application and with each other without significantly reducing the efficacy of the active ingredient. Some examples of pharmaceutically acceptable carrier (carrier) are cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as Stearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as Tween) , wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.

In this application, the term "pharmaceutical preparation" or "composition" may refer to a combination of a safe and effective amount of an active ingredient and a pharmaceutically acceptable carrier or excipient. For example, the "active ingredient" in the pharmaceutical composition described in the present application may refer to the vector described in the present application, such as viral vectors (including adeno-associated virus vectors). For example, the active ingredients, formulations and/or compositions described herein may be used to treat ocular disorders. For example, the "safe and effective amount" mentioned in this application means: the amount of the active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. For example, pharmaceutical formulations of the present application may be liquid or solid, such as powders, gels or pastes. For example, the pharmaceutical formulation can be a liquid, an injectable liquid. For example, the pharmaceutical formulation can be a dry powder, a lyophilized formulation. Suitable excipients will be known to those skilled in the art. For example, the "pharmaceutical formulation" or "composition" can be administered to the eye by subretinal or intravitreal administration. In either mode of administration, the "pharmaceutical formulation" or "composition" is provided as an injectable liquid. For example, injectable liquids may be provided as capsules or syringes. The compositions may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

Detailed description of the invention

recombinant nucleic acid

In one aspect, the present application provides a recombinant nucleic acid, which may comprise a nucleotide sequence encoding Frizzled receptor 4 (FZD4) or a truncation thereof. The Frizzled receptor 4 may comprise the amino acid sequence shown in SEQ ID NO.:3.

For example, the nucleotide sequence encoding Frizzled receptor 4 may comprise the nucleotide sequence in SEQ ID NO.:1.

For example, the nucleotide sequence encoding Frizzled receptor 4 may comprise a nucleotide sequence that is at least 90% homologous to SEQ ID NO.: 1, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to any one of the nucleotide sequences, and administration of said nucleotide sequence is capable of activating the Wnt signaling pathway And/or Norrin-β-catenin signaling pathway and/or increased expression of Frizzled receptor 4.

For example, the nucleotide sequence encoding Frizzled receptor 4 may comprise a nucleotide sequence that is completely complementary to a nucleotide sequence that is at least 90% homologous to SEQ ID NO.: 1, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to any one of the nucleotide sequences is completely complementary, and said The administration of the nucleotide sequence can activate the Wnt signaling pathway and/or the Norrin-β-catenin signaling pathway and/or increase the expression of Frizzled receptor 4.

For example, the nucleotide sequence encoding Frizzled receptor 4 may comprise a nucleotide sequence encoding a Frizzled receptor 4 truncation.

In one aspect, the nucleotide sequence encoding Frizzled receptor 4 may comprise a truncated body encoding Frizzled receptor 4. For example, the nucleotide sequence encoding Frizzled receptor 4 may comprise a nucleotide sequence encoding Frizzled receptor 4 domain A and domain B. For example, the domain A may be the Frizzled domain (CRD_FZ domain) of Frizzled receptor 4, and the domain A is used for binding to a ligand. For example, domain B may be the G protein-coupled receptor family 2 domain (7transmembrane receptor domain, 7tm_2 domain) of Frizzled receptor 4, and the domain B is used to transduce signals to downstream molecules of the pathway. For example, the nucleotide sequence encoding the domain A is the 40-483 nucleotide sequence shown in SEQ ID NO.:1, and the nucleotide sequence encoding the domain B is SEQ ID NO.: The nucleotide sequence at position 654-1509 shown in 1, and administration of said nucleotide sequence can activate Wnt signaling pathway and/or Norrin-β-catenin signaling pathway and/or increase the expression of Frizzled receptor 4.

In another aspect, the present application provides a recombinant nucleic acid, which may comprise DNA, cDNA and/or mRNA. For example, the recombinant nucleic acid comprises single-stranded recombinant nucleic acid and/or double-stranded recombinant nucleic acid.

In another aspect, the recombinant nucleic acid sequence of the present application may be DNA, RNA, cDNA or peptide nucleic acid (PNA). A nucleic acid sequence can be genomic, recombinant or synthetic. A nucleic acid sequence can be isolated or purified. A nucleic acid sequence can be single-stranded or double-stranded. For example, the nucleic acid sequence will encode Frizzled receptor 4 as described herein. Nucleic acid sequences can be derived by cloning, eg, using standard molecular cloning techniques including restriction, ligation, gel electrophoresis, eg as described in Sambrook et al. Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press). Nucleic acid sequences may be isolated, eg, using PCR techniques. Isolating means separating a nucleic acid sequence from any impurities and from other nucleic acid sequences and/or proteins that are naturally found in association with the nucleic acid sequence in its source. For example, nucleic acid molecules of the present application may also be free of cellular material, culture medium or other chemicals from purification/production processes. Nucleic acid sequences may be synthetic, for example produced by direct chemical synthesis. Nucleic acid sequences may be provided as naked nucleic acid, or may be provided complexed with proteins or lipids.

On the other hand, compared with the nucleotide sequence encoding the wild-type Frizzled receptor 4, the recombinant nucleic acid of the present application can increase the transcription level and significantly increase the expression level.

fusion nucleic acid

In one aspect, the present application provides a fusion nucleic acid comprising the recombinant nucleic acid encoding Frizzled receptor 4 or a truncation thereof described in the present application.

In another aspect, the fusion nucleic acid may also comprise one or more promoter sequences. For example, the promoter sequence may comprise CMV. For example, the promoter may be directly or indirectly linked to the nucleotide sequence of the recombinant nucleic acid. For example, the promoter may be operably linked to the nucleotide sequence of the recombinant nucleic acid. For example, the promoter may be operably linked to the nucleotide sequence of the recombinant nucleic acid. For example, the 3' end of the promoter may be directly or indirectly linked to the 5' end of the nucleotide sequence of the recombinant nucleic acid.

In another aspect, the fusion nucleic acid may also comprise one or more UTR sequences. For example, the UTR sequence may comprise a 5'-UTR sequence. For example, the UTR sequence may comprise a 3'-UTR sequence.

In another aspect, the promoter sequence may comprise one or more 5'-UTR sequences. For example, the 5'-UTR sequence may be directly or indirectly linked to the nucleotide sequence of the recombinant nucleic acid. For example, the 5'-UTR sequence can be operably linked to the nucleotide sequence of the recombinant nucleic acid. For example, the 3' end of the 5'-UTR sequence may be directly or indirectly linked to the 5' end of the nucleotide sequence of the recombinant nucleic acid.

In another aspect, the promoter sequence may comprise one or more 3'-UTR sequences. For example, the 3'-UTR sequence may be directly or indirectly linked to the nucleotide sequence of the recombinant nucleic acid. For example, the 3'-UTR sequence can be operably linked to the nucleotide sequence of the recombinant nucleic acid. For example, the 5' end of the 3'-UTR sequence may be directly or indirectly linked to the 3' end of the nucleotide sequence of the recombinant nucleic acid. For example, the 3'-UTR sequence may comprise a polyA sequence.

In another aspect, the fusion nucleic acid may also comprise a promoter sequence and a 5'-UTR sequence. For example, the promoter sequence and 5'-UTR sequence may be directly or indirectly linked to the nucleotide sequence of the recombinant nucleic acid. For example, the promoter sequence and 5'-UTR sequence can be operably linked to the nucleotide sequence of the recombinant nucleic acid. For example, the 3' end of the promoter sequence can be directly or indirectly connected to the 5' end of the nucleotide sequence of the recombinant nucleic acid, and the 3' end of the 5'-UTR sequence can be connected to the nucleotide sequence of the recombinant nucleic acid The 5' ends of the sequences can be linked directly or indirectly.

In another aspect, the fusion nucleic acid may also comprise a promoter sequence and a 3'-UTR sequence. For example, the promoter sequence and 3'-UTR sequence may be directly or indirectly linked to the nucleotide sequence of the recombinant nucleic acid. For example, the promoter sequence and 3'-UTR sequence can be operably linked to the nucleotide sequence of the recombinant nucleic acid. For example, the 3' end of the promoter sequence can be directly or indirectly connected to the 5' end of the nucleotide sequence of the recombinant nucleic acid, and the 5' end of the 3'-UTR sequence can be connected to the nucleotide sequence of the recombinant nucleic acid The 3' ends of the sequences can be linked directly or indirectly. For example, the 3'-UTR sequence may comprise a polyA sequence.

In another aspect, the fusion nucleic acid may further comprise a promoter sequence, a 5'-UTR sequence and a 3'-UTR sequence. For example, the promoter sequence, 5'-UTR sequence and 3'-UTR sequence may be directly or indirectly linked to the nucleotide sequence of the recombinant nucleic acid. For example, the promoter sequence, 5'-UTR sequence and 3'-UTR sequence may be operably linked to the nucleotide sequence of the recombinant nucleic acid. For example, the 3' end of the promoter sequence can be directly or indirectly connected to the 5' end of the nucleotide sequence of the recombinant nucleic acid, and the 3' end of the 5'-UTR sequence can be connected to the nucleotide sequence of the recombinant nucleic acid. The 5' end of the 3'-UTR sequence and the 3' end of the nucleotide sequence of the recombinant nucleic acid may be directly or indirectly connected. For example, the 3'-UTR sequence may comprise a polyA sequence.

In another aspect, said indirect linking can comprise linking through a linker. For example, the linker may comprise an oligonucleotide of length selected from the group consisting of 1-30 nucleotides, 1-15 nucleotides, and 3-6 nucleotides. For example, the linker may comprise an oligonucleotide of length selected from the group consisting of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, nucleotides, 8 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides.

For example, the linker may comprise a nucleotide linker sequence, which may be formed by restriction enzyme cleavage. For example, the restriction enzyme may comprise EcoRI and/or SalI.

carrier

In one aspect, the present application provides a vector, which may contain one or more whole, truncated and/or fragments of the recombinant nucleic acid and/or fusion nucleic acid described in the present application. For example, the vector may comprise a sequence encoding Frizzled receptor 4.

In another aspect, the vector may comprise one or more promoters. A promoter can mediate the expression of a nucleic acid sequence to which it is linked. Promoters can be constitutive or can be inducible. The promoter can direct non-specific expression in inner retinal cells, or neuron-specific expression. In the latter case, the promoter may direct cell type-specific expression, for example to optic ganglion cells. Suitable promoters will be known to those skilled in the art. For example, a suitable promoter may be selected from the group consisting of: L7, thy-1, recoverin, calbindin, human CMV, GAD-67, chicken beta actin, hSyn, Grm6, Grm6 enhancer SV40 fusion protein . Targeting can be achieved using cell-specific promoters, such as Grm6-SV40 for selective targeting to optic nerve cells. The Grm6 promoter is a fusion of the 200 base pair enhancer sequence of the Grm6 gene and the SV40 eukaryotic promoter. The Grm6 gene encodes the metabotropic glutamate receptor mGluR6 specific to optic nerve cells. The source of the Grm6 gene can be mouse and human. Ubiquitous expression can be achieved using pan-neuronal promoters, examples of which are known and available in the art. For example, it could be CAG. The CAG promoter is a fusion of the CMV early enhancer and the chicken β-actin promoter. For example, the promoter can be an immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. For example, the promoter can be elongation growth factor-1 alpha (EF-1 alpha). For example, other constitutive promoter sequences may also be used and may include, but are not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter , MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters may include, but are not limited to, actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Further, this application should not be limited to the use of constitutive promoters, inducible promoters may also be considered as part of this application. The use of an inducible promoter provides a molecular switch that turns on expression of a polynucleotide sequence operably linked to the inducible promoter when expression is desired, or turns off expression when no expression is desired. Examples of inducible promoters may include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. For example, one or more of said promoters may be the CMV, CAG and/or neural specific promoter SYN. For example the promoter may be CMV.

For example, the one or more promoter CMVs can be located anywhere in the vector, and the CMVs can promote the expression of the Frizzled receptor 4. For example, CMV may precede the sequence encoding Frizzled receptor 4.

In another aspect, the vector may comprise one or more enhancers. For example, the one or more enhancers can be located anywhere in the vector, and the enhancers can enhance the expression of Frizzled receptor 4. For example, the enhancer can be located upstream and/or downstream near the initiation site.

In another aspect, the vector may comprise one or more transcription termination signals, polyadenylation sequences, origins of replication, selectable markers, nucleic acid restriction sites and/or homologous recombination sites. For example, the vector may contain the gene sequence of one or more selectable markers to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance and green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

For example, one or more of said transcription termination signal, said polyadenylation sequence, said origin of replication, said selectable marker, said nucleic acid restriction site and/or said homologous recombination site may be associated with One or more promoters are operably linked to one or more of said enhancers. For example, the transcription termination signal, the polyadenylation sequence, the origin of replication, the selectable marker, the nucleic acid restriction site and/or the homologous recombination site may be located close to the promoter and /or upstream and/or downstream of an enhancer.

On the other hand, many expression vectors can be used to express Frizzled receptor 4 in mammalian cells (preferably human). For example, the vector may be selected from the group consisting of lentiviral vectors, DNA viral vectors, retroviral vectors, adenoviral vectors and adeno-associated viral vectors. For example, the vector may be selected from an adeno-associated virus. For example, the serotype of the vector may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and/or AAVNc80, or variants and combinations thereof. For example, the adeno-associated virus can be selected from AAV2, AAV5, AAV7 and/or AAV8, or combinations thereof. For example, the adeno-associated virus may be selected from AAV2.

For example, the AAV2 vector can be AAV2/2, AAV2/5, AAV2/8 or AAV2/9. For example, the AAV2 vector may comprise pAAV-RC5-Amp, RC8-cap, AAV2/8, AAV-helper-Amp, AAV-helper. For example, the viral vector comprises the plasmid AAV-MCS.

In another aspect, viral vectors can be modified to delete any non-essential sequences. For example, AAV viruses can be modified to delete all or part of the IX, Ela and/or Elb genes. Replication of wild-type AAV can be very inefficient in the absence of a helper virus such as adenovirus. For example, the replication gene and/or capsid gene of a recombinant adeno-associated virus can be provided in trans in the pRep/Cap plasmid. For example, only the 2ITR of the AAV genome is retained and packaged into the virion, while the required adenoviral genes are provided by the adenovirus or another plasmid. For example, the above-mentioned modifications can also be made to lentiviral vectors.

In another aspect, non-viral vectors such as plasmids can be used in conjunction with auxiliaries to facilitate uptake of the vector by target cells. For example, adjuvants may include polycationic agents. For example, delivery systems such as liposome-based delivery systems can be used. Vectors for use in this application may be suitable for use in vivo or in vitro, and may be suitable for use in humans.

host cell

In one aspect, the present application provides a host cell. For example, the host cell may contain one or more whole, truncation and/or fragments of recombinant nucleic acids, fusion nucleic acids described herein, and/or vectors. For example, one or more exogenous recombinant nucleic acids or fusion nucleic acids described in the present application are integrated in the chromosome of the host cell, truncated and/or fragments thereof.

In another aspect, the host cell comprises a mammalian cell. For example, the host cells comprise human cells. For example, the host cells can comprise 293T cells, photoreceptor cells, other visual cells and/or optic nerve cells. For example, the photoreceptor cells may comprise cone cells (cone cells) and/or rod cells (rod cells). For example, the other visual cells may include, but are not limited to, light-giving bipolar cells, light-withdrawing bipolar cells, horizontal cells, Muller cells, retinal ganglion cells, and/or amacrine cells.

Pharmaceutical formulations, treatments and preparations for pharmaceutical use

In one aspect, the present application provides a pharmaceutical preparation. For example, the pharmaceutical preparation may comprise one or more recombinant nucleic acids of the present application, one or more fusion nucleic acids of the present application and/or one or more carriers of the present application, and pharmaceutically acceptable carriers or excipients . A pharmaceutically acceptable carrier or excipient may refer to any ingredient that is not therapeutically active and has acceptable toxicity, such as buffers, solvents, tonicity agents, stabilizers, antioxidants that may be used in formulating pharmaceutical products , surfactants or polymers. For example, the formulation may be a liquid formulation.

For example, the pharmaceutical formulation may contain one or more carriers of the present application. For example, the vector may contain a recombinant nucleic acid and/or a fusion nucleic acid, and the recombinant nucleic acid may contain a nucleotide sequence encoding Frizzled receptor 4.

In another aspect, the pharmaceutical formulation comprises a carrier of the present application. For example, the content of the vector comprises 1×10 ⁹ -1×10 ¹⁶ viruses/ml. For example, the content of the vector comprises 1×10 ¹¹ -1×10 ¹³ viruses/ml. For example, the content of the vector comprises 2×10 ¹¹ -1×10 ¹² viruses/ml. For example, the content of the vector comprises 1×10 ⁹ viruses/ml, 1×10 ¹⁰ viruses/ml, 1×10 ¹¹ viruses/ml, 2×10 ¹¹ viruses/ml, 2×10 ¹¹ viruses /ml, 4× ¹⁰¹¹ viruses/ml, 6× ¹⁰¹¹ viruses/ml, 8×1011 viruses/ml, ⁹ × ¹⁰¹¹ viruses/ml, 1× ¹⁰¹² viruses/ml, 2× 10 ¹² viruses/ml, 3×10 ¹² viruses/ml, 5×10 ¹² viruses/ml, 1×10 ¹³ viruses/ml, 1×10 ¹⁴ viruses/ml, 1×10 ¹⁵ viruses/ml ml, 1×10 ¹⁶ viruses/ml.

For example, when the pharmaceutical preparation contains a carrier of the present application, the administration of the pharmaceutical preparation may be by administering a carrier to a cell or a subject. For example, when the pharmaceutical preparation contains two or more carriers of the present application, the administration of the pharmaceutical preparation may be to administer the two or more carriers to cells or subjects simultaneously. For example, when the pharmaceutical preparation contains two or more carriers of the present application, the administration of the pharmaceutical preparation may be to administer the two or more carriers to cells or subjects in any sequence. . For example, the pharmaceutical formulation can be injected intraocularly. For example, the formulation can be injected intravitreally.

In another aspect, the present application provides a method of restoring vision and/or treating an eye disease in a subject. For example, the eye disease can be retinopathy. For example, the retinopathy may be familial exudative vitreoretinopathy (FEVR). For example, a pharmaceutical formulation described herein is administered to a cell or a subject.

In another aspect, the present application provides a method of increasing the expression and/or activity of Frizzled receptor 4 (FZD4). For example, the drug is used to activate the Wnt signaling pathway and/or the Norrin-β-catenin signaling pathway. For example, a pharmaceutical formulation described herein is administered to a cell or a subject.

In another aspect, the present application provides a use of a vector, a recombinant nucleic acid, a fusion nucleic acid and/or a pharmaceutical preparation in the preparation of a medicament. For example, the medicament is used to restore vision and/or treat ocular diseases in a subject. For example, the eye disease can be retinopathy. For example, the retinopathy may be familial exudative vitreoretinopathy (FEVR). For example, the medicament is used to increase the expression and/or activity of Frizzled receptor 4 (FZD4). For example, the drug is used to activate the Wnt signaling pathway and/or the Norrin-β-catenin signaling pathway.

In another aspect, the present application provides a vector, recombinant nucleic acid, fusion nucleic acid and/or pharmaceutical preparation. For example, it is used to restore vision and/or treat ocular diseases in a subject. For example, the eye disease can be retinopathy. For example, the retinopathy may be familial exudative vitreoretinopathy (FEVR). For example, it is used to increase the expression and/or activity of Frizzled receptor 4 (FZD4). For example, the drug is used to activate the Wnt signaling pathway and/or the Norrin-β-catenin signaling pathway.

For example, the pharmaceutical preparation can cause long-term high expression of the Frizzled receptor 4 (FZD4) in retinal cells. For example, the high expression may mean that after administration of the pharmaceutical preparation described in the present application, the protein expression in cells or subjects may be more than 1.1 times, more than 1.3 times, more than 1.5 times, more than two times, or three times that of those without administration. more than five times more than ten times more than twenty times more than twenty-two times more than thirty times more than fifty times more than one hundred times more than five hundred times More than a hundred times. For example, the high expression may mean that after administration of the pharmaceutical preparation described in the present application, the protein expression level in cells or subjects may be more than 1.1 times, more than 1.3 times, more than 1.5 times, or twice the expression level after administration of the control vector. more than three times more than five times more than ten times more than twenty times more than twenty-two times more than thirty times more than fifty times more than one hundred times more than five hundred times Or more than fifteen hundred times. For example, the long-acting may refer to, after administration of the carrier or pharmaceutical preparation of the present application, compared with no administration or administration of a control vector in cells or subjects, it can be at least 7 days, at least 15 days, at least 20 days, at least Maintain the high expression level for 30 days, at least 45 days, at least 60 days, at least 90 days, or at least three months, at least four months, at least five months, at least six months.

For example, the pharmaceutical preparation can activate Wnt signaling pathway and/or Norrin-β-catenin signaling pathway in a long-lasting manner. For example, the activation may refer to that after administration of the pharmaceutical preparation described in the present application, the activation and/or inhibition level of the Wnt signaling pathway and/or Norrin-β-catenin signaling pathway downstream proteins in the cells or subjects may be that of the unadministered More than 1.1 times, more than 1.3 times, more than 1.5 times, more than twice, more than three times, more than five times, more than ten times, more than twenty times, more than twenty-two times, more than thirty times, more than fifty times, one More than a hundred times, more than five hundred times, more than a thousand times, or more than fifteen hundred times. For example, the activation may refer to the activation and/or inhibition level of Wnt signaling pathway and/or Norrin-β-catenin signaling pathway downstream proteins in cells or subjects after administration of the pharmaceutical preparation described in the present application. 1.1 times or more, 1.3 times or more, 1.5 times or more, 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 22 times or more, 30 times or more, 50 times or more of the expression amount More than one hundred times, more than five hundred times, more than one thousand times, or more than fifteen hundred times. For example, the long-acting may refer to, after administration of the carrier or pharmaceutical preparation of the present application, compared with no administration or administration of a control vector in cells or subjects, it can be at least 7 days, at least 15 days, at least 20 days, at least Maintain the Wnt signaling pathway and/or Norrin-β- Activation and/or inhibition levels of proteins downstream of the catenin signaling pathway.

For example, intraocular injection of the pharmaceutical formulation may not cause significant inflammatory reactions or other complications in the subject. For example, the inflammatory response or other complications may be increased intraocular pressure, conjunctival hyperemia, ocular inflammation, fundus hemorrhage or lens whitish damage, increased ocular discharge and/or endophthalmitis.

In this application, the subject may include humans and non-human animals. For example, the subject can include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys; for example, the subjects can include DBA/2J mice. In the present application, the cells may comprise bacterial cells (e.g., Escherichia coli), yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or myeloma cells, 293T cells.

Preparation

In one aspect, the present application provides a method for preparing Frizzled receptor 4. For example, the full-length nucleotide sequence or fragments of the recombinant nucleic acid and/or fusion nucleic acid of the present application can be obtained by PCR amplification, recombination or artificial synthesis. For example, for the PCR amplification method, primers can be designed according to the published relevant nucleotide sequences, especially the open reading frame sequence, and cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used to The library is used as a template to amplify related sequences. For example, when the sequence is long, two or more PCR amplifications can be performed, and then the amplified fragments can be spliced together in the correct order. For example, the DNA sequence encoding Frizzled receptor 4 can be obtained entirely through chemical synthesis, and then the DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. For example, a recombinant nucleic acid, fusion nucleic acid, vector or host cell of the present application can be isolated.

In another aspect, after obtaining the sequences of the present application, recombinant methods can be used to obtain said sequences in large quantities. For example, it can be cloned into a vector, then transformed into a host cell, and then the relevant sequence can be isolated from the proliferated host cell by conventional culture and isolation methods. For example, related sequences can also be synthesized by artificial synthesis, and fragments with very long sequences can be obtained by first synthesizing multiple small fragments and then connecting them. For example, the nucleic acid sequence of the present application can be obtained by amplifying DNA/RNA using PCR technology. Primers for PCR can be appropriately selected based on the sequence information of the present application disclosed herein, and can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

On the other hand, the nucleotide sequence encoding Frizzled receptor 4 of the present application can be introduced into host cells (such as mammalian cells) by conventional techniques. For example, it includes transducing the recombinant nucleic acid, fusion nucleic acid or vector of the present application into host cells. For example, the obtained host cells can be cultured by conventional methods to express Frizzled receptor 4 encoded by the gene of the present application. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter can be induced by a suitable method (such as temperature conversion or chemical induction), and the cells can be cultured for a period of time.

In another aspect, the Frizzled receptor 4 can be expressed in the host cell, or on the cell membrane, or secreted outside the cell. For example, Frizzled receptor 4 can be isolated and purified by various separation methods utilizing physical, chemical and other properties. Examples of these methods well known to those skilled in the art include, but are not limited to: conventional refolding treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic breakdown, ultratreatment, ultracentrifugation, molecular sieve chromatography (coagulation Gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The application also provides the following implementation modes:

1. A recombinant nucleic acid comprising a nucleotide sequence encoding Frizzled receptor 4 (FZD4) or a truncation thereof.

2. The recombinant nucleic acid according to embodiment 1, comprising a nucleotide sequence selected from the group consisting of:

a) the nucleotide sequence shown in SEQ ID NO.:1;

b) a nucleotide sequence having ≥99% homology to the nucleotide sequence shown in SEQ ID NO.:1;

c) a nucleotide sequence having ≥98% homology to the nucleotide sequence shown in SEQ ID NO.:1;

d) a nucleotide sequence having ≥95% homology to the nucleotide sequence shown in SEQ ID NO.:1;

e) a nucleotide sequence having ≥90% homology to the nucleotide sequence shown in SEQ ID NO.:1;

f) a nucleotide sequence having ≥85% homology to the nucleotide sequence shown in SEQ ID NO.:1; and

g) A nucleotide sequence having ≥80% homology to the nucleotide sequence shown in SEQ ID NO.:1.

3. The recombinant nucleic acid according to any one of the embodiments 1-2, which comprises a nucleotide sequence completely selected from the following group

Complementary Nucleotide Sequence:

a) the nucleotide sequence shown in SEQ ID NO.:1;

b) a nucleotide sequence having ≥99% homology to the nucleotide sequence shown in SEQ ID NO.:1;

c) a nucleotide sequence having ≥98% homology to the nucleotide sequence shown in SEQ ID NO.:1;

d) a nucleotide sequence having ≥95% homology to the nucleotide sequence shown in SEQ ID NO.:1;

e) a nucleotide sequence having ≥90% homology to the nucleotide sequence shown in SEQ ID NO.:1;

f) a nucleotide sequence having ≥85% homology to the nucleotide sequence shown in SEQ ID NO.:1; and

g) A nucleotide sequence having ≥80% homology to the nucleotide sequence shown in SEQ ID NO.:1.

4. According to the recombinant nucleic acid shown in embodiment 1, the truncated body comprises domain A and domain B, and the nucleotide sequence encoding domain A is the 40th- The nucleotide sequence at position 483, the nucleotide sequence encoding the structural domain B is the nucleotide sequence at position 654-1509 shown in SEQ ID NO.:1.

5. The recombinant nucleic acid according to any one of embodiments 1-4, which comprises DNA, cDNA and/or mRNA.

6. The recombinant nucleic acid according to any one of embodiments 1-5, which comprises single-stranded recombinant nucleic acid and/or double-stranded recombinant nucleic acid.

7. A fusion nucleic acid comprising the nucleotide sequence of the recombinant nucleic acid according to any one of embodiments 1-6.

8. The fusion nucleic acid according to embodiment 7, comprising a promoter sequence.

9. The fusion nucleic acid according to embodiment 8, said promoter comprising CMV.

10. The fusion nucleic acid according to any one of embodiments 8-9, the promoter is directly or indirectly linked to the nucleotide sequence of the recombinant nucleic acid.

11. The fusion nucleic acid according to any one of embodiments 8-10, the 3' end of the promoter is directly or indirectly connected to the 5' end of the nucleotide sequence of the recombinant nucleic acid.

12. The fusion nucleic acid according to any one of embodiments 7-11, comprising a UTR sequence.

13. The fusion nucleic acid according to any one of embodiments 7-12, comprising a 5'-UTR sequence.

14. The fusion nucleic acid according to embodiment 13, the 5'-UTR sequence is directly or indirectly linked to the nucleotide sequence of the recombinant nucleic acid.

15. The fusion nucleic acid according to any one of embodiments 13-14, the 3' end of the 5'-UTR sequence is directly or indirectly connected to the 5' end of the nucleotide sequence of the recombinant nucleic acid.

16. The fusion nucleic acid according to any one of embodiments 7-15, comprising a 3'-UTR sequence.

17. The fusion nucleic acid according to embodiment 16, the 3'-UTR sequence comprising a polyA sequence.

18. The fusion nucleic acid according to any one of embodiments 16-17, the 3'-UTR sequence is directly or indirectly linked to the nucleotide sequence of the recombinant nucleic acid.

19. The fusion nucleic acid according to any one of embodiments 16-18, the 5' end of the 3'-UTR sequence is directly or indirectly connected to the 3' end of the nucleotide sequence of the recombinant nucleic acid.

20. The fusion nucleic acid according to any one of embodiments 10-19, said indirect linking comprising linking via a linker.

21. The fusion nucleic acid according to embodiment 20, said linker comprising an oligonucleotide selected from the group of lengths of 1-30 nucleotides, 1-15 nucleotides and 3-6 nucleosides acid.

22. The fusion nucleic acid according to any one of embodiments 20-21, the linker comprising a nucleotide linker sequence formed by restriction endonuclease cleavage.

23. The fusion nucleic acid according to embodiment 22, said restriction enzyme comprising EcoRI and/or SalI.

24. A vector comprising the recombinant nucleic acid according to any one of embodiments 1-6 and/or the fusion nucleic acid according to any one of embodiments 7-23.

25. The vector of embodiment 24, comprising a promoter.

26. The vector according to embodiment 25, the promoter is operably linked to the nucleotide sequence of the recombinant nucleic acid and/or the fusion nucleic acid.

27. The vector according to any one of embodiments 24-26, comprising an enhancer, a transcription termination signal, a polyadenylation sequence, an origin of replication, a selectable marker, a nucleic acid restriction site and/or homologous recombination location.

28. The vector according to embodiment 27, said enhancer, said transcription termination signal, said polyadenylation sequence, said origin of replication, said selectable marker, said nucleic acid restriction site and/or Or said homologous recombination site is operably linked to said promoter.

29. The vector according to any one of embodiments 24-28, comprising a plasmid and/or a viral vector.

30. The vector of embodiment 29, wherein the viral vector is a lentiviral vector, a DNA viral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.

31. The vector according to any one of embodiments 29-30, wherein the viral vector is an adeno-associated virus having a serotype selected from the group consisting of: AAV1, AAV2, AAV5, AAV6, AAV7 , AAV8, AAV9 and AAVrh10.

32. The vector according to any one of embodiments 29-31, wherein the viral vector comprises plasmid AAV-MCS.

33. The vector according to any one of embodiments 24-32 for expressing Frizzled receptor 4.

34. An isolated cell comprising the recombinant nucleic acid according to any one of embodiments 1-6, the fusion nucleic acid according to any one of embodiments 7-23 and/or any one of embodiments 24-33 The vector described in Item 1, or the exogenous recombinant nucleic acid as described in any one of Embodiments 1-6 or the fusion nucleic acid described in any one of Embodiments 7-23 is integrated in the chromosome of the cell.

35. The cell of embodiment 34, comprising a mammalian cell.

36. The cell of embodiment 35, said mammal comprising a human.

37. The cell according to any one of embodiments 34-36, selected from the group consisting of 293T cells, photoreceptor cells, biganglionic cells and optic nerve cells.

38. The cell of embodiment 37, said photoreceptor cells comprising cone cells and/or rod cells.

39. The cell according to any one of embodiments 34-38, selected from the group consisting of rods, cones, light-giving bipolar cells, light-withdrawing bipolar cells, horizontal cells, retinal ganglion cells and amacrine cells.

40. A pharmaceutical preparation comprising the recombinant nucleic acid according to any one of embodiments 1-6, the fusion nucleic acid according to any one of embodiments 7-23 and/or any one of embodiments 24-33 The carrier, and a pharmaceutically acceptable adjuvant.

41. The pharmaceutical formulation according to embodiment 40, comprising a lyophilized formulation and/or a liquid formulation.

42. The pharmaceutical formulation according to any one of embodiments 40-41, wherein the content of the carrier comprises 1×10 ⁹ -1×10 ¹⁶ viruses/ml.

43. The pharmaceutical formulation according to any one of embodiments 40-42, wherein the content of the carrier comprises 1×10 ¹¹ -1×10 ¹³ viruses/ml.

44. The pharmaceutical formulation according to any one of embodiments 40-43, wherein the content of the carrier comprises 2×10 ¹¹ -1×10 ¹² viruses/ml.

45. The pharmaceutical formulation according to any one of embodiments 40-44, wherein the pharmaceutical formulation is for the treatment of an ocular disease comprising familial exudative vitreoretinopathy (FEVR).

46. The pharmaceutical formulation according to any one of embodiments 40-45, wherein said pharmaceutical formulation is used to increase the expression and/or activity of Frizzled receptor 4 (FZD4).

47. The pharmaceutical formulation according to any one of embodiments 40-46, wherein the pharmaceutical formulation is used to increase the expression of Frizzled receptor 4 (FZD4) to three-fold or more than the background expression.

48. The pharmaceutical formulation according to any one of embodiments 40-47, wherein the pharmaceutical formulation is used to increase the expression of Frizzled receptor 4 (FZD4) to five-fold or more than background expression.

49. A method for increasing the expression and/or activity of Frizzled receptor 4 (FZD4) for a long period of time, said method comprising administering a therapeutically effective amount of the recombinant protein described in any one of embodiments 1-6 to a subject in need thereof The nucleic acid, the fusion nucleic acid according to any one of embodiments 7-23, the carrier according to any one of embodiments 24-33 and/or the pharmaceutical preparation according to any one of embodiments 40-48.

50. The method of embodiment 49, wherein the vector comprises an adeno-associated viral vector.

51. The method according to any one of embodiments 49-50, which activates the Wnt signaling pathway and/or the Norrin-β-catenin signaling pathway.

52. An application comprising administering to a subject in need a therapeutically effective amount of the recombinant nucleic acid according to any one of embodiments 1-6, the fusion nucleic acid according to any one of embodiments 7-23, The carrier according to any one of embodiments 24-33 and/or the pharmaceutical preparation according to any one of embodiments 40-48.

53. The use according to embodiment 52 comprising the treatment of ocular diseases.

54. The use according to embodiment 53, said ocular disease comprising the treatment of familial exudative vitreoretinopathy (FEVR).

55. The use according to embodiment 54, wherein the familial exudative vitreoretinopathy is caused by Frizzled receptor 4 mutation.

56. The use according to any one of embodiments 52-55, wherein the vector comprises an adeno-associated virus vector.

57. The use according to any one of embodiments 52-56, the recombinant nucleic acid, the fusion nucleic acid, the vector and/or the pharmaceutical preparation are injected intraocularly.

58. The use according to embodiment 57, wherein the intraocular injection comprises intravitreal injection.

59. The use according to any one of embodiments 52-58, said subject comprising a human and a non-human mammal.

60. The use according to any one of embodiments 52-59, which increases the expression and/or activity of Frizzled receptor 4 (FZD4).

61. The use according to any one of embodiments 52-60, which increases the expression and/or activity of Frizzled receptor 4 (FZD4) for 3 months or more.

62. The use according to any one of embodiments 52-61, which increases the expression and/or activity of Frizzled receptor 4 (FZD4) for 6 months or more.

63. The use according to any one of embodiments 52-62, which activates the Wnt signaling pathway and/or the Norrin-β-catenin signaling pathway.

64. The recombinant nucleic acid according to any one of the embodiments 1-6, the fusion nucleic acid according to any one of the embodiments 7-23 and/or the carrier according to any one of the embodiments 24-33 in the preparation of drugs Use in , wherein the medicament is used to restore the vision of a subject and/or treat eye diseases.

65. The use according to embodiment 64, the ocular disease comprising retinopathy.

66. The use according to any one of embodiments 64-65, wherein the ocular disease comprises familial exudative vitreoretinopathy (FEVR).

67. A recombinant nucleic acid according to any one of embodiments 1-6, a fusion nucleic acid according to any one of embodiments 7-23 and/or a carrier according to any one of embodiments 24-33, It is used to restore vision and/or treat ocular diseases in a subject.

68. A recombinant nucleic acid according to any one of embodiments 1-6, a fusion nucleic acid according to any one of embodiments 7-23 and/or a carrier according to any one of embodiments 24-33, It is used in the treatment of retinopathy.

69. A recombinant nucleic acid according to any one of embodiments 1-6, a fusion nucleic acid according to any one of embodiments 7-23 and/or a carrier according to any one of embodiments 24-33, It is used in the treatment of familial exudative vitreoretinopathy (FEVR).

70. A method for producing Frizzled receptor 4, comprising culturing the cell according to any one of embodiments 34-39.

71. The method of embodiment 70, further comprising the step of isolating said Frizzled receptor 4.

Not intending to be limited by any theory, the following examples are only for explaining the fusion protein, preparation method and application of the application, and are not intended to limit the scope of the invention of the application.

Example

Example 1 Virus vector construction and virus packaging purification

This application optimizes the gene sequence of the wild-type applicant Frizzled protein receptor 4 (FZD4), and the expression level of the unoptimized wild DNA coding sequence is very low. Through the sequence optimization, analysis and test screening, the nucleotide sequence encoding human Frizzled receptor 4 of the present application was obtained. Wherein, the amino acid sequence of human Frizzled receptor 4 is shown in SEQ ID NO.: 3, and the nucleotide sequence of wild-type encoding human Frizzled receptor 4 is shown in SEQ ID NO.: 2. The sequence optimized in this application The nucleotide sequence is shown in SEQ ID NO.:1.

As shown in Figure 1, the sequence similarity between the recombinant nucleic acid of the present application and the wild-type nucleotide sequence after sequence comparison is 77.5%. As shown in FIG. 2 , a schematic diagram of the protein structure of the Frizzled receptor 4 transcript. Among them, domain A encodes the Frizzled domain (CRD_FZ domain) of Frizzled receptor 4, and domain B encodes the G protein-coupled receptor family 2 domain (7transmembrane receptor domain, 7tm_2domain) of Frizzled receptor 4.

Viral vector construction. The recombinant human Frizzled receptor 4 gene (nucleotide sequence shown in SEQ ID NO: 1) of the present application was added with two enzyme cutting sites, EcoRI and SalI, and primers were designed for the new gene and amplified by PCR to obtain The product was digested with the AAV-MCS plasmid vector with EcoRI and SalI respectively, the digested product was recovered, and ligated with T4 DNA ligase (Ligase) overnight. The ligation product was transformed into competent cells to obtain recombinant adeno-associated virus expression vector.

Screening and identification of recombinants. The recombinant adeno-associated virus expression vector obtained by spreading on the LB plate showed blue spots and white spots after culturing at 37°C, and the white ones were recombinant clones. White colonies were picked and added to LB liquid medium containing ampicillin (Amp) at a concentration of 100 mg/L, and cultured at 37° C. and 200 rpm for 8 hours. After culturing, the bacterial liquid was taken, and the plasmid was extracted. For the extraction steps of the plasmid, refer to the extraction kit (Biomiga) and instructions. Take 1 microliter of the plasmid as a template for PCR amplification. The PCR amplification program is shown in Table 1.

Table 1 PCR amplification program

The specific primers are:

Forward primer (1F): 5'-TTGGCAAAGAATTGGGATTC-3' (sequence shown in SEQ ID NO.:4);

Reverse primer (1R): 5'-TGCTCGAGAGATCTACGGGT-3' (sequence shown in SEQ ID NO.:5).

As shown in FIG. 3 , the PCR product was detected by electrophoresis, and a target band with a size of about 1614 bp was obtained. The identification result indicated that the clone contained the target gene.

Bacterial fluid preservation, PCR amplification and fragment sequencing. Take 1mL of the identified bacterial liquid and mix it with sterilized glycerol at a ratio of 1:3, store it at -80°C, and perform bacterial liquid sequencing. The sequence obtained by sequencing was compared with the recombinant human type II mitochondrial dynein-like GTPase gene. The recombinant adeno-associated virus expression vector AAV-MCS-rAAV2/2-rhFZD4 with the correct sequence was obtained successfully.

As shown in FIG. 4 , a schematic diagram of the structure of the expression vector AAV-MCS-rAAV2/2-rhFZD4.

Encapsulation of recombinant adeno-associated virus rAAV2/2-rhFZD4. The day before transfection, inoculate 293T cells in a 225cm ² cell culture flask at a seeding density of 3.0×10 ⁷ cells/mL. The medium is DMEM containing 10% bovine serum, and placed at 37°C containing 5% CO ₂ Cultivate overnight in the incubator; on the day of transfection, the medium of the cells was changed, and the culture was continued with fresh DMEM medium containing 10% bovine serum. When the cells grow to 80-90%, the medium is discarded, and the transfection is carried out with the PlasmidTrans II (VGTC) transfection kit (Invitrogen). The specific steps are: each transfection bottle takes pAdHelper (BioVector), pAAV-r2c5 (BioVector), the AAV-MCS-rAAV2/2-rhFZD4 plasmid of the present application were mixed according to the ratio required by the instructions, and mixed with PlasmidTrans II (VGTC) (transfection reagent) added to DMEM in a 1.5mL sterile Ep tube, the number is Reagent A, stand still at room temperature for 10-15 minutes; mix reagent A with 30mL DMEM containing 10% bovine serum, number reagent B; add reagent B evenly to the cell culture bottle, and place in an incubator at 37°C containing 5% CO ₂ 16 hours after transfection, the culture medium was changed to DMEM containing 10% bovine serum. After 48 hours of transfection, the cells were collected, resuspended in PBS, and repeatedly frozen and thawed 3 times to obtain the cells containing the viral vector rAAV2/2-rhFZD4 described in this application.

Purification and concentration of recombinant adeno-associated virus rAAV2/2-rhFZD4. Three steps of chloroform treatment-PEG/NaCl precipitation-chloroform extraction were used to separate, concentrate and purify the recombinant adeno-associated virus rAAV2/2-rhFZD4.

Virus purity verification. Fill SDS-PAGE separating gel and stacking gel, the concentration of separating gel is 10%. Add 15 μg to each sample well. After electrophoresis, stain with Coomassie Brilliant Blue and destain with corresponding destaining solution until clear bands with low background appear.

As shown in Figure 5, the recombinant adeno-associated virus rAAV2/2-rhFZD4 had clear bands with normal proportions, no visible bands, and a purity of over 99%.

The titer of recombinant adeno-associated virus rAAV2/2-rhFZD4 was determined by fluorescent quantitative PCR. Experimental materials: SYBRⅡ (takara); target fragment primer (20uM), accurate quantification of the target plasmid for packaging virus, fluorescent quantitative detection of the virus to be tested, PCR reaction conditions in the PCR eight-tube (Bio-red) are pre-denaturation: 95 °C for 10 minutes; cycle: 95°C for 15 seconds, 60°C for 1 minute. Finally, the genome titer of recombinant adeno-associated virus rAAV2/2-rhFZD4 was determined to be 1×10 ¹² vg/mL.

Referring to the above method, the viral vector rAAV2/2-hFZD4 expressing wild-type human Frizzled receptor 4 was constructed, isolated and purified.

Example 2 Rabbit Intravitreal Injection Experiment

Take 24 rabbits and divide them into 3 groups, divided into experimental group A (this application rAAV2/2-rhFZD4), experimental group B (wild type rAAV2/2-hFZD4) and control group (rAAV2/2-ZsGreen, source: Shandong Weiwei True Biotechnology Co., Ltd.), pipetting 50 microliters of 1×10 ¹² vg/mL rAAV2/2-rhFZD4, rAAV2/2-hFZD4 and rAAV2/2-ZsGreen to puncture the ciliary body 3 mm away from the limbus The flat part enters the vitreous cavity for injection into the vitreous cavity. Rabbits in each group were examined with slit lamp and intraocular pressure at 1, 3, 7, and 30 days after operation. All rabbits had no obvious abnormalities, no conjunctival hyperemia, discharge, endophthalmitis, and no increase in intraocular pressure. After 30 days of intravitreal injection, the retina was stripped, and RNA and protein were extracted for expression detection.

The expression of human Frizzled receptor 4 was detected by real-time fluorescent quantitative PCR. Using TRIZOL kit to extract the total RNA from rabbit retina of experimental group A (this application rAAV2/2-rhFZD4), experimental group B (wild-type rAAV2/2-hFZD4) and control group (rAAV2/2-ZsGreen) and synthesize by reverse transcription cDNA template. Then, according to the primer design principle of fluorescent quantitative PCR, primers were designed with primer premier 5 (rabbit-actin was used as an internal reference):

Rabbit-actin-forward primer: CCTTCTACAACGAGCTGCGC (sequence shown in SEQ ID NO.:6);

Rabbit-actin-reverse primer: TACAGGGACAGCACGGCC (sequence shown in SEQ ID NO.:7);

hFZD4-forward primer: CCTGAGCGCCCCATCATATT (sequence shown in SEQ ID NO.:8);

hFZD4-reverse primer: GAACAGGTTCTGCTGCCTCT (sequence shown in SEQ ID NO.:9);

rhFZD4-forward primer: ACTGGGCTGTGTTATGTCGG (sequence shown in SEQ ID NO.: 10);

rhFZD4-reverse primer: GTTTGTCGGTTTTCGTCCCG (sequence shown in SEQ ID NO.: 11).

Fluorescent quantitative PCR reaction. Fluorescent quantitative PCR was performed on a real-time fluorescent quantitative PCR instrument (Real-time PCR Detection System). Add 12.5 microliters of SYBR Green mix, 8 microliters of ddH ₂ O, 1 microliter of each pair of primers, 2.5 microliters of cDNA samples, and 25 microliters of the total system into a 0.2 mL PCR reaction tube. Each sample was used to amplify both the target gene and the internal reference gene rabbit-actin, and the amplification of each gene was performed in triplicate. In actual sample addition, in order to reduce errors, the common reagents in each PCR reaction tube can be added together and then divided. After sample addition, fluorescent quantitative PCR was performed. According to the reaction program of 95°C pre-denaturation for 1 second, 94°C denaturation for 15 seconds, 55°C annealing for 15 seconds, and 72°C extension for 45 seconds, the amplification was performed in a total of 40 cycles, and fluorescence signals were collected during the extension stage of each cycle. After the reaction, do the melting curve analysis at 94°C-55°C. The relative quantitative method is used to study the difference in gene expression. This method does not need to make a standard curve. The housekeeping gene rabbit-actin is used as the internal reference gene, and the analysis software that comes with the instrument can automatically generate the expression value.

As shown in Figure 6, the relative expression levels of gene mRNA in experimental group A and experimental group B were higher than those in the control group, and the relative expression level of mRNA in experimental group A was higher than that in experimental group B. It shows that the Frizzled receptor 4 expression level of the viral vector rAAV2/2-rhFZD4 of the present application in the retina is significantly higher than that of the wild type.

Western blot was used to detect the expression of human Frizzled receptor 4. The primary antibody is rabbit anti-human Frizzled receptor 4, from sigma, and the secondary antibody is goat anti-rabbit, from sigma. Separate the retinas of the rabbit eyeballs of experimental group A (this application rAAV2/2-rhFZD4), experimental group B (wild type rAAV2/2-hFZD4) and control group (rAAV2/2-ZsGreen), and use 100 microliters/50mg tissue Add the corresponding volume of RIPA lysate, homogenize with a homogenizer, and centrifuge to collect the supernatant. After the protein concentration was determined by the BCA method, the loading volume of each group was calculated based on 50 micrograms of total protein, and SDS-PAGE gel electrophoresis and western blot detection were performed. Chemiluminescent reagent (ECL) development was performed after antibody incubation.

As shown in Figure 7, the relative expression level of human Frizzled receptor 4 in experimental group A was significantly higher than that in experimental group B and the control group, with a significant difference P>0.05, indicating that the expression of human Frizzled receptor 4 on the retina of experimental group A The level was significantly improved, about 2 times and 5 times higher than that of the experimental group B and the control group, respectively.

The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of this application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

sequence listing

<110> Wuhan Neophth Biotechnology Co., Ltd.

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Claims (15)

1. A recombinant nucleic acid, the nucleotide sequence of which is shown in SEQ ID No. 1.

2. A vector comprising the recombinant nucleic acid of claim 1.

3. The vector of claim 2, wherein the vector is selected from the group consisting of a plasmid or a viral vector.

4. The vector of claim 3, wherein the vector is selected from a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a combination thereof.

5. The vector of claim 4, wherein the vector is an AAV vector.

6. The vector of claim 5, wherein the serotype of the vector is selected from the group consisting of AAV2, AAV5, AAV7, AAV8, or a combination thereof.

7. The vector of any one of claims 2 to 4, wherein the vector is selected from a DNA viral vector or a retroviral vector.

8. A pharmaceutical formulation, comprising:

(a) The vector of any one of claims 2-7, and

(b) A pharmaceutically acceptable carrier and/or excipient.

9. The pharmaceutical formulation of claim 8, wherein the pharmaceutical formulation is in a dosage form selected from the group consisting of: lyophilized formulations, liquid formulations, and combinations thereof.

10. A pharmaceutical formulation according to any of claims 8 to 9, wherein the carrier is present in the pharmaceutical formulation in an amount of 1 x 10 ⁹ -1×10 ¹⁶ Individual virus/ml.

11. Pharmaceutical preparation according to any of claims 8 to 9, characterised in that the carrier content in the pharmaceutical preparation is 1 x 10 ¹¹ -1×10 ¹³ One virus/ml.

12. A pharmaceutical formulation according to any of claims 8 to 9, wherein the carrier is present in the pharmaceutical formulation in an amount of 2 x 10 ¹¹ -1×10 ¹² Individual virus/ml.

13. A method for preparing recombinant human frizzled receptor 4, comprising the steps of: culturing a host cell comprising the vector of any one of claims 2-7, or having integrated into its chromosome an exogenous recombinant nucleic acid according to claim 1, thereby producing recombinant human frizzled receptor 4.

14. The method of claim 13, wherein the host cell is selected from 293T cells, photoreceptor cells, other visual cells, optic nerve cells, or a combination thereof.

15. The method of claim 14, wherein the photoreceptor cells are cone cells and/or rod cells and the other visual cells are binodal cells.

Images