CN112980819A - Construction method and application of retinitis pigmentosa animal model - Google Patents

Abstract

The invention provides a construction method and application of a retinitis pigmentosa animal model. Specifically, provided is a mutant of Cathepsin D (CTSD) gene, which is a C base repeat at position 262 of the CTSD gene. The invention also provides a CTSD mutant protein, compared with the wild CTSD protein, the 88 th amino acid of the mutant protein starts frame shift mutation; nucleic acid molecules and vectors encoding the muteins, and pharmaceutical compositions prepared using the muteins, nucleic acid molecules and vectors. The pharmaceutical composition can be used for inducing the retinal pigment degeneration disease by being applied to a non-human mammal, thereby being a convenient and stable non-human mammal model of the retinal pigment degeneration.

Description

Construction method and application of retinitis pigmentosa animal model

Technical Field

The invention belongs to biomedicine, and particularly relates to a construction method and application of a retinitis pigmentosa animal model.

Background

Retinitis Pigmentosa (RP) is a common type of inherited blinding disease, with clinical manifestations of night blindness, peripheral visual field loss, and progressive vision loss. RP primarily affects rod cells at the earliest, resulting in impaired night vision. Cone dysfunction and damage to the Retinal Pigment Epithelium (RPE) cells ensue. Typical ocular fundus changes include optic disc sallowness, retinal vascular thinning, mid-peripheral retinal salt and pepper-like changes, and/or osteocyte-like pigmentation. Patients with RP of different genetic patterns, etiology and age have widely varying clinical manifestations. At present, the pathogenesis and pathological mechanism of the disease are lack of deep system research, the pathogenic mechanism of mutant genes is not clear, and the disorder is brought to the targeted development of therapeutic intervention measures.

Therefore, there is a need in the art for an animal model of retinal pigment degeneration disease that is similar to clinical mutations.

Disclosure of Invention

The invention aims to provide a construction method of an animal model of a retinal pigment degeneration disease caused by CTSD mutation.

In a first aspect of the invention, there is provided a mutant of the human Cathepsin D (CTSD) gene, which mutant is a C base repeat at position 262 of the CTSD gene.

In another preferred embodiment, the mutation site of the mutant is located in exon three of the CTSD gene.

In another preferred embodiment, the mutant encodes a polypeptide having a frame shift mutation from the 88 th amino acid of the CTSD protein.

In another preferred embodiment, the mutant is a CTSD c.262dupc mutant.

In another preferred embodiment, the nucleic acid sequence of the mutant is shown in SEQ ID NO. 1:

atgcagccca gcagcctgct gcccctggcc ctgtgtctgc tggccgctcc tgccagcgcc ctggtgagaa tccctctgca caagttcacc tccatccgga ggaccatgag cgaggtgggc ggcagcgtgg aggacctgat cgctaaaggc cctgtgtcca agtatagcca ggcagtgccc gcagtgaccg agggccctat ccctgaagtg ctgaagaact acatggatgc ccagtattac ggagagattg gaattggcac cccccctcct gtgctgcaca gcaggctgag gcatggcctg ctgcagcctg tgggccctct gcaccctctg cagaccgccg gacacagact gctggatcct cctcaggtgc agcagaggca ggtgcagcat ctgagggaag agtggtacct ggtg。

in another preferred embodiment, the mutant occurs in a human or non-human mammalian cell.

In another preferred embodiment, the cell is selected from the group consisting of a somatic cell, a germ cell, an embryonic stem cell, and a combination thereof.

In another preferred embodiment, the somatic cell is selected from the group consisting of: HEK cells, photoreceptor cells (including cone cells and/or rod cells), other visual cells (such as bipolar cells, horizontal cells), (optic) nerve cells, or combinations thereof.

In a second aspect of the invention, there is provided a CTSD mutein characterized in that the mutein has a frame shift mutation starting from the 88 th amino acid compared to the wild-type CTSD protein.

In another preferred embodiment, the amino acid sequence of the mutein is as shown in SEQ ID No. 2: MQPSSLLPLALCLLAAPASALVRIPLHKFTSIRRTMSEVGGSVEDLIAKGPVSKYSQAVP AVTEGPIPEVLKNYMDAQYYGEIGIGTPPPVLHSRLRHGLLQPVGPLHPLQTAGHRLLD PPQVQQRQVQHLREEWYLV are provided.

In another preferred embodiment, the amino acid sequence of the CTSD mutant protein at positions 1-88 is the same as that of a wild-type CTSD protein, wherein the amino acid sequence of the wild-type CTSD protein is shown in SEQ ID NO. 3: MQPSSLLPLALCLLAAPASALVRIPLHKFTSIRRTMSEVGGSVEDLIAKGPVSKYSQAVP AVTEGPIPEVLKNYMDAQYYGEIGIGTPPQCFTVVFDTGSSNLWVPSIHCKLLDIACWI HHKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSQDTVSVPCQSASSASALGGVKVERQ VFGEATKQPGITFIAAKFDGILGMAYPRISVNNVLPVFDNLMQQKLVDQNIFSFYLSRD PDAQPGGELMLGGTDSKYYKGSLSYLNVTRKAYWQVHLDQVEVASGLTLCKEGCEAI VDTGTSLMVGPVDEVRELQKAIGAVPLIQGEYMIPCEKVSTLPAITLKLGGKGYKLSPE DYTLKVSQAGKTLCLSGFMGMDIPPPSGPLWILGDVFIGRYYTVFDRDNNRVGFAEAA RL are provided.

In another preferred embodiment, the amino acid sequence of the CTSD mutein is selected from the group consisting of:

(a1) an amino acid sequence comprising the sequence shown in SEQ ID NO. 2 by substitution, deletion or addition of preferably 1-10, more preferably 1-5, even more preferably 1-3, and most preferably 1 amino acid residue; and

(a2) and (3) a derivative protein which is similar to the structure or function of the protein shown in the sequence SEQ ID NO. 2.

In a third aspect of the invention, there is provided a polynucleotide encoding the mutein of the second aspect of the invention.

In another preferred embodiment, the polynucleotide sequence has the following mutations: (m) a repeat of C262.

In another preferred embodiment, the nucleotide sequence is selected from the group consisting of:

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

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

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

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

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

In a fourth aspect of the invention, there is provided a vector comprising the nucleic acid sequence of the mutant of the first aspect of the invention, and/or the polynucleotide of the third aspect.

In another preferred embodiment, the vector expresses human CTSD mutein.

In another preferred embodiment, the vector is a recombinant vector containing a nucleic acid sequence of the CTSD c.262dupc mutant; preferably, the nucleic acid sequence of the CTSD c.262dupC mutant is shown in SEQ ID No. 1.

In another preferred embodiment, the nucleic acid sequence of the vector is SEQ ID No. 6.

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

In another preferred embodiment, the vector comprises a plasmid or a viral vector.

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

In another preferred embodiment, the carrier is selected from the group consisting of: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector (AAV), or a combination thereof. Preferably, the vector is an AAV vector.

In a fifth aspect of the invention, there is provided a formulation comprising the following components:

(a) the protein of the second aspect, the vector of the fourth aspect, or a combination thereof; and/or (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the formulation comprises from 1% to 50% (w/v), preferably from 5% to 10% (w/v) of component (a).

In another preferred embodiment, the carrier (b) is present in the formulation in an amount of 1X 10⁹-1×10¹⁶Individual virus/ml, preferably 1X 10¹²-1×10¹³Individual virus/ml.

In another preferred embodiment, the formulation is in a dosage form selected from the group consisting of: a lyophilized formulation, a liquid formulation, or a combination thereof.

In another preferred embodiment, the administration form of the preparation comprises local administration and injection administration.

In another preferred embodiment, the formulation is a topical formulation selected from the group consisting of: a solution, a suspension, an eye drop, a spray formulation, an ointment, a gel, a cream, an emulsion, or a combination thereof.

In a sixth aspect of the invention, there is provided a use of the protein of the second aspect of the invention, the vector of the fourth aspect and/or the formulation of the fifth aspect, for the preparation of a composition for use in the preparation of a non-human mammalian model of a retinitis pigmentosa disease.

In another preferred embodiment, the pathological manifestations of retinitis pigmentosa disease are selected from the group consisting of: retinal pigment epithelial cell damage, choroidal atrophy, thinning of the outer nuclear layer of the retina, diminished corneal electrophysiological responses, thinning of retinal blood vessels, sallowness of the optic disc, retinal salt and/or osteocytic pigmentation, night blindness, peripheral visual field defects, progressive vision loss, or a combination thereof.

In another preferred embodiment, the pathological manifestations of retinitis pigmentosa disease are selected from the group consisting of: retinal pigment epithelial cell damage, choroidal atrophy, thinning of the outer nuclear layer of the retina, attenuation of corneal electrophysiological responses, or a combination thereof.

In a seventh aspect of the present invention, there is provided a method for preparing a non-human mammal model of retinitis pigmentosa disease, comprising:

(1) administering the protein of the second aspect of the invention, the vector of the fourth aspect, the formulation of the fifth aspect, or the composition of the sixth aspect to the sub-retinal space of a non-human mammalian model.

In another preferred embodiment, the human CTSD mutein is present in retinal pigment epithelial cells of said non-human mammalian model.

In another preferred embodiment, the mode of application is selected from: topical application, and injection application.

In another preferred embodiment, the non-human mammal is selected from the group consisting of rodents, primates.

In the eighth aspect of the invention, a non-human mammal model of retinal pigment degeneration diseases is provided, wherein a human CTSD mutant protein model exists in retinal pigment epithelial cells of the non-human mammal model.

In another preferred embodiment, the model is prepared by the method of the eighth aspect of the present invention.

The ninth aspect of the invention provides an application of the non-human mammal model of the retinal pigment degeneration disease, which is used for researching the retinal pigment degeneration disease and/or screening the medicine for treating the retinal pigment degeneration disease.

In a tenth aspect of the invention, there is provided a use of a mutant according to the first aspect, a protein according to the second aspect, or a polynucleotide according to the third aspect of the invention, for preparing a reagent for detecting whether a CTSD mutation has occurred in a sample or whether a CTSD mutein exists in a sample.

In another preferred embodiment, the reagent is used for screening or determining potential patients with retinitis pigmentosa disease or typing detection.

In another preferred embodiment, the test sample is derived from isolated human or non-human mammalian tissue or cells.

In another preferred embodiment, the tissue is selected from the group consisting of: blood, amniotic fluid, mucosa, hair, body fluid, eyeball, muscle, or a combination thereof.

In another preferred embodiment, the cells include somatic cells, amniotic cells and embryonic stem cells.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The present inventors have conducted extensive and intensive studies and have unexpectedly found that c.262dupc mutation occurs in the CTSD gene in RP patients for the first time. More surprisingly, the viral vector rAAV/CTSD-MUT is introduced into retina, and the pathological manifestations of retinitis pigmentosa are generated in mice 4-8 weeks later, which indicates that the CTSD mutant protein may have retinal toxicity, so that the preparation method of the non-human mammal model with retinitis pigmentosa can be established. On this basis, the inventors have completed the present invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.

Fig. 1 is a schematic diagram of CTSD mutant plasmid construction and target sequence design provided in the embodiment of the present invention.

FIG. 2 shows the success of the transfection of CTSD-MUT cells provided by the examples of the present invention.

FIG. 3 shows that the rAAV/CTSD-MUT specifically infects retinal pigment epithelial cells provided by the embodiment of the invention. Panel A shows the expression of the tag protein HA-tag in the RPE (pigment epithelial cell) -choroid-sclera complex after virus injection, whereas no viral infection was seen in the retina; immunofluorescent staining was also confirmed in panel B.

FIG. 4 is a comparison of retinal electrograms in normal mice and mice dark adapted at 4 weeks and 8 weeks after rAAV/CTSD-MUT injection. The wave amplitudes of a wave and b wave of the eyes injected with the recombinant virus are obviously reduced, and the ERG result of the RP patient is met.

FIG. 5 shows the fundus images and OCT of normal mice and mice at 4 weeks and 8 weeks after rAAV/CTSD-MUT injection.

FIG. 6 shows the homology analysis of the amino acid sequence of human murine CTSD protein.

Description of the terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

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

Retinitis Pigmentosa (RP)

As used herein, "retinitis pigmentosa," "RP," all refer to Retinitis Pigmentosa (RP), a common class of inherited blinding diseases that are clinically manifested as night blindness, peripheral visual field loss, and progressive visual deterioration. RP primarily affects rod cells at the earliest, resulting in impaired night vision. Cone dysfunction and damage to the Retinal Pigment Epithelium (RPE) cells ensue. Typical ocular fundus changes include sallowness of the optic disc, retinal vessel thinning, mid-peripheral retinal pepper-like changes, and/or osteocyte-like pigmentation. Patients with RP of different genetic patterns, etiology and ages have very different clinical manifestations. At present, the etiology and the pathological mechanism of the disease are lack of deep system research, the pathogenic mechanism of the mutant gene is not clear, and the targeted development of therapeutic intervention measures is hindered.

RP is highly heterogeneous in genetics, and it has been found that at least 87 genes can cause RP (see RetNet website: http:// sph. uth. edu/RetNet /). Mutations in these genes can result in autosomal dominant inheritance, autosomal recessive inheritance, or X-linked genetic RPs, respectively. More than 60% of the mutations were concentrated in 6 genes, i.e., CYP4V2, RHO, USH2A, RPGR, CRB1, and RP 2. More pathogenic mutant genes can be used as the basis for identifying the mutation, but further genetic research on some genes with few mutation reports is also necessary.

CTSD gene and protein thereof

As used herein, "Cathepsin D", "CTSD", "Cathepsin D" all refer to Cathepsin D, the CTSD gene being located at 11p15.5, and 9 exons. The CTSD protein is a main lysosome endopeptidase and has an important function on degradation of endogenous proteins. Studies have shown that homozygous deletion of CTSD can lead to early-onset, progressive neurodegenerative diseases (e.g., neuronal ceroid lipofuscinosis); heterozygous missense mutations in CTSD have been shown to cause early onset dyskinesia, brain atrophy and progressive ataxia syndrome. The access of the human CTSD protein is CAG33228.1, the GI 48146011 has 412 amino acids in total, and the amino acid sequence is shown in SEQ ID NO. 3.

The human mouse CTSD protein has high homology, the access of the mouse CTSD protein is NP-034113.1, GI 6753556, and has 410 amino acids in total, and the amino acid sequence is shown as SEQ ID NO. 4; the complete nucleic acid sequence of human CTSD is shown in SEQ ID No. 5.

CTSD mutant protein and coding sequence thereof

As used herein, the terms "mutein", "mutant polypeptide", "CTSD (c.262dupC) mutant", "CTSD (c.262dupC)" are used interchangeably to refer to the C base repeat at position 262 of the human CTSD gene sequence, resulting in a frameshift mutation, i.e., CTSD: NM-001909: exon3: c.262dupC: p.P88fs.

The nucleic acid sequence of the c.262dupC mutant CTSD fragment is shown in SEQ ID No. 1.

The amino acid sequence of the produced mutant protein is shown in SEQ ID No. 2.

The invention unexpectedly discovers that the CTSD gene in the RP patient has c.262dupC mutation for the first time, and no other research reports about the mutation. In one embodiment of the present invention, it was unexpectedly found that the pathological modification of RP was also induced by subretinal injection of artificially constructed recombinant viral vector of c.262dupc mutant CTSD gene. Since the role of protein encoded by CTSD gene in retina is less studied, the pathological mechanism of mutant protein is not clear. It is suspected that mouse RPE cell death is caused firstly by the retina toxic effect generated by mutant protein, normal CTSD protein can not be compensated, and then photoreceptor cell dysfunction even death is caused, thereby simulating the pathological process of RP.

In another preferred embodiment, the sequences of the muteins of the present invention further include those derived from the muteins of the present invention, which are obtained by substituting, deleting or adding one or more amino acid residues into the protein represented by SEQ ID No. 2, and which have substantially the function of the muteins of the present invention. It will be readily appreciated by those of ordinary skill in The art that altering a small number of amino acid residues in certain regions, e.g., non-critical regions, of a polypeptide does not substantially alter biological activity, e.g., that The sequence resulting from appropriate substitution of certain amino acids does not affect its activity (see Watson et al, Molecular Biology of The Gene, fourth edition, 1987, The Benjamin/Cummings pub. Co. P224). Thus, one of ordinary skill in the art would be able to effect such a substitution and ensure that the resulting molecule still possesses the desired biological activity.

For example, the amino acid sequence of the mutein of the invention (a) is as shown in SEQ ID NO 3; or (b) a protein derived from (a) comprising a sequence of (a) defined by substitution, deletion or addition of one or more amino acid residues, preferably 1 to 10, more preferably 1 to 5, more preferably 1 to 3, most preferably 1 amino acid residue, and having substantially the function of the protein defined in (a).

In the present invention, the derivative protein of the mutein of the present invention includes mutants in which at most 10, preferably at most 5, still more preferably at most 3, still more preferably at most 2, and most preferably at most 1 amino acid has been replaced with an amino acid having a similar or analogous property, as compared with the mutein having an amino acid sequence shown in SEQ ID NO 3. These conservative variant mutants can be generated by amino acid substitution, for example, as shown in the following table.

Initial residue	Representative substituted residue	Preferred substituent residues
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Adeno-associated virus

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

The recombinant adeno-associated virus (rAAV) is derived from non-pathogenic wild adeno-associated virus, is considered to be one of the most promising gene transfer vectors due to the characteristics of good safety, wide host cell range (divided and non-divided cells), low immunogenicity, long time for expressing foreign genes in vivo and the like, and is widely applied to gene therapy and vaccine research worldwide. Over 10 years of research, the biological properties of recombinant adeno-associated viruses have been well understood, and many data have been accumulated, especially in terms of their utility in various cells, tissues and in vivo experiments. In medical research, rAAV is used in the study of gene therapy for a variety of diseases (including in vivo, in vitro experiments); meanwhile, the gene transfer vector is used as a characteristic gene transfer vector and is widely applied to the aspects of gene function research, disease model construction, gene knock-out mouse preparation and the like.

In a preferred embodiment of the invention, the vector is a recombinant AAV vector. AAV is a relatively small DNA virus that can integrate into the genome of cells that they infect in a stable and site-specific manner. They are capable of infecting a large series of cells without any effect on cell growth, morphology or differentiation, and they do not appear to be involved in human pathology. AAV genomes have been cloned, sequenced and characterized. AAV contains an Inverted Terminal Repeat (ITR) region of approximately 4700 bases and containing approximately 145 bases at each end, which serves as the viral origin of replication. The remainder of the genome is divided into two important regions with encapsidation functions: a left part of the genome comprising a rep gene involved in viral replication and viral gene expression; and the right part of the genome comprising the cap gene encoding the viral capsid protein.

As used herein, "rAAV/CTSD-MUT", "pscAAV/CTSD-MUT-HA", "recombinant vector", "recombinant viral vector", "recombinant adeno-associated viral vector" are interchangeable and refer to a recombinant adeno-associated viral vector carrying a nucleic acid sequence as shown in SEQ ID No. 1 for a CTSD mutation site and an HA-tag, and in a preferred embodiment of the invention, the recombinant vector nucleic acid sequence is shown in SEQ ID No. 6.

Expression vector and recombination technology

Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. The expression vector may be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In general, any plasmid and vector may be used as long as it can replicate and is stable in the host.

One of ordinary skill in the art can use well-known methods to construct expression vectors containing the genes described herein. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. When the gene of the invention is used for constructing a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added in front of the transcription initiation nucleotide.

As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences of different origin. For example, a promoter is foreign to a gene of interest if the combination of the promoter and the sequence of the gene of interest is not normally found in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.

Pharmaceutical formulations and compositions

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. This term in relation to pharmaceutical compositions is intended to encompass products comprising the active ingredient and the inert ingredient as constituting carriers, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention include any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" or "acceptable" is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. As pharmaceutically acceptable carriers, binders, glidants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavoring agents, and the like may be used for oral administration; buffers, preservatives, analgesics, solubilizers, isotonizing agents, stabilizers and the like may be mixed for injection; bases, excipients, lubricants, preservatives and the like may be used for topical administration.

It is to be understood that the terms "administering", "administering" and/or "administering" a compound refer to providing, distributing, administering, distributing a compound of the invention to an individual or subject in need of treatment by any suitable means, such as oral, parenteral, rectal, transdermal, and the like. In the present invention, it is preferably administered by topical administration and/or injection.

The technical scheme of the invention has the following beneficial effects:

1. the invention provides a mutant of CTSD gene, namely CTSD c.262dupC mutant, corresponding mutant protein and an expression recombinant vector thereof, and unexpectedly discovers that RP pathological change can be caused by c.262dupC mutant CTSD gene recombinant virus vector injected under retina, so a retinitis pigmentosa disease model can be established by applying the vector containing the mutant sequence or the mutant protein.

2. The non-human mammal model of retinitis pigmentosa provided by the invention has the characteristics of high controllability, timely morbidity after treatment, obvious and stable disease symptoms, can be used for researching and screening the retinitis pigmentosa diseases and drugs for treating the retinitis pigmentosa diseases, and has wide application prospect.

The CTSD protein has high homology in human and mouse, the mutation site provided by the invention can be used as an index for screening the retinitis pigmentosa disease of children patients, and based on the mutation site, the application of the CTSD protein in preparing reagents for systematically detecting CTSD mutation at gene and protein levels is developed.

The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, without specific conditions being noted in the following examples, are generally performed according to conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

EXAMPLE 1 screening of CTSD mutant sites

The RP diagnosis is determined through ophthalmologic examination (including fundus photography, autofluorescence, OCT, electrophysiology and the like), peripheral blood samples of patients and family members of the patients are extracted and subjected to whole exome sequencing, mutation sites are determined through bioinformatics analysis, variation frequency is analyzed by utilizing a db SNP database, a thousand-human genome and an Ex AC database, and a Sanger sequencing method is used for verification to screen CTSD (c.262pC) gene pathogenic mutation sites which are co-separated from diseases and are not carried by normal family members.

EXAMPLE 2 construction of CTSD mutant plasmids and packaging of AAV viruses

The viral vectors were obtained by the plasmid cotransfection method. The auxiliary plasmid containing AAV2 coat protein gene and gene for assisting AAV replication and AAV/CTSD-MUT plasmid are cotransfected with HEK 293T cell to form recombinant adeno-associated virus vector. The construction of the plasmid rAAV/CTSD-MUT is shown in figure 1, the sequence of the plasmid rAAV/CTSD-MUT is shown in SEQ ID No. 1, the total length of the plasmid rAAV/CTSD-MUT is 4073bp, the plasmid rAAV/CTSD-MUT carries a nucleic acid sequence of a CTSD mutation site and an HA-tag label shown in SEQ ID No. 4, and the inserted nucleic acid sequence containing a c.262dupC mutation CTSD fragment is shown in SEQ ID No. 5.

After initial purification with ioxobutanol, further purification was carried out by ion exchange chromatography on fast protein liquid chromatography using 5ml-Hitrp Q sepharose as packing using an apparatus Pharmacia AKTA FPLC system (Amersham Biosciences, Piscataway, N.J.). The agarose gel column was then rinsed with 215mM NaCl, pH8.0, and the peaked recombinant viral vectors were collected. After the collected liquid was passed through a concentrator (100K concentrator, Millipore), the recombinant viral vector was concentrated by rinsing the concentrator with 0.014% Tween 20. DNA other than the viral particles was digested with DNase I, and the titer of the virus was determined by real-time fluorescence quantitative PCR. Finally, silver nitrate staining-SDS polyacrylamide gel electrophoresis is used to ensure that the virus vector particles are not polluted and do not contain endotoxin, and the virus vector particles are subpackaged and stored at-80 ℃.

As a result: the feature map of the viral vector rAAV/CTSD-MUT is shown in figure 1. FIG. 2 shows the success of the transfection of CTSD-MUT cells provided in this example.

Example 3 mouse retinitis pigmentosa model acquisition and characterization

1. The viral vector rAAV/CTSD-MUT constructed in example 2 was introduced into the retina by subretinal injection according to the following procedure

1) Anesthesia: the mice were deeply anesthetized with an intraperitoneal injection of 0.1ml of 10% chloral hydrate per 20g of mice.

2) The compound tropicamide eye drops are dripped into two eyes to disperse the pupils, and then the binomi eye drops are dripped into the two eyes to perform corneal surface anesthesia.

3)1:20 dilution of the amioda can disinfect periorbital skin and conjunctival sac.

4) The 2mm behind the sclera edge of the temporosuperior cornea of the right eye is selected as a needle inserting position, and under the guidance of an eye operation microscope sight glass, the conjunctiva of the position is locally cut off, and the sclera is exposed.

5) A 30G flat-head needle of a syringe needle with the diameter of 0.45mm is used for making a stoma, the syringe needle filled with rAAV/CTSD-MUT virus liquid is inserted into a vitreous cavity along the stoma, a full-retina mirror is placed on a cornea, the fundus is adjusted to be clear, blood vessels are avoided, and the injection of the retinal cavity is carried out.

6) And (4) coating antibiotic eye ointment.

2. Step 1 after treatment for 4 weeks and 8 weeks, identifying the model construction effect by a funduscopic and slice immunohistochemical staining method respectively:

obtaining eyeballs for slicing operation:

after the mice die at the dislocation of cervical vertebrae, quickly taking eyeballs, putting the eyeballs into 4% PFA, fixing the eyeballs on ice for 15min, cutting a cut on the cornea, and then continuously fixing the cut on ice. After 2h, the eyes were rinsed 3 times with PBS buffer, dehydrated in 30% sucrose solution for 2h, then dissected under the lens to remove the cornea and crystals, OCT embedded and quickly frozen in a freezer at-80 ℃. After about 10min, the OCT embedded eyeball is taken out and placed in a freezing microtome to be balanced at-25 ℃ for about 30min, and then the section can be obtained. The slice thickness was 12 μm.

After the slicing is completed, selecting the high-quality slices, placing the slices in an oven at 37 ℃ for 30min, then drawing circles on the part with the retinal tissue by using an immunohistochemical pen, washing the retina with PBS for three times to remove OCT, then sealing and permeating 5% of NGS (containing 0.25% Triton) for 2h, incubating primary antibody, and standing overnight at 4 ℃. On the next day, after washing twice with PBS, the corresponding fluorescent secondary antibody was incubated, and then washed twice with PBS, mounted, and observed.

As a result:

the staining results of the sections are shown in FIG. 3, and the rAAV/CTSD-MUT-treated mice were infected with pigment epithelial cells at 4 weeks and 8 weeks, and were atrophied compared to the normal group.

Fundus photography and OCT shows the results of fundus examination are shown in FIG. 5, where RPE andchoroidal atrophy 4 weeks after rAAV/CTSD-MUT treatment in mice and the outer nuclear layer of the retina is significantly thinned 8 weeks later (late).

Example 4 electrophysiological representation of the mouse model of retinitis pigmentosa

The specific electrophysiological method is as follows:

dark acclimation to mice over 12h, mice were deeply anesthetized with 0.1ml of 10% chloral hydrate i.p. injection under red light. The compound tropicamide eye drops are used for double eye drops to fully disperse the large pupils, and then the binomi eye drops are used for dropping the double eyes for corneal surface anesthesia. The reference electrode is arranged between the ears of the mouse and connected withThe ground electrode is positioned at the tail of the mouse, and the corner membrane electrode is directly contacted with the contact lens electrode (

Ganzfeld ERG; phoenix Research Labs, USA). Under weak red light, 3.0cd · s/m2 light intensity is selected, 10s are separated between every two stimulations, 10 stimulations are completed totally, and the superposed waveform is analyzed.

As a result: as shown in fig. 5, the rAAV/CTSD-MUT subretinal space treatment group ERG showed a significant drop in amplitude of the a-wave and b-wave and progressed over time.

Example 5 homology test for human murine CTSD proteins

According to NCBI protein database, the Accession of mouse CTSD protein is known as NP-034113.1, GI 6753556, and the total number of 410 amino acids, and the amino acid sequence is shown as SEQ ID NO. 6; CAG33228.1, GI 48146011, 412 amino acids in total, and the amino acid sequence is shown in SEQ ID NO. 2. The amino acid sequences of human murine CTSD proteins were selected and aligned at positions 15-410. sup. th of the sequences, and as a result, the human murine CTSD proteins have high homology as shown in FIG. 6.

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

Sequence listing

<110> first-person hospital in Shanghai City

<120> construction method of retinitis pigmentosa animal model and application thereof

<130> P2019-1381

<160> 6

<170> PatentIn version 3.5

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atgcagccca gcagcctgct gcccctggcc ctgtgtctgc tggccgctcc tgccagcgcc 60

ctggtgagaa tccctctgca caagttcacc tccatccgga ggaccatgag cgaggtgggc 120

ggcagcgtgg aggacctgat cgctaaaggc cctgtgtcca agtatagcca ggcagtgccc 180

gcagtgaccg agggccctat ccctgaagtg ctgaagaact acatggatgc ccagtattac 240

ggagagattg gaattggcac cccccctcct gtgctgcaca gcaggctgag gcatggcctg 300

ctgcagcctg tgggccctct gcaccctctg cagaccgccg gacacagact gctggatcct 360

cctcaggtgc agcagaggca ggtgcagcat ctgagggaag agtggtacct ggtg 414

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Met Gln Pro Ser Ser Leu Leu Pro Leu Ala Leu Cys Leu Leu Ala Ala

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Pro Ala Ser Ala Leu Val Arg Ile Pro Leu His Lys Phe Thr Ser Ile

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Arg Arg Thr Met Ser Glu Val Gly Gly Ser Val Glu Asp Leu Ile Ala

35 40 45

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

50 55 60

Gly Pro Ile Pro Glu Val Leu Lys Asn Tyr Met Asp Ala Gln Tyr Tyr

65 70 75 80

Gly Glu Ile Gly Ile Gly Thr Pro Pro Pro Val Leu His Ser Arg Leu

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Arg His Gly Leu Leu Gln Pro Val Gly Pro Leu His Pro Leu Gln Thr

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Ala Gly His Arg Leu Leu Asp Pro Pro Gln Val Gln Gln Arg Gln Val

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Gln His Leu Arg Glu Glu Trp Tyr Leu Val

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Met Gln Pro Ser Ser Leu Leu Pro Leu Ala Leu Cys Leu Leu Ala Ala

1 5 10 15

Pro Ala Ser Ala Leu Val Arg Ile Pro Leu His Lys Phe Thr Ser Ile

20 25 30

Arg Arg Thr Met Ser Glu Val Gly Gly Ser Val Glu Asp Leu Ile Ala

35 40 45

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

50 55 60

Gly Pro Ile Pro Glu Val Leu Lys Asn Tyr Met Asp Ala Gln Tyr Tyr

65 70 75 80

Gly Glu Ile Gly Ile Gly Thr Pro Pro Gln Cys Phe Thr Val Val Phe

85 90 95

Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Ile His Cys Lys Leu

100 105 110

Leu Asp Ile Ala Cys Trp Ile His His Lys Tyr Asn Ser Asp Lys Ser

115 120 125

Ser Thr Tyr Val Lys Asn Gly Thr Ser Phe Asp Ile His Tyr Gly Ser

130 135 140

Gly Ser Leu Ser Gly Tyr Leu Ser Gln Asp Thr Val Ser Val Pro Cys

145 150 155 160

Gln Ser Ala Ser Ser Ala Ser Ala Leu Gly Gly Val Lys Val Glu Arg

165 170 175

Gln Val Phe Gly Glu Ala Thr Lys Gln Pro Gly Ile Thr Phe Ile Ala

180 185 190

Ala Lys Phe Asp Gly Ile Leu Gly Met Ala Tyr Pro Arg Ile Ser Val

195 200 205

Asn Asn Val Leu Pro Val Phe Asp Asn Leu Met Gln Gln Lys Leu Val

210 215 220

Asp Gln Asn Ile Phe Ser Phe Tyr Leu Ser Arg Asp Pro Asp Ala Gln

225 230 235 240

Pro Gly Gly Glu Leu Met Leu Gly Gly Thr Asp Ser Lys Tyr Tyr Lys

245 250 255

Gly Ser Leu Ser Tyr Leu Asn Val Thr Arg Lys Ala Tyr Trp Gln Val

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His Leu Asp Gln Val Glu Val Ala Ser Gly Leu Thr Leu Cys Lys Glu

275 280 285

Gly Cys Glu Ala Ile Val Asp Thr Gly Thr Ser Leu Met Val Gly Pro

290 295 300

Val Asp Glu Val Arg Glu Leu Gln Lys Ala Ile Gly Ala Val Pro Leu

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Ile Gln Gly Glu Tyr Met Ile Pro Cys Glu Lys Val Ser Thr Leu Pro

325 330 335

Ala Ile Thr Leu Lys Leu Gly Gly Lys Gly Tyr Lys Leu Ser Pro Glu

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Asp Tyr Thr Leu Lys Val Ser Gln Ala Gly Lys Thr Leu Cys Leu Ser

355 360 365

Gly Phe Met Gly Met Asp Ile Pro Pro Pro Ser Gly Pro Leu Trp Ile

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Leu Gly Asp Val Phe Ile Gly Arg Tyr Tyr Thr Val Phe Asp Arg Asp

385 390 395 400

Asn Asn Arg Val Gly Phe Ala Glu Ala Ala Arg Leu

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Met Lys Thr Pro Gly Val Leu Leu Leu Ile Leu Gly Leu Leu Ala Ser

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Ser Ser Phe Ala Ile Ile Arg Ile Pro Leu Arg Lys Phe Thr Ser Ile

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Arg Arg Thr Met Thr Glu Val Gly Gly Ser Val Glu Asp Leu Ile Leu

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Lys Gly Pro Ile Thr Lys Tyr Ser Met Gln Ser Ser Pro Lys Thr Thr

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Glu Pro Val Ser Glu Leu Leu Lys Asn Tyr Leu Asp Ala Gln Tyr Tyr

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Gly Asp Ile Gly Ile Gly Thr Pro Pro Gln Cys Phe Thr Val Val Phe

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Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Ile His Cys Lys Ile

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Leu Asp Ile Ala Cys Trp Val His His Lys Tyr Asn Ser Asp Lys Ser

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Ser Thr Tyr Val Lys Asn Gly Thr Ser Phe Asp Ile His Tyr Gly Ser

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Gly Ser Leu Ser Gly Tyr Leu Ser Gln Asp Thr Val Ser Val Pro Cys

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Lys Ser Asp Gln Ser Lys Ala Arg Gly Ile Lys Val Glu Lys Gln Ile

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Phe Gly Glu Ala Thr Lys Gln Pro Gly Ile Val Phe Val Ala Ala Lys

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Phe Asp Gly Ile Leu Gly Met Gly Tyr Pro His Ile Ser Val Asn Asn

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Val Leu Pro Val Phe Asp Asn Leu Met Gln Gln Lys Leu Val Asp Lys

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Asn Ile Phe Ser Phe Tyr Leu Asn Arg Asp Pro Glu Gly Gln Pro Gly

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Gly Glu Leu Met Leu Gly Gly Thr Asp Ser Lys Tyr Tyr His Gly Glu

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Leu Ser Tyr Leu Asn Val Thr Arg Lys Ala Tyr Trp Gln Val His Met

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Asp Gln Leu Glu Val Gly Asn Glu Leu Thr Leu Cys Lys Gly Gly Cys

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Glu Ala Ile Val Asp Thr Gly Thr Ser Leu Leu Val Gly Pro Val Glu

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Glu Val Lys Glu Leu Gln Lys Ala Ile Gly Ala Val Pro Leu Ile Gln

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Gly Glu Tyr Met Ile Pro Cys Glu Lys Val Ser Ser Leu Pro Thr Val

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Tyr Leu Lys Leu Gly Gly Lys Asn Tyr Glu Leu His Pro Asp Lys Tyr

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Ile Leu Lys Val Ser Gln Gly Gly Lys Thr Ile Cys Leu Ser Gly Phe

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Met Gly Met Asp Ile Pro Pro Pro Ser Gly Pro Leu Trp Ile Leu Gly

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<212> DNA

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aactgcggcg tcatcccggc tataagcgca cggcctcggc gaccctctcc gacccggccg 60

ccgccgccat gcagccctcc agccttctgc cgctcgccct ctgcctgctg gctgcacccg 120

cctccgcgct cgtcaggtga agcctcaggg gccggggctc agggacgggc aggggtcgcg 180

gcgccgaggt cccggggcct gtggtgactt tcgcgctccc ctgtggcccc cacgagcccc 240

ttgcgccccc cgcgctggaa tgcacctgtg ccgccctgcg cggcctcctg cacggaccac 300

ccgcctacgg ggcgccgggc tccggaggtg caggggaccc ggggcagagg cgccagatgc 360

ctctccccca tatgccaccc tgggttgtac cttgaggact gcagactgac cgcagcctcc 420

ctggagacgg ggcggggcgg ggggaggtag tgctcattcg gggcaggtgg aattggggtc 480

tgtactgagc gcccttgttg ctggagacct aggtcaggcc tcagagcccc cgagtctggg 540

cgagtccatt tccttaggga cccctttacc acctgtgaac tgggggcttt aaaagtttgc 600

tccagcggct cttatcacag gccctgggct gggagacccc tcgagaccct aggagttccc 660

atgtccctga gagaggagga ggcatgggga gtgggtcggc tcacccaccc cgggcctggg 720

gttgtgctgt agtgaggccc acacgctcct caggccgatc ccctgtgcca ggtgaggcca 780

ccgattgggc ctggatggga tggggcccgg ccatgcctga ccagctgggc agaggagggc 840

catgctgcag tctgctttct tgaccccctc cccagccctt gcaaggcagc ccgcattccc 900

aggaggggta tgctgaccca tcccattggg cacctgcccc acccttgctc tgggcctttg 960

tgggagacct gggatctgcg atgggtccac tgccttttgg caggtgggtg aggtcagaag 1020

gctgcagggg ctggagctgg ctgggccagc tgggtaggac tgagcctcac caaaggctgt 1080

ggggaatggc ccgggggcgg gtagccccaa ttaaagtcgt tgtgggggag tagccacaag 1140

cctgagcctg ccttgacctt gccagcctat ccacaggcct cccctctcca aggaggacag 1200

acacagcaga ggggaaacga tcctggggct tcttggaggg aagggtagct gaatccaagc 1260

cctcacccga ttccagctct tgtgcgactg atactattac acctgcttcc tggtccctgg 1320

agggcgtgtc cctcccccag gacaaaacct ggagctcttc cagcccacca gctcttaggc 1380

aataatctca tcttccggga tcacgcccct gacaagccag gaaaagccag ctatgacctt 1440

gtactctcaa gtccctgggg cagggaagag gttttattta agtgattaaa agcccagggg 1500

agcttccttg gaacaaggag tgggttcaca ccaaggggaa ggccagtggc cctgggggag 1560

gagcagggac ccctctctct cttactcgct tcctgggttt agaactcagg accccgatct 1620

cagtctggag ctccctcctg caccctggct ggcggtgtgc tgggtgacag gactctggag 1680

gggtaccctg agtgcagctg tcggaggagg cagggcggtg gggggggcag cacagaagcc 1740

tctaaggccc caggtgcagt cctggacctc gtggagccgc atggagtgag gagaggtgcg 1800

gatgcccaga aacagatgtg ggatgagggc actgggcagc cacagggtcc atgtggagga 1860

ggacaggtag tcaaggaggg cttctggagg tggtgtggag ggcccatctg atggccagag 1920

gaggccaggc agagctgcca gtgccagcct ggaggtgggg ccaccttcgt gcaggtgtct 1980

gggggtggag agcaggtgtg atgggggctg ggtacagtgg gctgcctcag agcactttgg 2040

gcaggagtga caggtacccg ccagcaccct gagcagccat gctggccacc atcctggaag 2100

agaccagcgc aggtgcagag ggaggacggg agacccttgg gggctttgga gcctccagaa 2160

tggctgagga gaggagggtt gggcacactg gcccccaggt tgggtgtgtg gggctgaggt 2220

gggtggcggg tgaccacttc ttaggactgt ggcctgtgca acctggcggg gggcaatggg 2280

ttgccattca ctgacttggg ggagctgggc cagcagtttc caggtgacag gcaggagttt 2340

ggttttggct gtggcgactc tgagattccc caggggcctc caggtggatg tgcagcattg 2400

gcagcgtcgg ccggcaggcg ggagggcctc cctgatatgc cccgacccgt ggttgacagg 2460

atcccgctgc acaagttcac gtccatccgc cggaccatgt cggaggttgg gggctctgtg 2520

gaggacctga ttgccaaagg ccccgtctca aagtactccc aggcggtgcc agccgtgacc 2580

gaggggccca ttcccgaggt gctcaagaac tacatggacg tgagtatgag gtcttagccc 2640

tgctgcaagc ggagccactg tcaggagagc tccgtggcag tatggggaac attcccacct 2700

cctgttctca gcagcgcagt ttgagtggct cacctggcca cggtggcgtc ccggctcagc 2760

cacactcctt tcttccccat tctggaacct cctgccactg gcccctgtca ttgcagggtc 2820

ttggtcctgc tagggcctgg gagggtgatt gggagtggcc tcgggccttg gtgccagctc 2880

gcctgagggg gcggtccttg ggcctgcctg agtgtggccg gctgacctgg agcctttcac 2940

tgctgtcctg ctgggaggcc tgtggtgact cgtggcctcc ccagcccctc cccatcttct 3000

tcctctcgca acagcccctc ctgtgcccac tactccttca gggggaagca ggatccaagg 3060

tggagcactc tggaagccac ctagcaagct ggggcttggt cagcctggtc ccagctctat 3120

ggggtcagtt cgaggccagg gcctactgtc cagcctcggg gctctggccc actgtggggg 3180

agccttgccc tctgtcctgc ttggcccgag tcctggctgt gacaggaagc ccaagactca 3240

caggcatgtg actgggccag gggggccctg gggggaacca ggcgccgtgt gcctggtccc 3300

gactggccag ttcctgattg tcctagcgcg cgagcaagca gatgcacgca tgcgcacaca 3360

tgcacacaca cacatggaaa tttgctgagt gtccccctgc cagtggtcac ttctgtgggg 3420

ccatgagtca gcagctgctg ctgctccgtg aagccaggcg gttggagaaa cattgggctg 3480

ggctggtgcc aggaatctgg tggctgacgg tggcccaggc tccaatcctg ggggaagcgg 3540

gcgcccaggc gaccccaaac tccaggaccc tcttactctc tgcctcctga gaggctcggc 3600

ggcatgggac cccttgtact tgcctgatcc ctgagtcagc acccccacct gcgcctgcta 3660

tccctgtgta cacacgggga aactgaggcc ctgggccatt aaggcagggt tgctgggcag 3720

gcagtgattg taaggcttac cttctcccct tgaccagctg aacccctctg cctggaagcc 3780

ctccaagcct ggggttcacc ctggagggca gggcaggcac tgagacaccc agaatcagac 3840

cttgacatgg ccccaacctg gggaggaagt cacttccttt ctcagacctt ggactgcggg 3900

ccaccagggg agtcttccag gccgggcttt cctgcacccg gggctcaggc tgagggccac 3960

gtctgtcccc accctggctt gacctctggc cttgtctttt ccgtggggaa gtcctcagcc 4020

tcaccatgta ttgagcaggg gtaggtgaca gaagccaggg gtctagagac cccaagtacc 4080

cgtgggcatt aggaccgagg gctggggacc tggcccatct tccctgcagg ctgagcaggt 4140

gggagtgggt ggctgttggc agctgtgggc cccgtgatgc ccctccctcc agtatgggcc 4200

ttggctctgg ggacagccgg gccttctgag gcccagtggg gagatggggc cccctctccc 4260

atccctgacg gaccctgtcc cctgccaggc ccagtactac ggggagattg gcatcgggac 4320

gcccccccag tgcttcacag tcgtcttcga cacgggctcc tccaacctgt gggtcccctc 4380

catccactgc aaactgctgg acatcgcttg ctgtgagtca cgaaccctgg ccccgtcgcc 4440

caggtcctgc ccttccggga tgtcgctgca gggctgcctc aggaatcact tgggcaacgc 4500

aattctcctg cctcttggcc ccgtgagcca ggccagccct ccaccctgct ccagccactg 4560

actttctggg tgaaccacca gctgtggtct tgctctcagc agggctgggg ctggggtggc 4620

cacagaggga gccggctgtg gctgggaggg aggcccgggg tcacagccca cagtcccggg 4680

gctctggcat gatggtgggc ctcagatccc ctccaaatcc caccctgggg aggcagcttg 4740

gggggtgcgg gctccatggt agattgtagg gggatgggag ggctaaggcc gtggcgtggg 4800

ctgcgggatg accggcgggc ccccttgtcg cccggggcag ggatccacca caagtacaac 4860

agcgacaagt ccagcaccta cgtgaagaat ggtacctcgt ttgacatcca ctatggctcg 4920

ggcagcctct ccgggtacct gagccaggac actgtgtcgg tgagtccctc tggggccttt 4980

cccaggactc gagggtgcca gggtgtgggg ttcacccacc gtgggtatgt ggttgaaagg 5040

gagggctcgg tgatcccagc ccaaccccag ccctcaggtg gccggggcag tcagcagggt 5100

gaggaggggt ctgggaatgg gcctggttgt gtgcaggctg gagggacagc ctcaaaccca 5160

gggggtacag gggcaggggt ccccggagtc aggccacaat gagtgggagg gaggacaggg 5220

cagatcgatc gggctctttt tggcacattg ggtttgaggt gccagcaggt gttgagggct 5280

ggacctgggg ctgcacaggg tccctgcagc aggccgaggg cttggggagg cttggggttg 5340

ggaggatgta ccaggaaccc gctgtggggg ctgctggcgg agagtcaccg gcaggagcct 5400

gggagggcaa gggaggggga gcagcagtgg ctgttggggg ccccgcctgc atccaccatc 5460

ctcagggccc tgtgcagagc aactccctgc cctaggagag ggtgagctgg cccttgtcac 5520

cctgcctggg cctcagtgag ttctcacctc ggagctgtct gctgggggtg gaccaggcca 5580

caaggggttc aggagttacg ggatggtgac acagccccca gctctggagc gggccaggag 5640

ggcagcagag cccccctgca ggcccaggga cccgggaaga gggccccttc ctccagctga 5700

agctgctcct aacagttccc tgctgggctg gagtccagtc tgtactgggg gctcctcaga 5760

gccctcctgt ctggaccctg ctcagcctga acaggaaatt gccccgtctg ccctcttccg 5820

ccctcttctg ccccctccgc cccgtgtcag cctcaagctg tcgttccctt cccacatcct 5880

gctctggccg tgttctctct ctgcagcctc atccagggct gggggagggg acaggcagag 5940

aaggggaagg gggcagtatg gctgtgagct tgggatgggg tcggcaggtt ccccatctct 6000

cgggctcctg gcccaggctg tgtcttgttc ccagcgctga gggcaggagc aggacctgcc 6060

tgtcattggt ggtgggagta agaggtcgga gcagggagcg gagcaagggg cctgcccgtc 6120

tgtgctcctg cctggttccc atctcgtgta aaccgagccc tgatgacttc cacgaagagg 6180

gccccgccat accccgtgtc cccatcccca gcggtgttca gctcagggtt tccagccctt 6240

tcttctgggc cctctgggcc ccatcgtgtg tgggatgggc atccattgaa ctgggttttg 6300

tagcctcatg ctcagggagt ggtgtagggc tcagcctgtc tgctgcccac tgactctgcc 6360

ctggcctgca ggtgccctgc cagtcagcgt cgtcagcctc tgccctgggc ggtgtcaaag 6420

tggagaggca ggtctttggg gaggccacca agcagccagg catcaccttc atcgcagcca 6480

agttcgatgg catcctgggc atggcctacc cccgcatctc cgtcaacaac gtgctgcccg 6540

tcttcgacaa cctgatgcag cagaagctgg tggaccagaa catcttctcc ttctacctga 6600

gcaggtgggc gtgtgggttc cctctcgctc cgcgttgctg ggaggcaggg cggggctgga 6660

cggggagcct tctaggcacc ccctctcagt gctgccccct ccctgctgct gtgccagagc 6720

tcctgacctc tgacctcagg gcatccggga ggcgggggtt ggccgccctt ctgcagagga 6780

gtaagcggca gcacagagaa agctgtttgg ccggggtctc ccagtgggag gggctcgggc 6840

caggctgtgg gtcctgggac cttggcagct gggcctccct cctatgagaa tggacccagg 6900

tcaggggtcg tggcagttcc tcttggttca gtcggcccgt cggtccccag acctggtgat 6960

gggcacatgt ggtcctggtc ccggggttgc tgatggtgga gagggtcatg gtgcccaggg 7020

gaccggggat ccccggggag gtgaccttgg gtgcgtatgg gtccccggca ccacgctgcg 7080

agcagctctg tgggcgtccc cagcaggtgc cggctgccct cgaggaggaa catagggggc 7140

ccttccttct gggaaaaggg ttctccccca gggcccccac ctgcagccgc tgctccagct 7200

tgggtgaccc atggtcaggt gaccttggga aggggacgtc ggactcagtt agtttcctct 7260

tctcggggca gacctgagtg gagggttcca cataagaggg gtgactgggg gttgggggct 7320

tttgttttgg gggtgggcag cgtgtcagcc ggcggcctcc tgccctgagc tgcgcctggt 7380

cactgcccca cactctccag ggctgacagg ggcaggttta cctcacgtgg ctcacttggc 7440

acttggagtc tctgggcctg acccctaacc ttggatcgct tccggcagaa ttccggctgt 7500

agggctggct gggcctcttt gctctgcccc ttgcctgggc agtgaatact ccttagcaac 7560

acccagggct aagctgctca ctagaggcca ggacacaggt gaaccggctg ggccatttcc 7620

ccaggagcct tggggcacag gggaggcagc caggtgaaaa ggggtcccct gagggcaaga 7680

gggcatgcag gcatgagtgc ccacatgggg agggggcaca cagcctgagt acccaggtca 7740

gggaggggga cacaggcatg aatgcccatg tgggggaggg gcacacagca tgagtgccca 7800

tgtgggggag gggcacacgg tatgagtacc caggtcgggg gaggcataca caggcatgag 7860

tgccaggtgg aaggaggggc acacagagtg agtgcccagg tgggggaggt gcacatggtt 7920

gtgagtgccc aggtgtagtc tcaaggcagc agcttgggca ggaagtggag ccaggcagga 7980

gggagggtct gagggcttct gaaagcatgt ttggtgagga gaggaggggt gggaggcgct 8040

gatcaggttt ctacacttgg gattgcagag gtgttgacaa gaggcaaagg cggaggaggc 8100

tggaggaggg cggaggcccc aatcggtgtt gggaggactg ggtcaggcct ggcactgcct 8160

cgagtgacag gcagtgggat ggtggccagc ttagctgcag acgctctggc gggatggcag 8220

aaccgcccca gacacagaga gcttctctac caggaccggc aggatttgct gcgttgaaag 8280

ctgtacttga gcaatgttta gaaacaaacc cgggcgacat gggttgcagg tcctaggaag 8340

tgcagtgcgc tcctgcccag gagcaccttg gctggccatc agtggtctgg atgaggggga 8400

gatgagcgga cgtggctcgg ggatgcaggt ggagggtgtt cccaggagca gccagtgcag 8460

aggccctgcg gccagaacca gccatcccaa cttcccagat tgtgccatca ctccctcctg 8520

gaagccttct ttggtttttt cttccagaca gacagacagg gtcctcccca gtggagctcc 8580

tggcactcac ttccttgtct gccgctagcc ttgaccctga tgtctggctg aaccttggct 8640

cctgagcaga ccaggagaac ctgagggttg aggaaaacct cttctgggcc aggctgggcc 8700

cacgcagagc agccctaccc cgggaagaag ggagtctgtc cctcctcctg ccttctgggc 8760

ctttccatcc ctgcagtttc agaaaggccc ctccttcagg aagctctccc tgattgccac 8820

attcaccctc ttactcctga cctggcactc ttgtgcttgg ggggtggcgt ggcagctgac 8880

tcctcccttt tcctcctagg gacccagatg cgcagcctgg gggtgagctg atgctgggtg 8940

gcacagactc caagtattac aagggttctc tgtcctacct gaatgtcacc cgcaaggcct 9000

actggcaggt ccacctggac cagtgagtag tggctgcagt cggctcccct gggttctgtg 9060

ggcgggggcg gtgtgcggag accctggagg accccggttc tgcaggtggg ggttgcatgt 9120

ggggagtagt gggagctggg caagaaagag atggggtcag accagccctc catgcccctc 9180

cttgcccctc catgctcccc atcacctcca tcccctctat tccctctatc cctccatccc 9240

tccatttcct ccatgcctct gtgactctcc atgacccacc atcccttctg tccatccctc 9300

catgccctcc atcccctcca tcccctcatc cctctgtgac tcttcatgac cctccatctc 9360

ctccatccct ccatcccctc catccctcca tccctccatc ccctccatcc ctccatccct 9420

ccatccccac atccctctgt gactctccat gaccctccat cccctccatc tgtctatccc 9480

tccatccctc catccctcca tgcccctcca tcccttcatc ccctccatcc cctccatccc 9540

tctatgcccc tccatcccct ccatccctcc atgcccctca ccaccacctg agggtctccc 9600

accccctcta ccactctgtg tctcctctcc caccctcttc cctggagggc ttacagccgg 9660

ctgtgcttcc aggagccctg aggggaggag agtgcagccc agccagggga ggggctccca 9720

gggaggggca ctgggccccc agggcacact ccagtcccgg caggggcttc acgccctgac 9780

tccccgcagg gtggaggtgg ccagcgggct gaccctgtgc aaggagggct gtgaggccat 9840

tgtggacaca ggcacttccc tcatggtggg cccggtggat gaggtgcgcg agctgcagaa 9900

ggccatcggg gccgtgccgc tgattcaggg cgaggtgagc gccgggggct ggggctgggg 9960

ctggggctgg cagggggagc cccaaggcca ccactaccac cctgacactg ctgtgacccc 10020

tcttagtaca tgatcccctg tgagaaggtg tccaccctgc ccgcgatcac actgaagctg 10080

ggaggcaaag gctacaagct gtccccagag gactacacgc tcaaggtgag cgggcaatgg 10140

ggtgccgcac gccccaggtg agcgggcggt gagggggcgc acgctccagg tgagcgggca 10200

acaggtgggg gggcgggtgg tgctaggcct gggtactgac caccagggcc gtcccaggtg 10260

tcgcaggccg ggaagaccct ctgcctgagc ggcttcatgg gcatggacat cccgccaccc 10320

agcgggccac tctggatcct gggcgacgtc ttcatcggcc gctactacac tgtgtttgac 10380

cgtgacaaca acagggtggg cttcgccgag gctgcccgcc tctagttccc aaggcgtccg 10440

cgcgccagca cagaaacaga ggagagtccc agagcaggag gcccctggcc cagcggcccc 10500

tcccacacac acccacacac tcgcccgccc actgtcctgg gcgccctgga agccggcggc 10560

ccaagcccga cttgctgttt tgttctgtgg ttttcccctc cctgggttca gaaatgctgc 10620

ctgcctgtct gtctctccat ctgtttggtg ggggtagagc tgatccagag cacagatctg 10680

tttcgtgcat tggaagaccc cacccaagct tggcagccga gctcgtgtat cctggggctc 10740

ccttcatctc cagggagtcc cctccccggc cctaccagcg cccgctgggc tgagccccta 10800

ccccacacca ggccgtcctc ccgggccctc ccttggaaac ctgccctgcc tgagggcccc 10860

tctgcccagc ttgggcccag ctgggctctg ccaccctacc tgttcagtgt cccgggcccg 10920

ttgaggatga ggccgctaga ggcctgagga tgagctggaa ggagtgagag gggacaaaac 10980

ccaccttgtt ggagcctgca gggtggtgct gggactgagc cagtcccagg ggcatgtatt 11040

ggcctggagg tggggttggg attgggggct ggtgccagcc ttcctctgca gctgacctct 11100

gttgtcctcc ccttgggcgg ctgagagccc cagctgacat ggaaatacag ttgttggcct 11160

ccggcctccc ctc 11173

<210> 6

<211> 4073

<212> DNA

<213> synthetic sequence (Artificial sequence)

<220>

<223> pscAAV/CTSD-MUT-HA

<400> 6

agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60

acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120

tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180

ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct 240

cgagatctag aaagcttccc ggggggatct gggccactcc ctctctgcgc gctcgctcgc 300

tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag 360

tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctggaggg 420

gtggagtcgt gacctaggca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa 480

tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac 540

atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg 600

cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 660

agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca 720

ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta 780

gtgaaccgtc agatcgcctg gagacgccat ccggactcta aggtaaatat aaaattttta 840

agtgtataat gtgttaaact actgattcta attgtttctc tcttttagat tccaaccttt 900

ggaactgaat tccgcgggcc cggccaccat gcagcccagc agcctgctgc ccctggccct 960

gtgtctgctg gccgctcctg ccagcgccct ggtgagaatc cctctgcaca agttcacctc 1020

catccggagg accatgagcg aggtgggcgg cagcgtggag gacctgatcg ctaaaggccc 1080

tgtgtccaag tatagccagg cagtgcccgc agtgaccgag ggccctatcc ctgaagtgct 1140

gaagaactac atggatgccc agtattacgg agagattgga attggcaccc cccctcctgt 1200

gctgcacagc aggctgaggc atggcctgct gcagcctgtg ggccctctgc accctctgca 1260

gaccgccgga cacagactgc tggatcctcc tcaggtgcag cagaggcagg tgcagcatct 1320

gagggaagag tggtacctgg tgtacccata cgatgttcca gattacgctt gagctaggcc 1380

tcacctgcga tctcgatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 1440

cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt 1500

tcagggggag gtgtgggagg ttttttaaac tagtccactc cctctctgcg cgctcgctcg 1560

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 1620

gtgagcgagc gagcgcgcag agagggacag atccgggccc gcatgcgtcg acaattcact 1680

ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 1740

tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 1800

ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac 1860

gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct gctctgatgc 1920

cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 1980

tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 2040

gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt 2100

tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 2160

aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 2220

catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 2280

tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 2340

tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 2400

ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 2460

ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 2520

cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 2580

ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 2640

tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 2700

gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 2760

ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 2820

aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 2880

acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 2940

tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 3000

cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 3060

gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 3120

taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 3180

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 3240

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 3300

ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 3360

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 3420

cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 3480

cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 3540

tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 3600

taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 3660

gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 3720

agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 3780

ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 3840

acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 3900

caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 3960

tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 4020

tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aag 4073

Claims (10)

1. A mutant of the human Cathepsin D (CTSD) gene, which mutant is a C base repeat at position 262 of the CTSD gene.

2. A human CTSD mutein characterized in that the mutein has a frame shift mutation beginning with the 88 th amino acid relative to the wild-type CTSD protein.

3. A polynucleotide encoding the mutein of claim 2.

4. A vector comprising the nucleic acid sequence of the mutant of claim 1, and/or the polynucleotide of claim 3.

5. A formulation comprising the following components:

(a) the protein of claim 2, the vector of claim 4, or a combination thereof; and/or (b) a pharmaceutically acceptable carrier.

6. Use of the protein of claim 2, the vector of claim 4, or the pharmaceutical formulation of claim 5, for the preparation of a composition for use in the preparation of a non-human mammalian model of a retinitis pigmentosa disease.

7. A method for preparing a non-human mammal model of a retinitis pigmentosa disease, comprising:

(1) administering the protein of claim 2, the vector of claim 4, and/or the pharmaceutical composition of claim 5 to the sub-retinal space of a non-human mammalian model.

8. A non-human mammalian animal model of retinal pigment degeneration disease, wherein a human CTSD mutein model is present in retinal pigment epithelial cells of the non-human mammalian animal model.

9. The application of the non-human mammal model of the retinal pigment degeneration disease is characterized in that the model is used for researching the retinal pigment degeneration disease and/or screening the medicine for treating the retinal pigment degeneration disease.

10. Use of the mutant of claim 1, the protein of claim 2, or the polynucleotide of claim 3 to prepare a reagent for detecting the presence of a CTSD mutation or a CTSD mutein in a sample.

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