CN112481281B - AGBL5 nucleotide sequence for coding cytoplasmic carboxypeptidase protein 5 and application thereof - Google Patents

Abstract

The invention relates to the technical field of biomedical gene therapy, and discloses an AGBL5 nucleotide sequence for coding cytoplasmic carboxypeptidase protein 5 and application thereof. The nucleotide sequence of the invention has more than or equal to 95 percent of sameness with the nucleotide sequence shown in SEQ ID NO. 3. The invention proves that the efficiency of expressing protein by the AGBL5 coding sequence after codon optimization is higher than that of a wild type sequence, the AAV-AGBL5 plasmid transfected HEK cell containing the nucleotide sequence can express AGBL5 protein, and the protein can correctly function. Meanwhile, the AAV2/2.7M8-coagBL5 drug treatment can express protein in pathological eye of AGBL5 knockout mice, remarkably improve tubulin high-degree glutamination caused by AGBL5 defect, and has the effect of preventing or treating retinitis pigmentosa.

Description

AGBL5 nucleotide sequence for coding cytoplasmic carboxypeptidase protein 5 and application thereof

Technical Field

The invention relates to the technical field of biomedical gene therapy, in particular to an AGBL5 nucleotide sequence for coding cytoplasmic carboxypeptidase protein 5 and application thereof.

Background

AGBL5 encodes cytoplasmic carboxypeptidase-like protein 5 (Cytosalic carboxypeptidase-like protein 5), also known as CCP 5. It belongs to a metallocarboxypeptidase that mediates deglutamate modification of proteins. AGBL5 belongs to the CCP family of proteins and specifically catalyzes the de-glutamylation of branch point glutamate side chains, such as those produced by post-translational glutamylation of tubulin. It cannot act as a long chain de-glutaminase to shorten the long polyglutamic acid chain, a process catalyzed by AGTPBP1, AGBL1, AGBL2, AGBL3 and AGBL 4. Since AGBL5 is the only enzyme in the CCP family of proteins that recognizes a branched glutamation site, its absence in vivo may not be complemented by isoenzymes. AGBL5 also mediates desugarization of CGAS and modulates the antiviral activity of CGAS.

Mutations in AGBL5 cause retinitis pigmentosa 75(RP75), and RP75 is a retinal dystrophy and is a pigmentary retinopathy. Retinitis pigmentosa is characterized by visible retinal pigment deposition on the fundus, primary loss of rod cells, and secondary loss of cone cells. Patients often have night blindness and loss of the central peripheral visual field. As the condition progresses, the patient loses far peripheral vision and eventually central vision. Meanwhile, RP75 is inherited as an autosomal recessive inheritance.

AGBL5 catalyzes the de-glutamylation modification of tubulin, which is widely distributed in various tissue cells of the retina. For example, the transport cilia in the retina contain a large amount of tubulin, which plays an important role in the turnover of material in the extracellular segment of the retina, and therefore the AGBL5 deficiency is of great importance for the maintenance of the tubulin-containing structure in the retina. The RP75 symptoms caused by AGBL5 mutations are mainly manifested by visual cell degeneration, and in addition to the influence of metabolic turnover of the outer segment, abnormalities in tubulin function in various types of cells in various cell layers of the retina also affect changes in the overall microenvironment, ultimately leading to impairment of the visual cells.

Naturally occurring AAV serotypes are generally unable to transduce retinal tissue cells on the vitreous chamber side because of the presence of barriers that prevent the spread of AAV virions, internal limiting membranes, glial cells, and the like. Through constructing an AAV2 capsid protein coding sequence library, inserting a random 7 amino acid sequence at the position of loop4, injecting the mutated serotype into a mouse vitreous cavity for screening, and enriching to a main mutated subtype called AAV2/2-7M8, namely AAV 2-588 LALGETTRP. AAV2/2-7M8 serotype has strong tropism for retinal tissue, and fluorescent reporter protein packaged by the serotype can be detected in the whole retina by intravitreal injection into mouse eyes.

Disclosure of Invention

In view of this, the present invention aims to provide an AGBL5 nucleotide sequence encoding cytoplasmic carboxypeptidase protein 5, such that the nucleotide sequence can be optimized by multiple parameters, such as codon usage preference, DNA repeat sequence, mRNA secondary structure, GC content, etc., to increase expression efficiency of AGBL 5;

the invention also aims to provide a virus vector carrying the nucleotide sequence and having the function of preventing or treating retinitis pigmentosa caused by AGBL5 mutation;

another object of the present invention is to provide a pharmaceutical preparation comprising the above viral vector or nucleotide sequence, and having the effect of preventing or treating retinitis pigmentosa caused by AGBL5 mutation; (ii) a

Another object of the present invention is to provide related applications of the above nucleotide sequences, viral vectors and pharmaceutical preparations in the field of preventing or treating retinitis pigmentosa caused by AGBL5 mutation, including but not limited to preparation of related drugs and reagents and prevention or treatment methods;

it is another object of the present invention to provide a method for delivering the above pharmaceutical formulation by injecting the pharmaceutical formulation into the eye such as subretinal or intravitreal injection.

In order to achieve the purpose of the invention, the invention provides the following technical scheme:

an AGBL5 nucleotide sequence for coding cytoplasmic carboxypeptidase protein 5, which has more than or equal to 95 percent of homology with the nucleotide sequence shown in SEQ ID NO. 3.

Preferably, the nucleotide sequence has more than or equal to 98 percent of homology with the nucleotide sequence shown in SEQ ID NO. 3; more preferably, the nucleotide sequence has more than or equal to 99 percent of identity with the nucleotide sequence shown in SEQ ID NO. 3; in a specific embodiment of the invention, the sequence is shown in SEQ ID NO 3.

Preferably, the nucleotide sequence is a cDNA sequence.

Meanwhile, the invention also provides a virus vector which comprises the nucleotide sequence.

Preferably, the viral vector is an adeno-associated viral vector, a lentiviral vector, a retroviral vector or an adenoviral vector; in a specific embodiment of the invention, the invention employs an adeno-associated viral vector having a serotype of AAV2 wild type or AAV2/2.7M 8.

More specifically, the viral vector regulates the expression of AGBL5 protein by a promoter CMV (sequence shown as SEQ ID NO: 4).

In addition, the invention also provides a pharmaceutical preparation which comprises the nucleotide sequence or the viral vector.

Preferably, the pharmaceutical formulation is a liquid formulation; the pharmaceutical formulation may also include a pharmaceutically acceptable carrier or excipient.

According to the invention, codon optimization (codon optimization) is carried out on AGBL5 cDNA sequence to obtain coaGBBL 5, cell level expression efficiency detection is carried out on the sequence wtAGBL 5/coaGBBL 5 before and after optimization, and the expression efficiency of the optimized sequence is found to be obviously improved. HEK293 cells were then transfected with AAV-AGBL5 plasmid to verify the effectiveness of AGBL5 protein expression in vitro. Simultaneously, AGBL5 expression can be detected in the retina of mice by intravitreal injection of AAV-AGBL5 to AGBL5 knockout mice. The mouse retinal tubulin glutamation degree is detected after 6 months, and the mouse retinal tubulin glutamation degree treated by AAV2-AGBL5 is found to be obviously reduced compared with the control AAV treatment. The AAV2-AGBL5 medicine is proved to have the effect of preventing or treating retinitis pigmentosa.

Based on the excellent technical effects of the above items, the invention provides the following related applications:

the nucleotide sequence disclosed by the invention is applied to the preparation of a virus vector or a pharmaceutical preparation for preventing or treating eye diseases caused by AGBL5 mutation, or is applied to the prevention or treatment of eye diseases caused by AGBL5 mutation;

the application of the virus vector in preparing a pharmaceutical preparation for preventing or treating eye diseases caused by AGBL5 mutation, or the application in preventing or treating eye diseases caused by AGBL5 mutation;

the pharmaceutical preparation provided by the invention is applied to preventing or treating eye diseases caused by AGBL5 mutation.

Wherein the eye diseases caused by AGBL5 mutation are retinal pigment degeneration caused by AGBL5 mutation.

The invention also correspondingly provides a delivery method of the pharmaceutical preparation, which is used for injecting the pharmaceutical preparation to the eye, such as a subretinal position or a vitreous cavity position.

According to the technical scheme, the invention proves that the efficiency of the protein expression of the AGBL5 coding sequence after codon optimization is higher than that of a wild-type sequence, and the AAV-AGBL5 drug treatment can obviously improve the AGBL5 mutation to improve the retinal pathological symptoms of AGBL5 knockout mice. Through intravitreal injection of AAV-AGBL5, AGBL5 can be efficiently expressed in each tissue layer of retina, and can improve the high glutamation of retinal tubulin and restore the normal function of the tubulin in each cell type of retina. Therefore, the AAV-AGBL5 medicine has the effect of preventing or treating retinitis pigmentosa.

Drawings

FIG. 1-FIG. 3 show an alignment of the wtAGBL5 and coaGBBL 5 sequences; after optimization, the differential codon sequences are thickened and marked with underlines;

FIG. 4 shows an AAV-coaGGL 5 vector map (A) and an AAV-wtAGBL5 vector map (B); the vector comprises AAV 25 'ITR, CMV promoter, codon-optimized AGBL5 cDNA or wild type AGBL5 cDNA, bGH polyA sequence and AAV 23' ITR;

FIG. 5 shows the expression efficiency of AAV-coagBL5 and AAV-wtAGBL5 plasmids in HEK293 cells;

a: AAV-coagBL5 and AAV-wtaGBL5 plasmids are respectively transfected in HEK293 cells, the cells are lysed after 48 hours, and the expression level of AGBL5 protein is detected by Western blot;

b: HEK293 cells transfected by AAV-coaGGL 5 and AAV-wtAGBL5 plasmids express the relative abundance of AGBL5 protein;

FIG. 6 shows a functional validation of AAV-coagBL5 expression protein; respectively transfecting an AAV-coagBL5 plasmid and an AAV-GFP plasmid into HEK293 cells, cracking the cells after 48 hours, incubating the cracked products with porcine brain tissue tubulin at 37 ℃ for 5 hours, detecting the content of the glutamated protein in the porcine brain tissue tubulin in a drug group and a control group by using Western blot after the reaction is terminated;

FIG. 7 shows the measurement of retinal AGBL5 gene expression after intravitreal injection of AAV2/2.7M8-coAGBL5 (AAV-coAGBL5 in the figure); performing intravitreal injection on AGBL5 knockout mice by AAV2/2.7M8-coAGBL5 virus, and performing AGBL5 protein expression detection on mouse retinas by using Western Blot after injection for 16 weeks;

FIG. 8 shows the mean length measurement of mouse retinal glutamated tubulin after virus treatment with AAV2/2.7M8-coagBL5 (KO-AAV-coagBL 5 in the figure); and (3) taking the medicine of AGBL5 knockout mice at 16 weeks after injection, injecting the medicine into eyes and retinas of control eyes, carrying out immunofluorescence staining, and carrying out quantitative analysis on the average length of the glutamated tubulin.

Detailed Description

The invention discloses an AGBL5 nucleotide sequence for coding cytoplasmic carboxypeptidase protein 5 and application thereof, and can be realized by appropriately improving process parameters by taking the contents into account by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. The nucleotide sequences and uses of the invention have been described in terms of preferred embodiments, and it will be apparent to those skilled in the art that variations or appropriate alterations and combinations of the nucleotide sequences and uses of the invention described herein may be made to practice and use the techniques of the invention without departing from the spirit and scope of the invention.

The AAV2/2.7M8-coagBL5 medicine can effectively express AGBL5 in retinal cells and repair the retinal diseases caused by AGBL5 mutation of patients, the protein sequence coded by the cDNA of AGBL5 is shown in SEQ ID NO. 1, and the wild type AGBL5 cDNA sequence is shown in SEQ ID NO. 2.

According to the invention, an optimized sequence coAGBL5 (shown in figures 1-3) which is obviously different from the wtAGBL5 sequence is obtained by optimizing multiple parameters such as codon usage preference, DNA repetitive sequence, mRNA secondary structure, GC content and the like. The wt/co AGBL5 sequence is constructed into an AAV vector (figure 4), then the same amount of plasmid is transfected in HEK293 cells, expression of AGBL5 gene is detected, the expression level of AGBL5 protein after sequence optimization is found to be higher (figure 5), and codon optimization is proved to improve the efficiency of AGBL5 at the translation level and provide more protein with normal function for the cells, thereby better compensating the defect caused by gene mutation.

The invention not only proves that the expression efficiency of AGBL5 optimized by codon in vitro is better than that of a wild type sequence, but also confirms the function of AGBL5 protein through in vitro enzyme activity experiments, in order to further verify the effectiveness of AAV2/2.7M 8-coaGGL 5 medicaments in vivo, the invention packages AAV2/2.7M8 serotype AGBL5 virus medicaments, carries out intravitreal injection on AGBL5 knockout mice, and observes in vivo experimental results. The experimental result shows that the degree of the de-glutamylation of the tubulin in the retina of the eye treated by the injection of the medicine is higher, and the protein modification lesion caused by AGBL5 defect is improved.

The result is combined, the invention proves the therapeutic effect of the AAV2/2.7M8-coagBL5 gene therapy medicine on retinitis pigmentosa caused by AGBL5 mutation, and lays a foundation for further clinical application development.

The invention is further illustrated by the following examples.

Example 1: codon-optimized AGBL5 vector construction and expression verification

(1) Plasmid vector construction

1. The AAV-CMV plasmid skeleton, the coagBL5 fragment and the wtAGBL5 fragment are respectively and simultaneously subjected to double enzyme digestion by HindIII and XhoI, and then the enzyme-digested fragments are respectively connected with the skeleton.

2. And transforming the connecting product into escherichia coli, and selecting a single colony for enzyme digestion verification and sequencing verification.

(2) Cell transfection

1. One day prior to transfection, cells were trypsinized and counted, and cells were plated to a density of 90% on the day of transfection.

2. For each well of cells, 0.8. mu.g-1.0. mu.g of DNA was diluted with 50. mu.l of serum-free DMEM medium.

3. For each well of cells, 1. mu.l to 3. mu.l of LIPOFECTAMINE 2000 reagent was diluted with 50. mu.l of DMEM medium. LIPOFECTAMINE 2000 was diluted and mixed with the diluted DNA within 5 minutes.

4. The diluted DNA and diluted LIPOFECTAMINE 2000 were mixed and incubated at room temperature for 20 minutes.

5. Directly add the complex to each well, shake the plate and mix gently.

6. At 37 deg.C, 5% CO₂And culturing for 48 hours.

7. The culture medium is discarded, washed by PBS, digested by pancreatin, and centrifuged to collect cells for later use.

(3)Western Blot

1. And (3) preparing a protein sample, namely adding PMSF (prepared at present according to the dosage) into the lysate according to the proportion of 1: 100.

2. Cells were lysed using a strong lysis solution.

3. Protein concentration was determined using the BCA method.

4. Electrophoresis

a. Preparing corresponding separation gel (5 ml/block) according to the size of the detected protein, and solidifying the separation gel.

b. 5% concentrated gum (2 ml/block) was prepared, the glass plate was filled and a comb was inserted.

c. Mu.l of the prestained protein molecule marker SDS-PAGE was added to the wells, and 10. mu.l of 1 XSDS-PAGE protein loading buffer was added to the blank wells at the edges of the sample wells.

5. Rotary film

And placing a wet cushion layer on the film transferring white clamp, laying three pieces of wet filter paper which are overlapped together on the cushion layer, sequentially placing a wet pvdf film, glue, the filter paper, the cushion layer and a black clamp plate on the filter paper, placing the clamped plate into an electrophoresis tank filled with a film transferring buffer solution, and placing the film transferring tank in an ice bath for film transferring.

6. Sealing of

And after the membrane is completely transferred, rinsing for 1-2 minutes, completely absorbing the buffer solution by using a dropper, adding 5% of skimmed milk powder, slowly shaking on a side shaking table, and sealing for 15-60min at room temperature. TBS washing was added and the mixture was washed for 5 minutes. A total of 3 washes were performed.

7. Primary antibody incubation

Appropriate primary antibody was diluted in 5% skim milk/PBS + 2% BSA at the ratio and incubated overnight with slow shaking at 4 ℃ or for 2h on a side-shaking shaker at room temperature. After incubation, washing is carried out.

8. Incubation with a second antibody

Adding diluted secondary antibody, and slowly shaking and incubating for 40min-1h on a room temperature side shaking bed. After incubation, washing is carried out.

9. Protein detection

And (3) detecting the protein by using ECL reagents, uniformly mixing 1ml of each ECL reagent, dripping the mixture on the surface of the protein membrane, and incubating for 1-2min in a dark place. The protein film was placed neatly on plastic paper with tweezers and exposed on a gel imager. The results are shown in fig. 5A and fig. 5B, the expression level of AGBL5 protein after sequence optimization is higher, which proves that codon optimization improves the efficiency of AGBL5 at the translation level, and provides more proteins with normal functions for cells, thereby better compensating the defects caused by gene mutation.

Example 2: AAV-mediated AGBL5 in-vitro cell expression and function verification

(1) Cell transfection

1. One day prior to transfection, cells were trypsinized and counted, and cells were plated to a density of 90% on the day of transfection.

2. For each well of cells, 0.8. mu.g-1.0. mu.g of DNA was diluted with 50. mu.l of serum-free DMEM medium.

3. For each well of cells, 1. mu.l to 3. mu.l of LIPOFECTAMINE 2000 reagent was diluted with 50. mu.l of DMEM medium. LIPOFECTAMINE 2000 was diluted and mixed with the diluted DNA within 5 minutes.

4. The diluted DNA and diluted LIPOFECTAMINE 2000 were mixed and incubated at room temperature for 20 minutes.

5. Directly add the complex to each well, shake the plate, and mix gently.

6. At 37 deg.C, 5% CO₂And preserving the heat for 24-48 hours.

7. The cells are lysed 24-72 hours after the addition of the complex to the cells.

(2) Tubulin glutamation assay

1. The cell lysate was centrifuged at 40000g for 20min at 4 ℃.

2. 20ul of supernatant was incubated with 1ug of porcine tissue tubulin at 37 ℃ for 5 hours.

3. The reaction was stopped with a metal carboxypeptidase inhibitor.

(3)Western Blot

1. The above reaction product was denatured with the loading Buffer at 95 ℃ for 5 minutes.

2. Electrophoresis

a. Preparing corresponding separation gel (5 ml/block) according to the size of the detected protein, and solidifying the separation gel.

b. 5% concentrated gum (2 ml/block) was prepared, the glass plate was filled and a comb was inserted.

c. Mu.l of the prestained protein molecule marker SDS-PAGE was added to the wells, and 10. mu.l of 1 XSDS-PAGE protein loading buffer was added to the blank wells at the edges of the sample wells.

3. Rotary film

And placing a wet cushion layer on the film transferring white clamp, laying three pieces of wet filter paper which are overlapped together on the cushion layer, sequentially placing a wet pvdf film, glue, the filter paper, the cushion layer and a black clamp plate on the filter paper, placing the clamped plate into an electrophoresis tank filled with a film transferring buffer solution, and placing the film transferring tank in an ice bath for film transferring.

4. Sealing of

And after the membrane is completely transferred, rinsing for 1-2 minutes, completely absorbing the buffer solution by using a dropper, adding 5% of skimmed milk powder, slowly shaking on a side shaking table, and sealing for 15-60min at room temperature. TBS washing was added and the mixture was washed for 5 minutes. A total of 3 washes were performed.

5. Primary antibody incubation

Appropriate primary antibody was diluted in 5% skim milk/PBS + 2% BSA at the ratio and incubated overnight with slow shaking at 4 ℃ or for 2h on a side-shaking shaker at room temperature. After incubation, washing is carried out.

6. Incubation with a second antibody

Adding diluted secondary antibody, and slowly shaking and incubating for 40min-1h on a room temperature side shaking bed. After incubation, washing is carried out.

7. Protein detection

And (3) detecting the protein by using ECL reagents, uniformly mixing 1ml of each ECL reagent, dripping the mixture on the surface of the protein membrane, and incubating for 1-2min in a dark place. The protein film was placed neatly on plastic paper with tweezers and exposed on a gel imager.

The results are shown in fig. 6, AAV-coAGBL5 plasmid and AAV-GFP plasmid were transfected into HEK293 cells, the cells were lysed after 48 hours, the lysate and porcine brain tubulin were incubated for 5 hours at 37 ℃, and the content of glutamated protein in porcine brain tubulin in the experimental group and the control group was detected by Western blot after termination of the reaction, and it was found that the content of protein specifically bound by GT335 antibody in the experimental group was significantly reduced, and the protein specifically bound by poly e antibody was not different from the control group (fig. 6). The GT335 antibody can be specifically combined with the tubulin glutamizing branch site, and the difference of GT335 immune reaction of the experimental group and the control group indicates that the tubulin glutamizing branch site in the experimental group is partially degraded; the specific recognition of the long-chain glutamic acid by the PolyE antibody shows that the long-chain glutamic acid is not degraded because of no difference of the immune response of the PolyE in the experimental group and the PolyE in the control group. The function of AGBL5 is that the protein specifically catalyzes the branch point de-glutamylation modification of the microscopic protein, and the results prove that the protein expressed by AAV-coAGBL5 in vitro cells can perform correct functions.

Example 3: AAV2/2.7M8-coagBL5 gene therapeutic drug for improving mouse retinal tubulin glutamylation abnormality

(1) Virus package and virus medicine injection mouse

1. HEK293T cells with a degree of polymerization above 90% were treated as follows: and 3, a ratio transmission disc.

2. The serum-free medium is replaced 1-2h before plasmid transformation, and the target gene plasmid and the helper plasmid (containing AAV2.7M8 serotype elements) are transformed into HEK293T by using a transfection reagent.

3. After 24h of plasmid transformation, the new serum-free medium is replaced

4. The transfection is carried out for 72h for detoxification. Blowing down the cells with the culture medium, and centrifuging; the culture supernatant and the cell pellet were then harvested separately. The virus in the culture supernatant was precipitated with PEG8000, and the virus precipitate was collected overnight.

5. The virus mixture was purified by iodixanol density gradient centrifugation and then concentrated using an ultrafiltration tube.

6. AGBL5 knockout mice were constructed.

7. Ready 5 x 10¹²vg/ml of AAV2/2.7M8-coagBL5 drug and AAV2/2.7M 8-GFP.

8. 1 ul/eye of AAV2/2.7M8-coagBL5 drug and AAV2/2.7M8-GFP virus was injected intravitreally into the eyes of 6-8 week old mice.

9. At 16 weeks of age, mice were sacrificed and retinal tissue was isolated.

(2) Immunofluorescent staining of retinal tissue

1. The retinal tissue is taken for flaking, and washed 5min x 3 times with 0.01 MPBS.

2. Adding 10% normal goat serum dropwise, sealing at 37 deg.C for 45 min.

3. Excess liquid was aspirated, primary antibody (1: 100) was added, placed in a wet box, kept in a refrigerator at 37 ℃ for 1h and then kept overnight (in a wet box).

4.0.01M PBS washing 5min x 3 times.

5. Secondary antibody (1: 200) was added under dark conditions and incubated at 37 ℃ for 45 min.

6. The secondary antibody was discarded in the dark (note: no more rinse), and DAPI stain was added and allowed to react for 20min at room temperature.

7. Washing with 0.01MPBS under dark conditions for 5min × 6 times.

8. And sealing the film with an anti-fluorescence quencher under a dark condition, and observing under a fluorescence microscope.

The previous examples have confirmed that the expression efficiency of codon-optimized AGBL5 in vitro is superior to that of a wild-type sequence, and simultaneously the function of AGBL5 protein is confirmed through in vitro enzyme activity experiments, in order to further verify the effectiveness of AAV2/2.7M 8-coaGGL 5 drugs in vivo, the example packages AAV2.7M8 serotype AGBL5 virus drugs, carries out intravitreal injection on AGBL5 knockout mice, and observes in vivo experimental results. Firstly, AGBL5 was detected by extracting proteins from mouse retinal tissues in mouse eye expression, and it was found that AGBL5 protein could be detected in drug-injected eyes (AAV-coAGBL5) but AGBL5 could not be detected in control eyes (AAV-GFP) (FIG. 7), indicating that AAV2.7M8 serotype is suitable for intravitreal injection and can correctly deliver drugs to mouse retinal tissues. Then, the eye tissues of the mice are used for carrying out retina immunostaining analysis, and quantitative analysis on the glutamated tubulin shows that the length of the glutamated tubulin of the treated eyes is obviously smaller than that of the control eyes (figure 8), which shows that the degree of the glutamated tubulin of the treated eyes after the injection of the drug is higher, and the protein modification lesion caused by AGBL5 defect is improved.

By combining the results of the above embodiments, the invention proves that the AGBL5 protein expression level after sequence optimization is higher, and proves that codon optimization enables AGBL5 to improve the efficiency at the translation level, so that more proteins with normal functions are provided for cells, and the defects caused by gene mutation are better compensated; the AAV2/2.7M8-coagBL5 gene therapy medicament has an obvious therapeutic effect on retinitis pigmentosa caused by AGBL5 mutation, and lays a foundation for further clinical application development.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Wuhan Newcastle Biotechnology Ltd

<120> AGBL5 nucleotide sequence for coding cytoplasmic carboxypeptidase protein 5 and application thereof

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<400> 2

atggagctgc gctgtggggg attgctgttc agttctcgct ttgattcagg gaatctagcc 60

cacgtggaga aggtggaatc tttgtccagt gatggggaag gggtaggagg tggggcgtca 120

gccctgacca gtggcattgc ctcttcccct gactatgaat tcaacgtgtg gacccgacca 180

gactgtgctg aaacggaatt tgagaatggg aacaggtcat ggttctactt cagcgtccgg 240

ggaggaatgc caggaaaact catcaagatc aacattatga acatgaacaa gcagagcaag 300

ctgtattccc agggcatggc cccctttgtg cgcacactgc ccacccggcc acgctgggaa 360

cgcattcgag accggcccac ctttgagatg acagagacgc agtttgtgtt atcctttgtt 420

catcgtttcg tggagggccg tggggccacc accttcttcg ccttctgcta ccccttctcc 480

tacagtgact gccaggaact gctaaaccag ctagaccagc gctttccgga gaaccaccct 540

acccatagca gccccctgga taccatctat taccatcggg agctcctttg ctattctctg 600

gatggacttc gtgtagatct gctgacgatc acttcctgcc atgggcttcg agaagatcga 660

gagccccgtc tagagcagct atttcctgat accagcaccc ctcgaccatt ccgtttcgca 720

ggcaagagga tattcttctt aagcagtaga gtacacccag gggagactcc atctagcttt 780

gtcttcaatg gctttctgga cttcatcctc cgacctgatg atccccgggc ccaaaccctc 840

cgtcgcctct tcgtctttaa gctgattccc atgttgaacc ccgatggtgt ggtccgggga 900

cactaccgca cagactcacg tggagtgaat ctgaaccgtc agtacctgaa gcctgatgcc 960

gtcctgcacc cggccatcta tggggccaaa gctgtgcttc tctaccacca tgtgcactct 1020

cgtctgaact cccagagttc ctctgagcac cagcccagtt cctgtctccc tcctgatgct 1080

cctgtttctg acctggagaa agccaacaat ctccaaaatg aagctcagtg tgggcactca 1140

gctgacaggc ataacgctga agcctggaaa caaacagagc cagcagaaca gaagctcaac 1200

agtgtgtgga ttatgccaca acagtctgcg gggcttgaag agtcagcccc tgataccatc 1260

ccccccaaag agagtggcgt tgcttactat gtggacctgc atggacatgc ttccaaaagg 1320

ggctgcttca tgtacggaaa cagctttagt gatgagagca cccaggtgga aaacatgcta 1380

tatccaaagc tcatctcctt gaattcagcc cacttcgact tccagggctg caatttctca 1440

gagaagaata tgtatgcccg agaccgtaga gatggccagt ctaaagaggg aagcggccgt 1500

gttgcaatct acaaagcctc agggataatc cacagctaca cacttgaatg caactacaac 1560

actggacgct cagtaaacag catccctgct gcctgccatg acaatgggcg tgccagcccc 1620

cctcccccgc cggctttccc ctccagatac actgtggaac tatttgagca ggtgggacga 1680

gctatggcca ttgcagccct ggacatggcg gaatgtaatc cgtggccccg aattgtactg 1740

tcagagcaca gcagccttac taatctacgg gcctggatgc tgaaacatgt acgcaacagc 1800

cgaggcctaa gcagcactct gaatgtgggt gtcaacaaga agaggggcct tcgaactcca 1860

cccaaaagtc acaatgggtt gcctgtctcc tgctccgaaa acaccttgag tcgggcacga 1920

agttttagca ccggcacaag tgccggtggt agcagcagca gccaacaaaa ttctccacag 1980

atgaagaatt cccccagctt tccttttcat ggcagtcggc ctgcagggct gccaggcctg 2040

ggctctagta cccaaaaggt cacccaccgg gtgctgggcc ccgtcagaga gccccgaagc 2100

caggacagga gacggcagca gcagcccctg aaccatcgtc ctgcaggcag cctcgctcca 2160

tccccagctc ctactagttc tggcccagcc tcctcacaca agctgggctc ctgtctactg 2220

cctgattcat tcaacatacc agggagcagt tgctcactct tgtcctctgg agacaaacca 2280

gaggctgtca tggtaatcgg gaaaggtctg ctagggactg gagctcggat gccctgcatc 2340

aagactcgat tgcaggctag gcccaggttg ggccggggct caccgccgac tcgcagaggg 2400

atgaaaggct cttcaggccc cacatcccct accccccgga ccagggagag cagtgagctg 2460

gagctgggat cctgctctgc tacaccaggg ctgcctcagg ccaggccccc acggccccgc 2520

tctgcccctg ccttttctcc tatatcctgt agtctatctg actccccatc ctggaattgt 2580

tacagcaggg gtcccttggg ccaacctgag gtttgttttg tccctaaatc tcccccactg 2640

actgtttctc cccgggtctg a 2661

<210> 3

<211> 2661

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggaactcc gatgtggtgg cctgttgttc agctctagat ttgacagcgg gaaccttgcc 60

catgttgaga aagtggagag cttgagttcc gatggcgaag gtgtgggagg aggtgctagt 120

gcccttacca gcgggattgc ttcaagtcca gattatgaat ttaatgtatg gaccagacca 180

gattgtgccg agaccgaatt tgagaacggt aatcggtcat ggttctactt ctctgtgagg 240

ggagggatgc cgggcaaact gatcaagatc aatatcatga atatgaacaa gcagagcaaa 300

ctttattctc agggtatggc tcctttcgtg aggacattgc ctacaagacc tcgctgggag 360

aggattaggg accggcctac attcgagatg accgagaccc agttcgtgct gtcctttgtg 420

cataggttcg tcgaaggacg cggcgccaca actttcttcg ctttctgtta cccattttcc 480

tattccgatt gccaggagct gcttaaccag ctggaccagc gcttccctga gaatcacccc 540

acccactcct cccctttgga cacaatttat taccataggg agctcctgtg ctattccttg 600

gatggactga gggtcgatct tctcaccatt acatcatgcc atggcttgag ggaggatagg 660

gaacccaggt tggagcagct gtttcccgac accagcaccc caaggccgtt tcgattcgca 720

ggtaaaagaa tttttttcct gagctctcga gttcacccgg gggaaacacc ctcctccttc 780

gtattcaacg gattcctgga ttttattctg agacccgatg accctagagc ccagaccttg 840

cggagattgt tcgtgttcaa gctgatccca atgcttaacc cagatggggt ggtgcgggga 900

cactatagaa ccgatagtcg cggagtcaac ctgaaccggc agtacctgaa gccagatgcc 960

gtgttgcacc ccgccatata tggagcgaaa gcagtgctgc tctaccatca tgtccacagt 1020

aggctgaact cccaatctag ctccgagcac caaccctcct catgtctccc tccagatgcc 1080

ccagtcagcg acctggagaa ggcaaataac ctgcagaacg aagcacagtg cgggcactcc 1140

gctgacagac acaacgctga ggcctggaaa caaactgagc ccgccgagca gaagctgaat 1200

tctgtttgga tcatgccaca gcagtccgcc ggacttgaag agtccgctcc tgacacgatt 1260

cccccgaagg agagcggcgt agcttattat gtcgatttgc acggccatgc atcaaagcga 1320

gggtgcttca tgtacggcaa ttctttctct gacgaatcca cccaagtgga aaatatgctt 1380

tatccaaagc tgatatcact taattccgcc cacttcgact tccagggctg taacttctcc 1440

gaaaagaaca tgtacgcgcg agataggcgg gatggtcaga gcaaggaggg ctctgggagg 1500

gttgcaatat acaaggcctc tggtatcatc cactcttata ccctggaatg caactacaat 1560

accggtcgat ccgtcaattc tattccagct gcctgtcacg acaatggaag ggctagtcca 1620

cctccacctc ctgctttccc ctctagatat acggtcgaac tgttcgaaca ggttggccgc 1680

gccatggcaa tcgctgctct tgacatggct gagtgtaacc catggcctcg catcgttctc 1740

tcagagcata gttccctgac taacctcaga gcttggatgt tgaagcatgt caggaactcc 1800

aggggactca gcagcaccct gaatgttgga gtcaacaaaa aaaggggcct ccgaacccca 1860

cccaaatcac ataatggact gcctgtttca tgcagcgaga atacactgtc ccgggcccga 1920

agctttagca ccggtacctc cgcaggagga agcagttcca gtcagcagaa cagcccccag 1980

atgaagaact ccccctcctt ccctttccat gggagcagac cagcaggtct tccagggctg 2040

ggaagttcca cacagaaggt cacacaccgc gttctcggcc ccgtgagaga acctagatct 2100

caggatcgca gacgccagca gcaaccgctt aatcacaggc cagccggttc tttggcccct 2160

agtcctgcac ctacgtccag tggtcctgct tcttcccata aactgggtag ctgtctcctc 2220

cctgacagct ttaatatccc cggaagctcc tgttctctgc tctcaagcgg tgacaagcca 2280

gaggcggtca tggtgatagg taaggggctg ctgggtacgg gtgctaggat gccatgtatc 2340

aaaaccaggc tccaagctag acctcgattg ggaaggggat cacctccaac ccgcagaggg 2400

atgaagggat catctggccc aacctccccc actccaagga ctagggaatc tagtgagctc 2460

gagttgggtt cttgtagcgc cactcctgga ttgccacagg ccagaccgcc aagaccacga 2520

tctgctccgg cgttctcccc cattagctgt tccctttccg attcacctag ctggaattgc 2580

tatagtcggg gtcctctggg gcaaccagaa gtatgtttcg tccctaaatc tccaccactg 2640

acggtgtccc cacgggtatg a 2661

<210> 4

<211> 203

<212> DNA

<213> CMV promoter sequence (CMV promoter)

<400> 4

gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 60

ccaagtctcc accccattga cgtcaatggg agtttgtttt gcaccaaaat caacgggact 120

ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 180

gggaggtcta tataagcaga gct 203

Claims (11)

1. An AGBL5 nucleotide sequence encoding cytoplasmic carboxypeptidase protein 5,

the sequence is shown in SEQ ID NO. 3.

2. The nucleotide sequence of claim 1, wherein the nucleotide sequence is a cDNA sequence.

3. Use of a nucleotide sequence of any one of claims 1-2 for the preparation of a viral vector or a pharmaceutical formulation for the treatment of an ocular disorder caused by a mutation in AGBL5, wherein the ocular disorder caused by a mutation in AGBL5 is retinitis pigmentosa caused by a mutation in AGBL 5.

4. A viral vector comprising the nucleotide sequence of any one of claims 1-2.

5. The viral vector according to claim 4, wherein the viral vector is an adeno-associated viral vector, a lentiviral vector, a retroviral vector or an adenoviral vector.

6. The viral vector according to claim 5, wherein the serotype of the adeno-associated viral vector is AAV2 wild type or AAV2/2.7M 8.

7. The viral vector according to any one of claims 4 to 6, wherein the expression of AGBL5 protein is regulated by the promoter CMV.

8. Use of a viral vector according to any one of claims 4 to 7 for the preparation of a pharmaceutical formulation for the treatment of an ocular disorder caused by a mutation in AGBL5,

the eye diseases caused by the AGBL5 mutation are that the AGBL5 mutation causes retinitis pigmentosa.

9. A pharmaceutical preparation comprising a nucleotide sequence according to any one of claims 1 to 2 or a viral vector according to any one of claims 4 to 7.

10. The pharmaceutical formulation of claim 9, wherein the pharmaceutical formulation is a liquid formulation.

11. The pharmaceutical formulation of claim 9 or 10, further comprising a pharmaceutically acceptable carrier or excipient.

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