CN112553225B - PDE6B nucleotide sequence and application thereof - Google Patents

Abstract

The invention relates to the technical field of biomedical gene therapy, and discloses a PDE6B nucleotide sequence and application thereof. The invention proves that the efficiency of the protein expression of the coding sequence of PDE6B after codon optimization is higher than that of a wild-type sequence, and the retina pathological symptoms of mice with retinitis pigmentosa caused by PDE6B mutation can be obviously improved and the recovery of the retina function can be achieved by using AAV2/2.7m8-coPDE6B medicine treatment. By injecting AAV2/2.7m8-coPDE6B medicine, PDE6B can be efficiently expressed in the outer nuclear layer of retina and increase the thickness of the outer nuclear layer of retina. The electroretinogram showed that the mice in the drug-treated group of AAV2/2.7m8-coPDE6B responded strongly to the stimulus. Therefore, the AAV2/2.7m8-coPDE6B medicine has the effect of preventing or treating retinitis pigmentosa.

Description

PDE6B nucleotide sequence and application thereof

Technical Field

The invention relates to the technical field of biomedical gene therapy, in particular to a PDE6B nucleotide sequence and application thereof.

Background

The PDE6 protein is composed of two catalytic subunits, PDE6A and PDE6B, and two inhibitory subunits. The protein can regulate cGMP level in cytoplasm when the rod cells are stimulated by light, thereby hyperpolarizing the rod cell membrane and finally generating receptor potential. The PDE6B mutation results in inactivation of the PDE6 protein, disruption of the light transmission pathway, and a sharp increase in the cytoplasmic cGMP and calcium levels, causing apoptosis, resulting in degeneration of the visual cells. Since PDE6B is indispensable in the rod cell light transmission pathway, and mutation of PDE6B gene causes severe retinitis pigmentosa diseases, no effective cure method exists at present.

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 retinal tissue tropism, 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 a PDE6B nucleotide sequence, so that the nucleotide sequence can have higher expression efficiency of PDE6B through optimizing multiple parameters such as codon usage bias, DNA repeat sequence, mRNA secondary structure, GC content in equilibrium sequence, etc.;

another object of the present invention is to provide a viral vector carrying the above nucleotide sequence and having the effect of preventing or treating retinitis pigmentosa caused by the mutation of PDE 6B;

it is another object of the present invention to provide a pharmaceutical preparation comprising the above viral vector or nucleotide sequence, and having an effect of preventing or treating retinitis pigmentosa caused by PDE6B mutation; (ii) a

It is another object of the present invention 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 PDE6B mutation, including but not limited to the preparation of related drugs and reagents and methods for preventing or treating retinitis pigmentosa;

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 above purpose, the invention provides the following technical scheme:

PDE6B nucleotide sequence has 95% identity to 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 that is serotype AAV2 wild type or AAV2/2.7M 8.

More specifically, the viral vector regulates the expression of PDE6B protein by promoter CAG (sequence shown in 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.

In the invention, codon optimization (codon optimization) is carried out on a PDE6B cDNA sequence to obtain copE 6B, cell level expression efficiency detection is carried out on the sequences wtpDE 6B/copE 6B before and after optimization, and the expression efficiency of the optimized sequence is found to be obviously improved. In-vivo experiments in mice prove that the expression efficiency of the CAG promoter is higher than that of the sCBA + AT2R Intron 1 promoter and the sCBA promoter. The expression of PDE6B was detected in retinal tissue cells of mice by intravitreal or subretinal injection of AAV2/2.7m8-coPDE6B in PDE6B mutant rd10 mice. The mouse outer nuclear retina layer (ONL) thickness and the state of visual cell apoptosis were measured after 3 weeks, and it was found that the AAV2/2.7m8-coPDE6B treated mouse outer nuclear retina layer thickness was significantly increased and visual cell apoptosis was improved compared to control AAV treatment. Meanwhile, the results of the electroretinogram show that rd10 mouse treatment eyes have strong response to the stimulation. The structure and the function of the mouse retina treated by the AAV2/2.7m 8-copE 2 6B are remarkably restored, and the AAV2/2.7m 8-copE 6B medicament has the effect of preventing or treating retinitis pigmentosa.

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

the application of the nucleotide sequence of the invention in preparing a viral vector or a pharmaceutical preparation for preventing or treating eye diseases caused by PDE6B mutation, or in preventing or treating eye diseases caused by PDE6B mutation;

the application of the virus vector in preparing a pharmaceutical preparation for preventing or treating eye diseases caused by PDE6B mutation, or the application in preventing or treating eye diseases caused by PDE6B mutation;

the application of the pharmaceutical preparation provided by the invention in preventing or treating eye diseases caused by PDE6B mutation.

Wherein, the eye diseases caused by the PDE6B mutation are retinal pigment degeneration caused by the PDE6B 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 codon-optimized PDE6B coding sequence is higher than that of a wild-type sequence, and the retina pathological symptoms of rd10 mice with retinitis pigmentosa caused by PDE6B mutation can be remarkably improved and the recovery of the retina function can be achieved by using AAV2/2.7m8-coPDE6B drug treatment. By injecting AAV2/2.7m8-coPDE6B medicine into vitreous cavity or subretinal space, PDE6B can be expressed at high efficiency in outer nuclear layer of retina, and the thickness of the outer nuclear layer of retina is increased. Meanwhile, the electroretinogram showed that rd10 mice in the drug-treated group of AAV2/2.7m8-coPDE6B responded strongly to the stimulus compared to the control group. Therefore, the AAV2/2.7m8-coPDE6B medicine has the effect of preventing or treating retinitis pigmentosa.

Drawings

FIGS. 1-3 show an alignment of the wtPDE6B and coPDE6B sequences; after optimization, the differential codon sequences are thickened and marked with underlines;

FIG. 4 shows the AAV 2-copOD 6B vector map (A) and AAV2-wtPDE6B vector map (B); the vector comprises AAV 25 'ITRs, CAG promoter (CMV enhancer, chicken β actin promoter, chimera intron), codon optimized PDE6B cDNA or wild type PDE6B cDNA, bGH polyA sequence and AAV 23' ITRs;

FIG. 5 shows the expression efficiency of AAV2-coPDE6B and AAV2-wtPDE6B plasmids in HEK293 cells;

a: AAV2-coPDE6B and AAV2-wtpDE6B plasmids are respectively transfected in HEK293 cells, the cells are lysed after 48 hours, and the expression level of PDE6B protein is detected by Western blot;

b: AAV2-coPDE6B and AAV2-wtPD 6B plasmids transfect HEK293 cells to express the relative abundance of PDE6B protein;

FIG. 6 shows the comparison of the expression efficiency of CAG promoter, sCBA + AT2R Intron 1 promoter and sCBA promoter in rd10 mice; AAV2/2.7m8-CAG-coPDE6B, AAV2/2.7m8-sCBA + AT2R Intron 1-coPDE6B and AAV2/2.7m8-sCBA-coPDE6B virus vitreous cavity or subretinal injection rd10 mice, 3 weeks later, the mouse eyeball tissue lysis cells are taken to extract RNA, and the mRNA level of PDE6B is analyzed by qPCR;

FIG. 7 shows that after AAV2/2.7m8-CAG-coPDE6B virus treatment, rd10 mice treated eyes (rd10-AAV2/2.7m8-CAG-coPDE6B) and untreated eyes (rd10-AAV2/2.7m8-CAG-GFP) and wild type mice (WT) retina outer nuclear layer thickness assay and apoptosis status assay;

a: taking treated eyes and untreated eyes of rd10 mice and retinas of eyes of wild type mice 3 weeks after injection, carrying out immunofluorescence staining, and carrying out quantitative analysis on the thicknesses of upper and lower outer nuclear layers which are different in length from optic nerves;

b: taking treated eyes and untreated eyes of rd10 mice and retinas of eyes of wild type mice 3 weeks after injection, and detecting apoptosis degree of visual cells by using TUNEL experiment after slicing;

FIG. 8 shows electroretinogram measurements of mice after treatment with AAV2/2.7m8-CAG-coPDE6B virus; electroretinograms were performed 3 weeks after injection on the treated eyes of rd10 mice (rd10-AAV2/2.7m 8-CAG-cote 6B) and untreated eyes (rd10-AAV2/2.7m8-CAG-GFP) as well as wild type mice (WT), given different intensities of light stimuli in the dark, and recorded the a-wave and b-wave amplitudes of the eyes of each mouse (n 10) under light stimuli.

Detailed Description

The invention discloses a PDE6B nucleotide sequence and application thereof, and can be realized by appropriately improving process parameters by a person skilled in the art with reference to the content. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. 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.

AAV2/2.7m8-CAG-coPDE6B can effectively express PDE6B in retinal cells by intravitreal or subretinal injection, repair retinal diseases caused by PDE6B mutation, and the protein sequence coded by the cDNA of PDE6B is shown in SEQ ID NO. 1, and the wild-type PDE6B cDNA sequence is shown in SEQ ID NO. 2.

The invention obtains an optimized sequence coPDE6B (figures 1-3) which is obviously different from a wtPDE6B sequence by optimizing a plurality of parameters such as codon usage preference, a DNA repetitive sequence, mRNA secondary structure, GC content and the like. The wt/co PDE6B sequence was constructed into AAV vector (FIG. 4), then the same amount of two plasmids were transfected in 293 cell, and the expression of PDE6B gene was tested, and the expression efficiency of PDE6B after sequence optimization was found to be higher (FIG. 5), which proves that codon optimization can improve the expression level of protein without changing protein sequence, and the supplement protein with higher expression level can theoretically provide better therapeutic effect under the condition that the cell can not provide enough dosage due to mutation or completely lacks normal functional protein.

According to the invention, rd10 mice are injected into a vitreous cavity or under a retina of a virus medicament packaged by PDE6B cDNA plasmids driven by different promoters, and mouse eye tissues are extracted after 3 weeks to detect the expression level of PDE6B mRNA, so that the PDE6B driven by the CAG promoter expresses more mRNA in rd10 mice eyes (figure 6), and the expression efficiency of the CAG promoter is obviously higher than that of sCBA + AT2R Intron 1 and sCBA promoters in rd10 mice. Therefore, the CAG promoter is a better choice for PDE6B gene therapy of rd10 mice than other promoters.

In addition, in order to demonstrate the therapeutic effect of AAV-coPDE6B gene therapy drugs on retinitis pigmentosa caused by PDE6B mutation, in vivo experiments were performed using PDE6B mutant mouse rd 10. The improvement of the eye lesions of the mice by the drug treatment is observed after 3 weeks by injecting the drug into the vitreous cavity or under the retina. First, eye tissues from rd10 mice receiving drug injections were used for retinal immunostaining analysis, and quantitative analysis of the thickness of the outer nuclear layer in different areas showed that the number of nuclear layers in the outer nuclear layer was significantly greater in the treated eyes than in the control eyes (fig. 7A). Subsequently, the TUNEL method examined the degree of chromosomal DNA fragmentation in eye cells of mice to reflect the apoptotic state of the cells, and we found that the number of positively labeled cells in treated eyes was significantly lower than that in untreated eyes (fig. 7B), indicating that there was no chromosomal DNA fragmentation in most cells of rd10 mice after drug treatment, and that apoptosis was alleviated. The above results indicate that PDE6B expressed in the treated eye after drug injection retained more visual cells, maintaining the basic structure of the retina. Meanwhile, the invention utilizes electroretinogram analysis to evaluate the function of drug-treated eyes (rd10-AAV2/2.7m8-CAG-coPDE6B) and control eyes (rd10-AAV2/2.7m8-CAG-GFP) of rd10 mice. The a-wave amplitude and B-wave amplitude were found to be significantly higher in the treated eyes under different intensities of external light stimulation in the dark than in the control eyes (fig. 8A,8B), indicating that drug treatment significantly improved ocular function.

By combining the results, the invention proves the therapeutic effect of the AAV-coPDE6B gene therapeutic drug on retinitis pigmentosa caused by PDE6B mutation, and lays a foundation for further clinical application development.

The invention is further illustrated by the following examples.

Example 1: codon-optimized PDE6B vector construction and expression validation

(1) Plasmid vector construction

1. The AAV2-CAG plasmid skeleton and the coPDE6B fragment or the wtpDE6B fragment are subjected to double digestion by HindIII and XhoI respectively, and then the 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

1.293 cells were plated in cell culture well plates until the cells grew to 70-80% confluence.

2. The medium was changed to DMEM +1 XGlutamax.

3. Respectively diluting the plasmid and the PEI reagent with a culture medium, uniformly mixing in a ratio of 1:2, standing at room temperature for 20min, adding the mixture into a cell culture solution, and gently shaking.

4. Place the cell culture plate in CO at 37 ℃₂Culturing in an incubator for 48 h.

(3)Western Blot

1. And (3) preparing a protein sample, namely adding PMSF (PMSF for use in preparation according to the amount) into the lysate according to the proportion of 1: 100.

2. Cells were lysed using a strong lysate on ice.

3. Protein concentration was determined using 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 pre-stained protein molecule marker SDS-PAGE was added to the wells and 10. mu.l of SDS-PAGE protein loading buffer at 1 Xwas added to the blank wells at the sides of the sample wells.

5. Rotary film

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 splint on the filter paper, placing the splint 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

After the membrane is completely transferred, rinsing for 1-2 minutes, sucking up the buffer solution by a dropper, adding 5% of skimmed milk powder, slowly shaking on a side-swinging shaking table, and sealing at room temperature for 15-60 min. TBS washing was added and the mixture was washed for 5 minutes. The total number of washes was 3.

7. Primary antibody incubation

Appropriate primary antibody was diluted with 5% nonfat dry milk/PBS + 2% BSA in proportion and incubated either overnight at 4 ℃ with slow shaking or for 2h at room temperature on a side shaker with slow shaking. 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 the ECL reagents, dripping the ECL reagents on the surface of the protein membrane, and incubating for 1-2min in a dark place. The protein film is placed on the plastic paper in order by tweezers, and then the plastic paper is placed on a gel imager for exposure. The results are shown in fig. 5A and fig. 5B, the expression efficiency of PDE6B after sequence optimization is higher, which proves that codon optimization can improve the expression level of protein without changing the protein sequence, and the supplement protein with higher expression level can theoretically provide better therapeutic effect under the condition that the cell can not provide enough dosage or completely lacks normal functional protein due to mutation.

Example 2: the CAG promoter has higher expression efficiency in rd10 mice

(1) Virus medicine injection mouse

1. Ready 5 x 10¹²vg/ml of AAV2/2.7m8-CAG-coPDE6B, AAV2/2.7m8-sCBA + AT2R Intron 1-coPDE6B and AAV2/2.7m8-sCBA-coPDE 6B.

2. 1 ul/eye of each of the three viruses described above was injected into the eye of rd10 mice, a different age appropriate, through the vitreous cavity or subretinally.

3. At 3 weeks after mouse injection, mice were sacrificed and retinal tissue was isolated.

(2) qPCR detection of PDE6B mRNA expression level

1. The mortar was pre-cooled with liquid nitrogen and the mouse eye tissue was added to the mortar and ground well to a powder.

2. Transferring the powder into an EP tube filled with Trizol lysate, shaking vigorously, standing at room temperature for 5min, centrifuging at 10000rpm at 4 ℃ for 10 min.

3. The supernatant was pipetted and transferred to a new EP tube, 200ul chloroform/isoamyl alcohol was added per ml lysate, mixed vigorously by inversion, centrifuged at 10000rpm at 4 ℃ for 10 min.

4. Transferring supernatant, adding equal volume of isopropanol, precipitating at-20 deg.C for 1hr, centrifuging at 12000rpm, and 4 deg.C for 10 min.

5. Washed twice with 75% ethanol, dried and dissolved in 50ul of ddH 2O.

6. Reverse transcription was performed according to the TAKARA kit instructions.

7. The qPCR reaction solution was prepared according to the system shown in Table 1 below.

TABLE 1

sample	volume(ul)
		SYBR Green Mix MMaster	10
Upstream primer	0.5
		Downstream primer	0.5
ddH2O	8
		Form panel	1

8. And operating the PCR program on the computer, and analyzing the result.

Viral drugs packaged by PDE6B cDNA plasmids driven by different promoters are injected into rd10 mice through a vitreous cavity or under retina, and after 3 weeks, eye tissues of the mice are extracted to detect the expression level of PDE6B mRNA, and as a result, the PDE6B driven by the CAG promoter expresses more mRNA in eyes of rd10 mice (figure 6), which shows that the expression efficiency of the CAG promoter is remarkably higher than that of sCBA + AT2R Intron 1 and sCBA promoters in rd10 mice. Therefore, the CAG promoter is a better choice for PDE6B gene therapy of rd10 mice than other promoters.

Example 3: AAV-coPDE6B gene therapeutic medicine for improving eye function of rd10 mouse and repairing retina structure

(1) Virus medicine injection mouse

1. Ready 5 x 10¹²vg/ml of AAV2/2.7m8-CAG-coPDE6B drug and AAV2/2.7m 8-CAG-GFP.

2. 1 ul/eye of AAV2/2.7m8-CAG-coPDE6B drug and AAV2/2.7m8-CAG-GFP virus were injected intravitreally or subretinally into the eyes of age-appropriate mice.

3. At 3 weeks after injection, mice were sacrificed and retinal tissue was isolated and prepared into sections for use.

(2) Electroretinogram analysis

1. The mice were anesthetized and pupil dilated while a 2.5% hypromellose liquid containing electrodes was dropped into the eyes and corneal potential responses were recorded.

2. Mice were allowed to adapt to darkness overnight under dark adaptation conditions, and were given brief flash stimuli of different intensities with LED lamps, and dark adaptation ERG was recorded.

(3) Immunofluorescent staining of retinal tissue

1. Retinal tissue was sectioned and washed 5min × 3 times with 0.01M PBS.

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. Wash 5min X6 times in 0.01M PBS under dark conditions.

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

(4) TUNEL method for detecting apoptosis

1. Frozen tissue sections were fixed in 10% neutral formaldehyde at room temperature for 10min and washed 2 times with PBS for 5min each time. Adding ethanol: treating in a solution of acetic acid (2:1) at-20 deg.C for 5 min; PBS wash 2 times for 5min each.

2. Adding PBS containing 2% hydrogen peroxide, and reacting at room temperature for 5 min; PBS wash 2 times for 5min each.

3. Absorbing the excessive liquid by using filter paper, adding 2 drops of TdT enzyme buffer solution on the slices, and standing for 1-5min at room temperature; excess liquid was aspirated off, 54ul of TdT enzyme reaction solution was added dropwise, and the reaction mixture was placed in a wet box and reacted at 37 ℃ for 1 hr.

4. The sections were placed in a staining jar, preheated wash and stop solution was added, incubated at 37 ℃ for 30min, and the slide was lifted off every 10min to gently stir the liquid.

Washing with PBS for 5min for 3 times; dripping two drops of peroxidase-labeled digoxin antibody, and reacting in a wet box at room temperature for 30 min; PBS wash 5 times, each for 5 min.

6. Dripping a newly prepared 0.05% DAB solution, and developing for 3-6min at room temperature; washing with distilled water for 1-5min for 4 times.

7. Counterstaining with methyl green for 10min at room temperature. The glass slide is washed by distilled water for 3 times, lifted and laid down for 10 times in the first 2 times, and finally kept still for 30 s. The column was washed 3 times with 100% n-butanol in the same manner.

8. Dehydrating with xylene for 2min for 3 times, sealing, drying, and observing under microscope.

To demonstrate the therapeutic effect of AAV-codPDE 6B gene therapy drugs on retinitis pigmentosa induced by PDE6B mutations, in vivo experiments were performed using PDE6B mutant mouse rd 10. The improvement of the eye lesions of the mice by the drug treatment is observed after 3 weeks by injecting the drug into the vitreous cavity or under the retina. First, eye tissues from rd10 mice receiving drug injections were used for retinal immunostaining analysis, and quantitative analysis of the thickness of the outer nuclear layer in different areas showed that the number of nuclear layers in the outer nuclear layer was significantly greater in the treated eyes than in the control eyes (fig. 7A). Subsequently, the TUNEL method examined the degree of chromosomal DNA fragmentation in eye cells of mice to reflect the apoptotic state of the cells, and we found that the number of positively labeled cells in treated eyes was significantly lower than that in untreated eyes (fig. 7B), indicating that chromosomal DNA fragmentation did not occur in most cells of rd10 mice after drug treatment, as a result of which apoptosis was alleviated. The above results indicate that PDE6B expressed in the treated eye after drug injection retained more visual cells, maintaining the basic structure of the retina. At the same time, we used electroretinograms to evaluate the function of drug-treated eyes (rd10-AAV2/2.7m8-CAG-coPDE6B) and control eyes (rd10-AAV2/2.7m8-CAG-GFP) of rd10 mice. The a-wave amplitude and B-wave amplitude were found to be significantly higher in the treated eyes under different intensities of external light stimulation in the dark than in the control eyes (fig. 8A,8B), indicating that drug treatment significantly improved ocular function.

By combining the results, the invention proves the therapeutic effect of the AAV-coPDE6B gene therapeutic drug on retinitis pigmentosa caused by PDE6B 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> PDE6B nucleotide sequence and application thereof

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850

<210> 2

<211> 2565

<212> DNA

<213> wild-type PDE6B cDNA sequence (wild-type PDE6B cDNA)

<400> 2

atgagcctca gtgaggagca ggcccggagc tttctggacc agaaccccga ttttgcccgc 60

cagtactttg ggaagaaact gagccctgag aatgtggccg cggcctgcga ggacgggtgc 120

ccgccggact gcgacagcct ccgggacctc tgccaggtgg aggagagcac ggcgctgctg 180

gagctggtgc aggatatgca ggagagcatc aacatggagc gcgtggtctt caaggtcctg 240

cggcgcctct gcaccctcct gcaggccgac cgctgcagcc tcttcatgta ccgccagcgc 300

aacggcgtgg ccgagctggc caccaggctt ttcagcgtgc agccggacag cgtcctggag 360

gactgcctgg tgccccccga ctccgagatc gtcttcccac tggacatcgg ggtcgtgggc 420

cacgtggctc agaccaaaaa gatggtgaac gtcgaggacg tggccgagtg ccctcacttc 480

agctcatttg ctgacgagct cactgactac aagacaaaga atatgctggc cacacccatc 540

atgaatggca aagacgtcgt ggcggtgatc atggcagtga acaagctcaa cggcccattc 600

ttcaccagcg aagacgaaga tgtgttcttg aagtacctga attttgccac gttgtacctg 660

aagatctatc acctgagcta cctccacaac tgcgagacgc gccgcggcca ggtgctgctg 720

tggtcggcca acaaggtgtt tgaggagctg acggacatcg agaggcagtt ccacaaggcc 780

ttctacacgg tgcgggccta cctcaactgc gagcggtact ccgtgggcct cctggacatg 840

accaaggaga aggaattttt tgacgtgtgg tctgtgctga tgggagagtc ccagccgtac 900

tcgggcccac gcacgcctga tggccgggaa attgtcttct acaaagtgat cgactacatc 960

ctccacggca aggaggagat caaggtcatt cccacaccct cagccgatca ctgggccctg 1020

gccagcggcc ttccaagcta cgtggcagaa agcggcttta tttgtaacat catgaatgct 1080

tccgctgacg aaatgttcaa atttcaggaa ggggccctgg acgactccgg gtggctcatc 1140

aagaatgtgc tgtccatgcc catcgtcaac aagaaggagg agattgtggg agtcgccaca 1200

ttttacaaca ggaaagacgg gaagcccttt gacgaacagg acgaggttct catggagtcc 1260

ctgacacagt tcctgggctg gtcagtgatg aacaccgaca cctacgacaa gatgaacaag 1320

ctggagaacc gcaaggacat cgcacaggac atggtccttt accacgtgaa gtgcgacagg 1380

gacgagatcc agctcatcct gccaaccaga gcgcgcctgg ggaaggagcc tgctgactgc 1440

gatgaggacg agctgggcga aatcctgaag gaggagctgc cagggcccac cacatttgac 1500

atctacgaat tccacttctc tgacctggag tgcaccgaac tggacctggt caaatgtggc 1560

atccagatgt actacgagct gggcgtggtc cgaaagttcc agatccccca ggaggtcctg 1620

gtgcggttcc tgttctccat cagcaaaggg taccggagaa tcacctacca caactggcgc 1680

cacggcttca acgtggccca gacgatgttc acgctgctca tgaccggcaa actgaagagc 1740

tactacacgg acctggaggc cttcgccatg gtgacagccg gcctgtgcca tgacatcgac 1800

caccgcggca ccaacaacct gtaccagatg aagtcccaga accccttggc taagctccac 1860

ggctcctcga ttttggagcg gcaccacctg gagtttggga agttcctgct ctcggaggag 1920

accctgaaca tctaccagaa cctgaaccgg cggcagcacg agcacgtgat ccacctgatg 1980

gacatcgcca tcatcgccac ggacctggcc ctgtacttca agaagagagc gatgtttcag 2040

aagatcgtgg atgagtccaa gaactaccag gacaagaaga gctgggtgga gtacctgtcc 2100

ctggagacga cccggaagga gatcgtcatg gccatgatga tgacagcctg cgacctgtct 2160

gccatcacca agccctggga agtccagagc aaggtcgcac ttctcgtggc tgctgagttc 2220

tgggagcaag gtgacttgga aaggacagtc ttggatcagc agcccattcc tatgatggac 2280

cggaacaagg cggccgagct ccccaagctg caagtgggct tcatcgactt cgtgtgcaca 2340

ttcgtgtaca aggagttctc tcgtttccac gaagagatcc tgcccatgtt cgaccgactg 2400

cagaacaata ggaaagagtg gaaggcgctg gctgatgagt atgaggccaa agtgaaggct 2460

ctggaggaga aggaggagga ggagagggtg gcagccaaga aagtaggcac agaaatttgc 2520

aatggcggcc cagcacccaa gtcttcaacc tgctgtatcc tgtga 2565

<210> 3

<211> 2565

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgagcttgt cagaggaaca ggcccggtcc tttttggatc agaatccaga ttttgcacga 60

cagtactttg gtaagaaact ttcacccgaa aacgtggcag ccgcgtgtga ggacggatgt 120

ccaccagatt gcgattcact gcgcgacctt tgccaggttg aggaatcaac tgctcttttg 180

gagctggtgc aggacatgca agaaagcatt aatatggaac gcgtggtttt taaagttctt 240

agacggctct gcaccctgct tcaagctgat cggtgttccc tcttcatgta tcggcaacgg 300

aatggtgtgg ccgaattggc aaccagactc ttcagcgtgc aacctgactc tgtcctcgag 360

gattgcctcg tcccacccga tagtgagatc gtctttcctc tcgatatcgg ggtggtggga 420

cacgttgctc aaacaaaaaa gatggtgaat gttgaagacg tggcagagtg tccacacttc 480

tctagttttg ccgatgagct gaccgactat aagaccaaga acatgctggc tactcccatc 540

atgaacggta aggatgttgt agcagttatc atggccgtta acaagctgaa tggacccttt 600

tttaccagtg aagacgagga tgtctttctg aaatatctca atttcgccac cttgtacctg 660

aaaatttatc atctcagtta tctgcacaac tgtgagacaa gaagggggca ggtgctcctt 720

tggagcgcca acaaagtgtt cgaggaactt acagacattg agaggcagtt ccataaagca 780

ttctacaccg ttcgcgccta tctgaactgt gagaggtatt ccgttggact gcttgacatg 840

accaaagaga aggaattttt cgatgtttgg agcgtgctca tgggagagtc ccaaccttac 900

tctgggcccc gcactcccga tggtagagag atcgtgtttt acaaggtcat tgactacata 960

ctccacggga aagaagagat taaagtaatc ccaacacctt cagccgatca ttgggctctg 1020

gccagtgggc tccctagcta tgtagccgaa agcgggttta tttgcaacat tatgaacgcc 1080

agtgcagacg aaatgtttaa attccaggaa ggggcactgg atgactccgg ctggctcatc 1140

aagaatgttt tgtcaatgcc cattgtgaac aagaaagagg aaatcgtggg ggtggcaacg 1200

ttttataatc gcaaagatgg aaaaccattt gacgagcagg acgaggtctt gatggagtct 1260

ctgactcagt tcctgggatg gagcgtcatg aacaccgaca cgtacgacaa gatgaacaaa 1320

ctggagaacc gcaaagacat tgcccaggat atggtgctgt atcacgttaa atgtgacagg 1380

gacgaaatcc agctgatact gcctacccga gctagattgg gtaaagagcc agcggactgt 1440

gacgaggatg aactggggga gatcctgaag gaggaactgc cgggccccac cacatttgat 1500

atatacgagt ttcacttttc cgatctcgaa tgcacagagc tggacctcgt gaagtgcggc 1560

attcagatgt attatgaact cggtgtggtt agaaagttcc aaattcctca ggaagtgctg 1620

gtacgcttcc tgttttcaat ctccaagggc tatagaagga ttacatatca taactggcgg 1680

cacgggttta acgtggcgca aacgatgttt accttgctga tgaccggcaa gctgaagtca 1740

tactacactg acctggaggc tttcgcaatg gtcacggctg gactctgtca cgacatagat 1800

caccggggga ccaataattt gtatcagatg aagagtcaaa atccactggc caaacttcat 1860

ggatccagta tactcgaacg gcatcacctg gagtttggta agttcctgct cagcgaagaa 1920

accctcaaca tatatcagaa cctgaataga cgccagcatg agcatgtgat tcatctgatg 1980

gatatcgcta tcattgcaac ggacctggct ctgtacttta aaaaacgcgc catgttccag 2040

aagatcgtgg acgagtcaaa aaattatcag gacaaaaaga gctgggttga gtatctttca 2100

ctcgagacca ctcgcaagga gattgttatg gctatgatga tgactgcttg tgatctctca 2160

gccatcacca agccctggga ggtccaaagc aaagttgctc tgctcgttgc tgccgaattc 2220

tgggaacagg gcgatctgga gagaaccgtg ctggaccagc aacccattcc gatgatggac 2280

cgcaacaagg ctgccgagct gcctaagttg caggtgggct tcattgactt cgtatgtacc 2340

ttcgtataca aagaattttc ccggttccat gaggagatcc tgccaatgtt tgatcgcctg 2400

caaaacaaca ggaaggagtg gaaagcgctc gcagacgagt acgaggcgaa ggtgaaggcc 2460

ctcgaagaaa aagaggaaga ggagagggtc gcagccaaga aggtgggaac agaaatctgt 2520

aacggaggtc ctgctcccaa gtcttccacg tgctgcatcc tgtga 2565

<210> 4

<211> 1676

<212> DNA

<213> CAG promoter sequence (CAG promoter)

<400> 4

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct 420

cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga 480

tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg 540

gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 600

cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg 660

gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc 720

cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 780

cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa 840

gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg 900

tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct 960

gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg 1020

gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt 1080

gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc 1140

cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg 1200

gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg 1260

ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg 1320

gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc 1380

agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc 1440

cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg 1500

gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc 1560

ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga 1620

ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacag 1676

Claims (11)

1. PDE6B nucleotide sequence, characterized in that 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 according to any one of claims 1-2 for the preparation of a viral vector or a pharmaceutical formulation for the treatment of an ocular disease caused by a mutation in PDE6B, wherein the ocular disease caused by a mutation in PDE6B is retinitis pigmentosa caused by a mutation in PDE 6B.

4. A viral vector comprising a nucleotide sequence according to any one of claims 1 to 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 PDE6B protein is regulated by the promoter CAG.

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 disease caused by a mutation in PDE6B, wherein the ocular disease caused by a mutation in PDE6B is retinitis pigmentosa caused by a mutation in PDE 6B.

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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