阅读说明：本技术 一种编码人受体酪氨酸激酶Mer的核苷酸序列及其应用 (Nucleotide sequence for coding human receptor tyrosine kinase Mer and application thereof ) 是由 李斌 任盛 于 2020-08-05 设计创作，主要内容包括：本发明涉及生物医药基因治疗技术领域,公开了一种编码人受体酪氨酸激酶Mer的核苷酸序列及其应用。本发明所述核苷酸序列与SEQ ID NO:3所示核苷酸序列有≥95％相同性。本发明证实使用AAV-MERTK药物处理可以显著改善MERTK突变导致视网膜色素变性的大鼠的视网膜病理症状,以及视网膜功能的恢复。玻璃体腔注射AAV-MERTK药物,MERTK可以在视网膜色素上皮层高效表达,并且增加视网膜外核层厚度。同时,视网膜电位图显示,和对照组相比,AAV-MERTK药物处理组大鼠对刺激反应强烈。因此此AAV-MERTK药物具有预防或治疗视网膜色素变性的作用。(The invention relates to the technical field of biomedical gene therapy, and discloses a nucleotide sequence for coding human receptor tyrosine kinase Mer 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 pathological symptoms of retina of a rat with retinitis pigmentosa caused by MERK mutation and the recovery of the function of the retina can be obviously improved by using AAV-MERK drug treatment. AAV-MERK drugs are injected in the vitreous cavity, and the MERK can be efficiently expressed in the retinal pigment epithelium layer and can increase the thickness of the retinal outer nuclear layer. Meanwhile, the electroretinogram showed that the AAV-MERK drug-treated rats responded strongly to the stimulus as compared with the control group. Therefore, the AAV-MERK medicament has the effect of preventing or treating retinitis pigmentosa.)

1. A nucleotide sequence encoding human receptor tyrosine kinase Mer, which is characterized by having at least 95% identity with the nucleotide sequence shown in SEQ ID NO. 3, preferably having at least 98% identity with the nucleotide sequence shown in SEQ ID NO. 3, and more preferably having at least 99% identity with the nucleotide sequence shown in SEQ ID NO. 3.

2. The nucleotide sequence of claim 1, wherein the sequence is set forth in SEQ ID NO 3.

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

4. Use of the nucleotide sequence of any one of claims 1-3 for the preparation of a viral vector or a pharmaceutical formulation for the prevention or treatment of an ocular disease caused by a merk mutation, or for the prevention or treatment of an ocular disease caused by a merk mutation.

5. The use of claim 4, wherein the ocular disease caused by the merk mutation is retinitis pigmentosa caused by the merk mutation.

6. A viral vector comprising a nucleotide sequence according to any one of claims 1 to 3.

7. The viral vector according to claim 6, wherein the viral vector is an adeno-associated viral vector.

8. The viral vector according to claim 7, wherein the serotype of the adeno-associated viral vector is AAV2 wild type or AAV2-7M 8.

9. The viral vector according to any one of claims 6 to 8, wherein the expression of the merk protein is regulated by the promoter hRPE 65.

10. Use of the viral vector of any one of claims 6 to 9 for the preparation of a pharmaceutical formulation for the prevention or treatment of an ocular disease caused by a merk mutation, or for the prevention or treatment of an ocular disease caused by a merk mutation.

11. The use of claim 10, wherein the ocular disease caused by the merk mutation is retinitis pigmentosa caused by the merk mutation.

12. A pharmaceutical preparation comprising a nucleotide sequence according to any one of claims 1 to 3 or a viral vector according to any one of claims 6 to 9.

13. The pharmaceutical formulation of claim 12, wherein the pharmaceutical formulation is a liquid formulation.

14. The pharmaceutical formulation of claim 12 or 13, further comprising a pharmaceutically acceptable carrier or excipient.

15. The pharmaceutical formulation of any one of claims 12-14 for use in preventing or treating an ocular disease caused by a merk mutation.

16. The use of claim 15, wherein the ocular disease caused by the merk mutation is retinitis pigmentosa caused by the merk mutation.

17. A method of delivering a pharmaceutical formulation, wherein the pharmaceutical formulation of any one of claims 12-14 is injected into the eye.

Technical Field

The invention relates to the technical field of biomedical gene therapy, in particular to a nucleotide sequence for coding human receptor tyrosine kinase Mer and application thereof.

Background

Merks encode the receptor tyrosine kinase Mer, which typically transduces extracellular matrix signals into the cytoplasm by binding to several ligands, including LGALS3, TUB, TULP1, or GAS 6. In this manner, receptor tyrosine kinases are able to regulate a number of physiological processes including cell survival, migration, differentiation and phagocytosis of apoptotic cells (effector cells). In retinal pigment epithelial cells, merks function to mediate their phagocytosis of shed debris from the extracellular segment of the rod, maintaining normal metabolic turnover of the rod.

Merk mutations cause retinitis pigmentosa 38(RP38), where RPE cells are unable to normally phagocytose optic cell outer segment fragments due to protein loss as a result of merk mutations, and there is a large debris accumulation between the inner and outer segments; at the same time, retinal blood vessels develop density and structural abnormalities, which are manifested by an increased number of decellularized capillaries, with Muller cell fibers extending through the retina into the subretinal space. Retinal pigment deposition is visible to patients on a fundus examination, with initial presentation of loss of rods followed by cone loss. Patients often have night blindness and a loss of the peripheral visual field. As the disease progresses, the far peripheral vision field is lost, and eventually central vision is lost.

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 mutant serotype into a mouse vitreous cavity for screening, and enriching to a main mutant 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 the above, the present invention aims to provide a nucleotide sequence encoding human receptor tyrosine kinase Mer, which can optimize multiple parameters such as codon usage bias, DNA repeat, mRNA secondary structure, GC content, etc., to increase the efficiency of merk expression;

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

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 merk 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 merk mutation, including but not limited to the preparation of related drugs, reagents and methods for preventing or treating;

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:

a nucleotide sequence for coding human receptor tyrosine kinase Mer has more than or equal to 95% 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 as 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; in a specific embodiment of the invention, the serotype of the adeno-associated viral vector is AAV2 wild type or AAV2-7M 8.

More specifically, the viral vector regulates the expression of MERKT protein by a promoter hRPE65 (shown as SEQ ID NO: 4).

In addition, the invention also provides a pharmaceutical preparation which comprises the nucleotide sequence or the virus 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 the MERTK cDNA sequence to obtain the coMERTK, and cell level expression efficiency detection is carried out on the sequence wtMERTK/coMERTK before and after optimization, so that the optimized sequence expression efficiency is obviously improved. Then AAV2/2-7M8-comeRTK medicine is used to infect retinal pigment epithelial cell line APRE-19, and the merRTK protein can be effectively expressed. The expression of the MERKs can be detected in retinal pigment epithelial cells of rats by injecting AAV2/2-7M 8-MERKs into the vitreous cavity of the rats with the MERKs mutation. The thickness of the outer nuclear layer of the retina (ONL) of the rat was measured after 6 months and it was found that the thickness of the outer nuclear layer of the retina of the rat treated with AAV2/2-7M 8-MERK was significantly increased compared to the control AAV treatment, while electroretinogram results showed that the rat responded strongly to the stimulus. The structure and the function of the rat retina treated by the AAV2/2-7M 8-MERKs are remarkably restored, and the AAV2/2-7M 8-MERKs 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 viral vector or a pharmaceutical preparation for preventing or treating eye diseases caused by MERK mutation, or is applied to the prevention or treatment of eye diseases caused by MERK mutation;

the invention also provides an application of the virus vector in preparing a pharmaceutical preparation for preventing or treating eye diseases caused by MERK mutation, or an application of the virus vector in preventing or treating eye diseases caused by MERK mutation;

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

Wherein the eye disease caused by the MERK mutation is retinitis pigmentosa caused by the MERK 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 subretinal injection or vitreous cavity injection.

As can be seen from the above technical solutions, the present invention demonstrates that the use of AAV-MERK drug treatment can significantly improve the improvement of retinal pathological symptoms of rats with MERK mutation causing retinitis pigmentosa, and the recovery of retinal function. AAV-MERK drugs are injected in the vitreous cavity, and the MERK can be efficiently expressed in the retinal pigment epithelium layer and can increase the thickness of the retinal outer nuclear layer. Meanwhile, the electroretinogram showed that the AAV-MERK drug-treated rats responded strongly to the stimulus as compared with the control group. Therefore, the AAV-MERK medicament has the effect of preventing or treating retinitis pigmentosa.

Drawings

FIGS. 1-3 show the wtMERTK and the corMERTK sequence alignments; the optimized differential codon sequences are thickened and marked with underlines;

FIG. 4 shows an AAV-comeRTK vector map (A) and an AAV-wtmerK vector map (B); the vector comprises AAV25 'ITR, hRPE65 promoter, codon optimized MERK cDNA or wild type MERK cDNA, bGH polyA sequence and AAV 23' ITR;

FIG. 5 shows the expression efficiency of AAV-comeRTK and AAV-wtmerRTK plasmids in ARPE-19 cells;

a: respectively transfecting AAV-comeRTK and AAV-wtmerK plasmids in ARPE-19 cells, cracking the cells after 48 hours, and detecting the expression level of merk protein by Western blot;

b: AAV-comeRTK and AAV-wtmerK plasmid are transfected into ARPE-19 cells to express relative abundance of merk protein;

FIG. 6 shows that AAV2/2-7M8-comeRTK virus can efficiently infect APRE-19 cells;

ARPE-19 cells were infected with AAV-coMERRTK viruses encapsidated with different serotypes of protein (MOI 10)⁴) After 48 hours, the cells were lysed and the content of MERTKmRNA in the different serotype infected and uninfected cells was determined using qPCR;

FIG. 7 shows the detection of retinal MERK gene expression following intravitreal injection of AAV2/2-7M8-comeRTK virus;

performing vitreous chamber injection on RCS rats (MERK defective rats) by AAV2/2-7M8-comeRTK virus, and performing MERK protein expression detection on treated eyes (AAV-comeRTK) and untreated eyes (AAV-GFP) by using Western Blot by taking rat retinas 6 months after injection;

FIG. 8 is a graph showing the measurement of the thickness of the outer nuclear layer of the retina in RCS rats and wild type rats after AAV2/2-7M8-comeRTK virus treatment;

taking drug injection eyes and control eye retinas of RCS rats and wild rats 6 months after injection, carrying out immunofluorescence staining, and carrying out quantitative analysis on the thicknesses of the upper segment outer nuclear layer and the lower segment outer nuclear layer which are different in distance from the optic nerve head;

FIG. 9 is a graph showing electroretinogram measurements in rats after treatment with AAV2/2-7M8-comeRTK virus;

electroretinogram analysis of drug-injected and control eyes and wild-type rats in RCS rats 6 months after injection, light stimulation at a specific intensity was given in the light and dark conditions, and the b-wave amplitude was recorded for each rat (n ═ 10) drug-injected and control eyes under light stimulation.

Detailed Description

The invention discloses a nucleotide sequence for coding human receptor tyrosine kinase Mer and application thereof, and can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. 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 AAV-MERK drug can effectively express the MERK in retinal cells and repair retinal diseases caused by the mutation of the MERK, and the protein sequence coded by the cDNA of the MERK is shown in SEQ ID NO. 1.

According to the invention, by optimizing multiple parameters such as codon usage preference, DNA repetitive sequence, mRNA secondary structure, GC content and the like, an optimized sequence comeRTK (coded optimized human MERK cDNA) which is obviously different from a wtMERK (sequence shown as SEQ ID NO: 2) sequence is obtained, and the optimized sequence and wild type sequence pair are shown in figures 1-3. The wt/co MERK sequence is constructed to an AAV vector, a plasmid map is shown in figure 4, then the same amount of plasmid is transfected in ARPE-19 cells, the expression of the MERK gene is detected, the expression efficiency of the MERK gene after sequence optimization is higher, the codon optimization is proved to be capable of improving the expression level of the protein under the condition of not changing the protein sequence, more proteins with normal functions are provided for the cells, and therefore the defects caused by gene mutation are well compensated.

The effect of codon optimization and AAV2/2-7M8 serotype combination on improving the MERK expression efficiency is tested through in vitro experiments, and an RCS rat model (MERK defect type) is utilized to carry out in vivo experiments, so that the treatment effect of AAV-MERK gene treatment drugs on retinitis pigmentosa caused by MERK mutation is further proved. The improvement of the eye lesion of the rat by the drug treatment is observed after 6 months by injecting the drug into the vitreous cavity.

The invention is further illustrated by the following examples.