Nucleotide sequence for coding human receptor tyrosine kinase Mer and application of nucleotide sequence

A nucleotide sequence and tyrosine kinase technology, applied in the field of biomedical gene therapy, can solve the problem of transducing retinal tissue cells

Active Publication Date: 2020-10-27
WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Naturally occurring AAV serotypes are generally unable to transduce retinal tissue cells on the side of the vitreous cavity due to the presence of barriers such as the inner limiting membrane, glial cells, etc. that prevent the spread of AAV virions

Method used

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  • Nucleotide sequence for coding human receptor tyrosine kinase Mer and application of nucleotide sequence
  • Nucleotide sequence for coding human receptor tyrosine kinase Mer and application of nucleotide sequence
  • Nucleotide sequence for coding human receptor tyrosine kinase Mer and application of nucleotide sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Codon-optimized MERTK vector construction and expression verification

[0046] (1) Plasmid vector construction

[0047] 1. The AAV-hRPE65 plasmid backbone, coMERTK fragment and wtMERTK fragment were digested with HindIII and XhoI at the same time, and then the digested fragments were ligated to the backbone respectively.

[0048] 2. The ligation product was transformed into E. coli, and a single colony was picked for enzyme digestion verification and sequencing verification.

[0049] (2) Cell transfection

[0050] 1. One day before transfection, cells were trypsinized and counted, and the cells were plated so that the density on the day of transfection was 90%.

[0051] 2. For each well of cells, use 50 μl of serum-free DMEM medium to dilute 0.8 μg-1.0 μg of DNA.

[0052] 3. For each well of cells, use 50 μl of DMEM medium to dilute 1 μl-3 μl of LIPOFECTAMINE 2000 reagent. After dilution of LIPOFECTAMINE 2000, mix with the diluted DNA within 5 minutes.

...

Embodiment 2

[0075] Example 2: AAV2 / 2-7M8-MERTK efficiently infects ARPE-19 cells

[0076] Different protein shells were packaged to form different serotypes of AAV-coMERTK viruses, including AAV2 / 2-7M8, AAV2 / 5, AAV2 / 8 and AAV2 / 9, and the four serotypes were infected with ARPE-19 cells (MOI =10 4 ), ARPE-19 cells not infected with the virus were used as controls. Cells were lysed 48 hours after infection, and the content of MERTK mRNA in cells infected with different serotypes and uninfected cells was determined by qPCR method. The specific implementation methods are as follows:

[0077] (1) Virus packaging, virus infection of ARPE-19 cells

[0078] 1. ARPE-19 cells with a degree of polymerization above 90% are transferred in a ratio of 1:3.

[0079] 2. About 1-2 hours before transfection, replace with serum-free medium, and use transfection reagent to transfer the target gene plasmid and helper plasmid into ARPE-19.

[0080] 3. After 24 hours of plasmid transfection, replace with new ...

Embodiment 3

[0100] Example 3: AAV-coMERTK gene therapy drug improves eye function and repairs retinal structure in RCS rats

[0101] (1) Rats injected with virus drugs

[0102] 1. Prepare 5*10 12 AAV-coMERTK drug and AAV-GFP at vg / ml.

[0103] 2. Inject 1 ul / eye of AAV-coMERTK drug and AAV-GFP virus into the eyes of age-appropriate rats through the vitreous cavity.

[0104] 3. When the rats were 6 months old, the rats were sacrificed, the retinal tissues of the rats were separated, and stained to detect the number of retinal visual cells and the content of the target protein.

[0105] (2)Western Blot

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Abstract

The invention relates to the technical field of biomedical gene treatment, and discloses a nucleotide sequence for coding human receptor tyrosine kinase Mer and application thereof. The nucleotide sequence disclosed by the invention and the nucleotide sequence shown in SEQ ID NO: 3 have more than or equal to 95% identity. The application proves that by using an AAV-MERTK drug for treatment, the retinal pathology symptoms of rats with retinitis pigmentosa caused by MERTK mutation can be remarkably improved, and retinal function recovery can be remarkably improved. The AAV-MERTK drug is injectedinto a vitreous cavity, MERTK can be efficiently expressed in a retinal pigment epithelium layer, and the thickness of a retinal outer nuclear layer is increased. Meanwhile, a retinal potential diagram shows that compared with a control group, the rats in an AAV-MERTK drug treatment group have strong response to stimulation. Therefore, the AAV-MERTK drug has the effect of preventing or treating the retinitis pigmentosa.

Description

technical field [0001] The invention relates to the technical field of biomedical gene therapy, and more specifically relates to a nucleotide sequence encoding human receptor tyrosine kinase Mer and its application. Background technique [0002] MERTK encodes Mer, a receptor tyrosine kinase that normally transduces signals from the extracellular matrix into the cytoplasm by binding to several ligands, including LGALS3, TUB, TULP1 or GAS6. In this way, receptor tyrosine kinases are able to regulate many physiological processes including cell survival, migration, differentiation and phagocytosis of apoptotic cells (effector cells). In retinal pigment epithelial cells, the function of MERTK is to mediate its phagocytosis of extracellular segmental fragments and maintain the normal metabolic renewal of cone and rod cells. [0003] Mertk mutation can cause retinitis pigmentosa 38 (RP38). Due to the loss of function of the protein due to MERTK mutation, RPE cells cannot normally ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N9/12C12N15/864A61K48/00A61P27/02
CPCC07K14/71C12N9/12C12N15/86A61K48/0008A61P27/02C12N2800/22C12N2750/14143A61K2039/54
Inventor 李斌任盛
Owner WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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