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Nucleotide sequence encoding human receptor tyrosine kinase mer and application thereof

A tyrosine kinase and polynucleotide technology, applied in the field of biomedical gene therapy, can solve problems such as transduction of retinal tissue cells, and achieve the effects of preventing or treating retinitis pigmentosa, increased thickness and strong stimulation response

Active Publication Date: 2022-05-27
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 encoding human receptor tyrosine kinase mer and application thereof
  • Nucleotide sequence encoding human receptor tyrosine kinase mer and application thereof
  • Nucleotide sequence encoding human receptor tyrosine kinase mer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0046] (1) Construction of plasmid vector

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

[0048] 2. The ligation product was transformed into Escherichia coli, and a single colony was picked for 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 was 90% on the day of transfection.

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

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

[0053] ...

Embodiment 2

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

[0076] Different protein coats were packaged to form AAV-coMERTK viruses of different serotypes, including AAV2 / 2-7M8, AAV2 / 5, AAV2 / 8 and AAV2 / 9, and the four serotype viruses were infected with ARPE-19 cells (MOI =10 4 ), and ARPE-19 cells without virus infection served 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. The specific implementation method is as follows:

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

[0078] 1. ARPE-19 cells with a degree of polymerization of more than 90% were plated at a ratio of 1:3.

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

[0080] 3. 24h after plasmid transfection, change to a new serum-free medi...

Embodiment 3

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

[0101] (1) Virus drug injection in rats

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

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

[0104] 3. At the age of 6 months, the rats were sacrificed, the retinal tissues of the rats were separated, and the number of retinal visual cells and the content of target protein were detected by staining.

[0105] (2)Western Blot

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Abstract

The invention relates to the technical field of biomedical gene therapy, and discloses a nucleotide sequence encoding human receptor tyrosine kinase Mer and its application. The nucleotide sequence of the present invention has ≥95% identity with the nucleotide sequence shown in SEQ ID NO:3. The present invention proves that AAV-MERTK drug treatment can significantly improve retinal pathological symptoms and restore retinal function in rats with retinitis pigmentosa caused by MERTK mutation. Intravitreal injection of AAV‑MERTK drugs can highly express MERTK in the retinal pigment epithelium and increase the thickness of the outer nuclear layer of the retina. At the same time, the electroretinogram showed that compared with the control group, the rats in the AAV‑MERTK drug treatment group responded strongly to the stimulus. Therefore, this AAV‑MERTK drug has the effect of preventing or treating retinitis pigmentosa.

Description

technical field [0001] The invention relates to the technical field of biomedical gene therapy, and more particularly to a nucleotide sequence encoding human receptor tyrosine kinase Mer and its application. Background technique [0002] MERTK encodes the receptor tyrosine kinase Mer, which normally transduces signals from the extracellular matrix into the cytoplasm by binding to several ligands, including LGALS3, TUB, TULP1, or GAS6. In the above manner, 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 the phagocytosis of shed fragments from the outer segments of the photoreceptor cells and maintain the normal metabolic renewal of cone photoreceptor cells. [0003] MERTK mutation can cause retinitis pigmentosa 38 (RP38). Due to the loss of function of the protein due to ...

Claims

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Application Information

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Patent Type & Authority Patents(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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