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A kind of pde6b nucleotide sequence and its application

A nucleotide sequence and sequence technology, applied in the PDE6B nucleotide sequence and its application fields, can solve the problems of transduction of retinal tissue cells, etc., and achieve the prevention or treatment of retinitis pigmentosa, strong stimulation response, and retinal structure and function recovery. Effect

Active Publication Date: 2022-07-15
WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] 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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  • A kind of pde6b nucleotide sequence and its application
  • A kind of pde6b nucleotide sequence and its application
  • A kind of pde6b nucleotide sequence and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Codon-optimized PDE6B vector construction and expression verification

[0044] (1) Construction of plasmid vector

[0045] 1. The AAV2-CAG plasmid backbone and the coPDE6B fragment or the wtPDE6B fragment were digested simultaneously with HindIII and XhoI, respectively, and then the digested fragments were connected to the backbone respectively.

[0046] 2. The ligation product was transformed into Escherichia coli, and a single colony was picked for digestion verification and sequencing verification.

[0047] (2) Cell transfection

[0048] 1. 293 cells were plated in a cell culture well plate, and the cells were grown to a concentration of 70-80%.

[0049] 2. Change the medium to DMEM+1XGlutaMAX.

[0050] 3. Dilute the plasmid and PEI reagent with the medium respectively and mix them in a ratio of 1:2. After mixing, let stand for 20 minutes at room temperature, add the mixture to the cell culture medium, and shake gently.

[0051] 4. Place the cell cultu...

Embodiment 2

[0070] Example 2: CAG promoter has higher expression efficiency in rd10 mice

[0071] (1) Mice injected with virus drugs

[0072] 1. Prepare 5*10 12 vg / ml of AAV2 / 2.7m8-CAG-coPDE6B, AAV2 / 2.7m8-sCBA+AT2RIntron 1-coPDE6B and AAV2 / 2.7m8-sCBA-coPDE6B drugs.

[0073] 2. 1ul / eye of the above three viruses were injected into the eyes of rd10 mice of different ages through the vitreous cavity or subretinal respectively.

[0074] 3. At 3 weeks after the mice were injected, the mice were sacrificed, and the retinal tissues of the mice were isolated.

[0075] (2) qPCR detection of PDE6B mRNA expression level

[0076] 1. Pre-cool the mortar with liquid nitrogen, add the mouse eye tissue into the mortar and grind it into powder.

[0077] 2. Transfer the powder into an EP tube containing Trizol lysis solution, shake vigorously and then stand at room temperature for 5 minutes, centrifuge at 10,000 rpm and 4°C for 10 minutes.

[0078] 3. Transfer the supernatant to a new EP tube, add 200...

Embodiment 3

[0087] Example 3: AAV-coPDE6B gene therapy drug improves ocular function and repairs retinal structure in rd10 mice

[0088] (1) Mice injected with virus drugs

[0089] 1. Prepare 5*10 12 vg / ml of AAV2 / 2.7m8-CAG-coPDE6B drug and AAV2 / 2.7m8-CAG-GFP.

[0090] 2. 1ul / eye of AAV2 / 2.7m8-CAG-coPDE6B drug and AAV2 / 2.7m8-CAG-GFP virus were injected into the eyes of age-appropriate mice by intravitreal or subretinal injection.

[0091] 3. At 3 weeks after the mice were injected, the mice were sacrificed, and the retinal tissues of the mice were separated and prepared into slices for use.

[0092] (2) Electroretinogram analysis

[0093] 1. Mice were treated with anesthesia and pupil dilation, and 2.5% hypromellose liquid containing electrodes was instilled into the eyes at the same time, and the corneal potential response was recorded.

[0094] 2. Under dark-adapted conditions, the mice were allowed to adapt to the dark overnight, and were given short-term flash stimulation of diffe...

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Abstract

The invention relates to the technical field of biomedical gene therapy, and discloses a PDE6B nucleotide sequence and application thereof. The present invention confirms that the protein expression efficiency of the codon-optimized PDE6B coding sequence is higher than that of the wild-type sequence, and the use of AAV2 / 2.7m8-coPDE6B drug treatment can significantly improve the retinal pathological symptoms of mice with retinitis pigmentosa caused by PDE6B mutation and achieve retinal Restoration of functionality. Injection of AAV2 / 2.7m8‑coPDE6B drug, PDE6B can be highly expressed in the retinal outer nuclear layer and increase the retinal outer nuclear layer thickness. Potentiometric retinal images showed that mice in the AAV2 / 2.7m8‑coPDE6B drug-treated group responded strongly to stimulation. Therefore, AAV2 / 2.7m8‑coPDE6B drugs can prevent or treat retinitis pigmentosa.

Description

technical field [0001] The invention relates to the technical field of biomedical gene therapy, and more particularly to a PDE6B nucleotide sequence and application thereof. Background technique [0002] PDE6 protein consists of two catalytic subunits, PDE6A and PDE6B, and two inhibitory subunits. This protein can regulate the level of cGMP in the cytoplasm of rod photoreceptor cells when they are stimulated by light, thereby hyperpolarizing the rod photoreceptor membrane and finally generating receptor potential. PDE6B mutation can lead to the inactivation of PDE6 protein, the destruction of the phototransduction pathway, the sharp increase of cGMP and calcium ion levels in the cytoplasm, and the apoptosis and the degeneration of visual cells. Since PDE6B is indispensable in the light transduction pathway of rod cells, mutations in the PDE6B gene can cause severe retinitis pigmentosa, and there is currently no effective cure. [0003] Naturally occurring AAV serotypes are...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/864A61K48/00A61K38/46A61P27/02
CPCC12N9/16C12N15/86A61K48/005A61K48/0008A61P27/02C12Y301/04035C12N2750/14143C12N2800/107C12N2800/22A61K38/00
Inventor 李斌任盛
Owner WUHAN NEUROPHTH BIOTECHNOLOGY LTD CO
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