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A kind of isolated nucleic acid molecule and its use

A technology of nucleic acid molecules and uses, applied in the field of biomedicine, can solve the problems of affecting lipid decomposition, ineffective treatment methods, and unclear specific signs and symptoms of BCD.

Active Publication Date: 2021-02-09
CHIGENOVO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The CYP4V2 gene is one of the proteins in the cytochrome P450 superfamily. The protein encoded by the CYP4V2 gene is involved in the process of fatty acid metabolism. It is generally believed that the mutation of the CYP4V2 gene in crystal-like retinal degeneration destroys its enzyme function involved in fatty acid metabolism, thereby affecting lipid breakdown.
However, how CYP4V2 causes the specific signs and symptoms of BCD is unclear
At present, the treatment of BCD mainly refers to the treatment of retinitis pigmentosa (RP). Although the research of CYP4V2 gene mutation provides the possibility of gene therapy in the future, there is no effective treatment at present.

Method used

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  • A kind of isolated nucleic acid molecule and its use
  • A kind of isolated nucleic acid molecule and its use
  • A kind of isolated nucleic acid molecule and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0368] 1. Use of CYP4V2 and RdCVF in the preparation of medicaments for treating, alleviating and / or preventing diseases or conditions associated with retinal pigment epithelium (RPE) atrophy.

[0369] 2. The use according to embodiment 1, wherein the disease or condition comprises crystalline retinal degeneration.

[0370] 3. The use according to any one of embodiments 1-2, wherein the CYP4V2 is human CYP4V2.

[0371] 4. The use according to any one of embodiments 1-3, wherein the CYP4V2 comprises the amino acid sequence shown in any one of SEQ ID NO: 76-82.

[0372] 5. The use according to any one of embodiments 1-4, wherein the polynucleotide encoding CYP4V2 comprises the nucleic acid sequence shown in any one of SEQ ID NO:62-68.

[0373] 6. The use according to any one of embodiments 1-5, wherein the RdCVF is human RdCVF.

[0374] 7. The use according to any one of embodiments 1-6, wherein the RdCVF comprises the amino acid sequence shown in any one of SEQ ID NO: 83-89.

...

Embodiment 1

[0447] Example 1. Validation of promoter strength by expression of CYP4V2

[0448] (1) The full-length CYP4V2 promoter sequence is 2000bp upstream of the coding region of human genome CYP4V2 (HGNC:23198), synthesized by Suzhou Jinweizhi Company.

[0449] (2) Design the primer sequences of different length promoters CYP4V2-Pf, CYP4V2-P1, CYP4V2-P2, CYP4V2-P3, CYP4V2-P4, CYP4V2-P5, CYP4V2-P6, and use the nucleic acid molecules obtained in step (1) as templates PCR amplification, and gel electrophoresis to recover the amplified product. The primer sequences are shown in SEQ ID NO: 30-37.

[0450] The PCR reaction system (50 μl) is as follows:

[0451] h 2 O (Invitrogen, 10977015) 20μl

[0452] Primer F (10pmol / μl) 2μl

[0453] Primer R (10pmol / μl) 2μl

[0454] primeSTAR MAX (Takrara, R045R) 25μl

[0455] CYP4V2-pro-2000bp 5ng

[0456] Table 2 PCR reaction conditions.

[0457]

[0458] (3) The pAV-CAG-CYP4V2-P2A-EGFP vector (purchased from Shandong Weizhen Biotechnolog...

Embodiment 2

[0477] Example 2. Using EGFP as a reporter gene to verify promoter strength

[0478] (1) PCR amplification of promoters EF1a, EFS, OPEFS. The EF1a primer sequence is shown in SEQ ID NO: 40-41, and the EFS primer sequence is shown in SEQ ID NO: 42-43. The OPEFS promoter was based on the universal promoter EFS, and primers were designed to transform EFS into OPEFS by two rounds of PCR. The primers used in the first round of PCR reactions are EFS forward primers (as shown in SEQ ID NO: 42) and OPEFS-overlapR1 (as shown in SEQ ID NO: 44), and the primers used in the second round of PCR reactions are EFS forward primers ( shown in SEQ ID NO: 42) and OPEFS-overlapR2 (shown in SEQ ID NO: 45). Amplified products were recovered by gel electrophoresis. The PCR reaction system (50 μl) is as follows:

[0479] H2O (Invitrogen, 10977015) 20μl

[0480] Primer F (10pmol / μl) 2μl

[0481] Primer R (10pmol / μl) 2μl

[0482] primeSTAR MAX (Takrara, R045R) 25μl

[0483] pAV-CAG-CYP4V2-P2A-E...

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Abstract

The present application relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a CAG promoter, a nucleotide sequence encoding a first protein, a nucleotide sequence encoding a second protein and a nucleoside encoding a BGH signal site An acid sequence, wherein the first protein and the second protein are each differently selected from the group consisting of CYP4V2 protein and RdCVF protein, wherein the nucleotide sequence encoding the CYP4V2 protein comprises the nucleotide sequence of SEQ ID NO:168, The nucleotide sequence encoding the RdCVF protein comprises the nucleotide sequence of SEQ ID NO:167. The present application also relates to the use of the nucleic acid molecule in the preparation of a medicament for treating, alleviating and / or preventing diseases or conditions associated with retinal pigment epithelium (RPE) atrophy.

Description

technical field [0001] This application relates to the field of biomedicine, in particular to an isolated nucleic acid molecule and its use. Background technique [0002] Bietti's crystalline dystrophy (BCD) is a rare form of retinal degeneration in which symptoms primarily include crystalline (clear coverings) in the cornea, small, yellow or white crystal-like deposits deposited in the light-sensitive tissue of the retina objects, and progressive atrophy of the retina, choroidal capillaries, and choroid. The deposits can damage the retina, causing gradual loss of vision. People with crystalline retinal degeneration usually begin to notice vision problems in their teens or twenties, and vision problems can worsen at different rates in each eye, even within the same family Progression varies widely among individuals, yet most sufferers become blind by the age of forty or fifty. Crystalline retinal degeneration is estimated to affect 1 in 67,000 people worldwide and is more...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/864A61K48/00A61K38/18A61K38/44A61P27/02A61P9/10
CPCC12N9/0081C12Y114/15006C07K14/475C12N15/86A61K48/005A61K38/18A61K38/44A61P27/02A61P9/10C12N2750/14143C07K2319/02A61K2300/00C12N2830/50C07K14/47C12N15/625C12N9/0071C12Y114/14A61K38/00
Inventor 杨丽萍陈邵宏贾睿璇张天赋张凡曾巧莉和赛超史天永郝丹丹姜尚伟裴红杰王旭东曾露颖
Owner CHIGENOVO CO LTD
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