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RNA interference fragment and its use

A technology of RNA interference and fragments, which is applied in the field of RNA interference fragments, can solve problems such as unsatisfactory treatment effects, and achieve the effect of solving imbalance problems and reducing hemolytic symptoms

Active Publication Date: 2009-12-09
SHANGHAI JIAO TONG UNIV AFFILIATED CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned methods have defects and cannot achieve satisfactory therapeutic effect.

Method used

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  • RNA interference fragment and its use

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Design of RNAi fragments

[0027] Apply some principles in RNAi design (Berns K, Nature. 2004; 428(6981): 431-7): (1) start from the AUG start codon of the transcript (mRNA), look for the "AA" double sequence, and Write down the 19-23 base sequence of its 3' end as a potential siRNA target site. (2) Compare potential sequences with corresponding genomic databases (human or mouse, rat, etc.) and exclude those that are homologous to other coding sequences / ESTs, e.g. using BLAST (www.ncbi.nlm.nih .gov / BLAST / ); (3) select the appropriate target sequence for synthesis, and artificially design three RNAi fragments targeting the α chain:

[0028] α1: GAC CTA CTT CCC GCA CTT C

[0029] α2: GTT CCT GGC TTC TGT GAG C

[0030] α3: ATA CCG TTA AGC TGG AGC C

[0031] These three RNAi fragments are all 19bp in length, and since the synthesis of such short fragments is easy in the prior art, details will not be described here.

Embodiment 2

[0032] Example 2 Construct expression vector

[0033] The three RNAi fragments α1, α2 and α3 designed above were used to construct expression vectors, and the specific steps were as follows:

[0034] 1. Acquisition of PH carrier

[0035] The H1 promoter dependent on RNA polymerase III was amplified from human embryonic kidney 293T cells (Shanghai Institute of Cells, Chinese Academy of Sciences) by nested PCR, as follows:

[0036] PCR primers:

[0037] 1: 5'-TATCTAGAGAATTCGAACGCTGACG-3';

[0038] 2: 5'-TGAAGCTTGTGGTCTCATACAGAAC-3';

[0039] 3: 5'-TCTAGACTGACGTCATCAACCCGCTCCA-3' (antisense strand).

[0040] Among them, primers 2 and 3 have XbaI and HindIII restriction sites respectively.

[0041] Amplification conditions:

[0042] First expansion: 94°C 4min, 1 cycle; 94°C 30second, 53-57°C 45second, 72°C 30second, 30 cycles; 72°C 10min, 1 cycle;

[0043] Second expansion: 94°C 4min, 1 cycle; 94°C 30second, 55°C 45second, 72°C 30second, 32 cycles; 72°C 10min, 1 cycle.

...

Embodiment 3

[0048] Example 3 Transfect human erythroleukemia cell line K562 and establish mixed clones

[0049]1. Transfection: K562 cells (purchased from Shanghai Institute of Cells, Chinese Academy of Sciences) were cultured in DMEM medium (GIBCO) with 15% fetal bovine serum at 37°C and 5% CO 2 Conditioned cultivation. Liposome 2000 (Invitrogen) was used for cell transfection, and the method can be completely according to the manufacturer's instructions. For transfection, K562 cells were transfected with 1 μg of RNAi vectors Pα1, Pα2 and Pα3 (10 5 / hole, 24-well culture plate), the ratio of plasmid to liposome is 1ug:2.5ul.

[0050] 2. Establishment of mixed clonal cell lines: due to the low transfection efficiency, in order to exclude the influence of negative cells (untransfected interference plasmids), after 48 hours of transfection, hygromycin (hygromycin) with a concentration of 600-800 μg / ml was added. Primer, sigma company) was screened for more than two weeks to obtain posi...

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Abstract

The invention opened a RNAi segment against mRNA of the ª‡-globin for the ª‰-thalassemia. The carrier formed by the segment and the K562 mixing clone generated by the segment can decrease the expressing amount of theª‡-globin above 30%. So the RNAi segment and the carrier have the high value in the clinical gene curing.

Description

technical field [0001] The invention belongs to the field of molecular genetics and gene therapy, in particular, the invention relates to an RNA interference fragment capable of inhibiting the alpha-chain of beta-thalassemia and its application. Background technique [0002] Thalassemia is a hemolytic anemia caused by a decrease in the synthesis rate of one or some protein chains, resulting in a lack of some peptide chains and a relative increase in other peptide chains, resulting in an imbalance in the number of peptide chains. Beta-thalassemia (beta thalassemia) is one of these types, and there are hundreds of different types of beta thalassemia worldwide. [0003] A large number of research data show that, except for a small number of β-thalassemias caused by gene deletions, most of them are caused by different types of point mutations in the β-globin gene (including single base substitutions, insertions or deletions of individual bases). Sincerely. These point mutation...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63A61K48/00A61P7/06C12N15/113
Inventor 曾溢滔谢书阳张敬之黄淑帧
Owner SHANGHAI JIAO TONG UNIV AFFILIATED CHILDRENS HOSPITAL
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