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Preparation method of recombinant adeno-associated virus expressing hsa-miR-21<*> and application in treatment of hypertension

A technology of hsa-mir-21 and anti-hsa-mir-21, which is applied in the preparation of recombinant adeno-associated virus expressing hsa-miR-21* and its application in the treatment of hypertension, can solve the problem of unsuitable Clinical application, short expression time of adenovirus vector, and worrying about biological safety, etc.

Active Publication Date: 2013-03-13
汪道文 +2
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Problems solved by technology

[0006] At present, adenovirus and lentivirus are mostly used as miRNA expression vectors, but adenovirus vectors have a short expression time and are immunogenic to the body. Most lentiviruses are transformed from leukemia virus or HIV, and their biological safety is worrying, so they are not suitable for the future. clinical application

Method used

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  • Preparation method of recombinant adeno-associated virus expressing hsa-miR-21&lt;*&gt; and application in treatment of hypertension
  • Preparation method of recombinant adeno-associated virus expressing hsa-miR-21&lt;*&gt; and application in treatment of hypertension
  • Preparation method of recombinant adeno-associated virus expressing hsa-miR-21&lt;*&gt; and application in treatment of hypertension

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Screening of aortic miRNA expression in spontaneously hypertensive rats

[0024] 1. In this experiment, 4-week-old spontaneously hypertensive rats (SHR) and control rats (Wistar) were used, fed for 8 weeks, and the blood pressure of the tail artery of the rats was monitored. The results showed that the basal blood pressure of SHR was significantly higher than that of normal control rats, and as the week age increased, the blood pressure level of SHR rats continued to rise ( Figure 1A ).

[0025] 2. At the end of the experiment (week), the rat aortic RNA was routinely extracted:

[0026] Add 1ml TRIZOL reagent per 100mg of tissue, and homogenize with an electric homogenizer. After standing at room temperature for 5 minutes, add 0.2ml of chloroform, mix well, leave at room temperature for 5 minutes, and centrifuge at 12,000g at 4°C for 15 minutes. Transfer the upper aqueous phase to a new Ep tube, add 0.5ml of isopropanol, mix well, place at room temperature fo...

Embodiment 2

[0057] Example 2 Construction of recombinant adeno-associated virus

[0058] 1. Insert synthesis

[0059] Based on hsa-miR-21 ＊ base sequence ( figure 2 ), respectively designed and synthesized to express hsa-miR-21 ＊ and reverse complementary hsa-anti-miR-21 ＊ The two reverse complementary strands were dissolved with TE. The sequence is as follows:

[0060] hsa-miR-21 ＊ :

[0061] Upstream primer: 5'GATCCacagcccatcgactggtgttgTTCAAGAGAcaacaccagtcgatgggctgtCCGC 3'

[0062] Downstream primer: 3'GtgtcgggtagctgaccacaacAAGTTCTCTgttgtggtcagctacccgacaGGCGCCGG 5'

[0063] hsa-anti-miR-21 ＊ :

[0064] Upstream primer: 5'GATCCcaacaccagtcgatgggctgtTTCAAGAGAacagcccatcgactggtgttgCCGC 3'

[0065] Downstream primer: 3'GgttgtggtcagctacccgacaAAGTTCTCTtgtcgggtagctgaccacaacGGCGCCGG 5'

[0066] 2. Carry out the reaction according to the system and temperature in the instructions:

[0067] Nuclease-Free Water 36μl

[0068] Annealing Buffer for DNA Oligos (5X) 10μl

[0069] DNA olig...

Embodiment 3

[0155] Example 3 Expression of hsa-miR-21 ＊The therapeutic effect of recombinant adeno-associated virus on hypertension

[0156] 1. SHR rat aorta miR-21 ＊ Expression detection:

[0157] We used 4-week-old SHR rats and injected rAAV-miR-21 through the tail vein respectively. ＊ and rAAV-anti-miR-21 ＊ , virus titer 1×10 11 PFU / per rat, at the end of the experiment (after 8 weeks), real-time PCR was used to detect miR-21 in the aorta of SHR rats ＊ The expression of rAAV-miR-21 ＊ Treatment can significantly increase aortic miR-21 ＊ The expression of rAAV-anti-miR-21 ＊ Treatment can significantly reduce aortic miR-21 ＊ expression( Figure 5 ).

[0158] 2. SHR rat blood pressure monitoring:

[0159] Tail artery blood pressure was measured every 2 weeks, and the results showed that: rAAV-miR-21 ＊ Treatment can significantly reduce the systolic blood pressure level of SHR rats in a time-dependent manner, while rAAV-anti-miR-21 ＊ The treatment can significantly increase the...

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Abstract

The invention relates to a construction and preparation method of a recombinant adeno-associated virus recombinant (rAAV-miRNA-21<*> / rAAV-anti-miRNA-21<*>) that is able to regulate human miRNA-21<*>, more specifically to cloning of hsa-miR-21<*> and its antisense sequence hsa-anti-miR-21<*>, a packaging preparation method of recombinant adeno-associated virus recombinants containing hsa-miR-21<*> and hsa-anti-miR-21<*>, as well as pharmaceutical application of the recombinant adeno-associated virus recombinants. With a length of 21nt, the hsa-miR-21<*> is non-coding RNA. A chemical synthesis method is utilized to construct pAAV-D(+)-miR-21<*> and pAAV-D(+)-anti-miR-21<*> expression plasmids. A three-plasmid calcium phosphate cotransfection method is utilized to prepare target segment-containing recombinant adeno-associated viruses and purify them, wherein the pAAV-D(+)-miR-21<*> can obviously lower the blood pressure level of spontaneously hypertensive rats and improve the heart function simultaneously. Therefore, the hsa-miR-21<*> expression sequence-containing recombinant adeno-associated virus is conformed to have the possibility to be used as a drug for clinical treatment of hypertension and other related diseases.

Description

1. Technical field [0001] The present invention relates to the ability to regulate human miRNA-21 ＊ Recombinant adeno-associated virus recombinant (rAAV-miRNA-21 ＊ / rAAV-anti-miRNA-21 ＊ ) construction and preparation methods, more specifically related to hsa-miR-21 ＊ and antisense sequence hsa-anti-miR-21 ＊ clones and respectively containing hsa-miR-21 ＊ and hsa-anti-miR-21 ＊ The packaging preparation method of the recombinant adeno-associated virus recombinant, and the pharmaceutical application of the recombinant adeno-associated virus recombinant. 2. Background technology [0002] microRNA (miRNA, miR) is a newly discovered endogenous small molecule non-coding single-stranded RNA with a length of about 22 nucleotides derived from endogenous hairpin transcripts. The untranslated region (3'untranslated region, 3'UTR) specifically binds to promote the degradation of the target mRNA, or inhibits the translation of the target mRNA, and reduces the level of the encoded pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/864C12N15/113A61K48/00A61P9/12
Inventor 汪道文陈琛王峰杨盛兰马飞
Owner 汪道文
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