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Application of recombinant adeno-associated virus of target silent KLF4 gene

A technology targeting silencing and genes, applied in the fields of molecular biology and biomedicine, to improve the systolic function of the right ventricle, inhibit pulmonary hypertension, and prevent and treat pulmonary hypertension.

Pending Publication Date: 2019-05-03
AFFILIATED HOSPITAL OF ZUNYI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of recombinant adeno-associated virus targeting the KLF4 gene for targeted prevention or treatment of pulmonary arterial hypertension in vivo has not yet been reported in the literature

Method used

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  • Application of recombinant adeno-associated virus of target silent KLF4 gene
  • Application of recombinant adeno-associated virus of target silent KLF4 gene
  • Application of recombinant adeno-associated virus of target silent KLF4 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Construction of recombinant adeno-associated virus targeting silencing of the KLF4 gene:

[0031] 1. Construction of recombinant plasmid pHBAAV-r-KLF4shRNA-GFP

[0032]According to the sequence of the rat KLF4 gene (GenBank Accession Number: NM_053713.1) and referring to the siRNA target sequence selection principle proposed by Tuschl et al., a specific siRNA sequence against KLF4 was designed: CACCCACACTTGTGACTAT. Cut the blank vector (pHBAAV-U6-MCS-CMV-EGFP) with EcoRI and BamHI, and then insert the siRNA target sequence into the U6 promoter to regulate its expression. The adenoviral vector contains EGFP gene expression regulated by the CMV promoter. The expression of green fluorescent protein (GFP) is controlled by the CMV promoter, respectively, as a sign of transduction efficiency. Entrusted Shanghai Hanheng Biotechnology Co., Ltd. to synthesize the complete shRNA sequence (annealing of shRNA template, preparation of linearized expression vector, ligati...

Embodiment 2

[0048] Example 2: The preventive effect of recombinant adeno-associated virus rAAV1 expressing KLF4-shRNA on pulmonary hypertension:

[0049] 1. Preparation of pulmonary arterial hypertension rat model, observation of rAAV1-KLF4-shRNA transfection and targeted silencing of pulmonary vascular KLF4 gene:

[0050] Male SD rats were randomly divided into 3 experimental groups exposed to cigarette smoke and 1 healthy control group (Sham) not exposed to smoke. Referring to the method of Jiang X et al. who exposed cigarette smoke for 4 months to construct a pulmonary hypertension model (references Jiang X, Yuan L, Li P, Wang J, Wang P, Zhang L, Sun B, Sun W. Effect of Simvastatinon 5-HT and 5-HTT in a Rat Model of Pulmonary Artery Hypertension. Cell Physiol Biochem. 2015; 37(5):1712-24.). One month before receiving smoke exposure, rAAV1-KLF4-shRNA (1.8×10 11 vg / 200 μl / mouse) the other two groups were injected with equal volumes of rAAV1-control vector (control virus, rAAV1 packagin...

Embodiment 3

[0057] Example 3: Therapeutic effect of recombinant adeno-associated virus rAAV1 expressing KLF4-shRNA on pulmonary arterial hypertension

[0058] 1. Targeted silencing effect of rAAV1-KLF4-shRNA injected through the airway on pulmonary vascular KLF4 gene

[0059] Male SD rats were randomly divided into 3 experimental groups exposed to cigarette smoke and 1 healthy control group (Sham) not exposed to smoke. A pulmonary hypertension model was constructed by exposure to cigarette smoke (the method is the same as in Example 2). After 3 months of smoke exposure, a group of rats exposed to cigarette smoke were injected with rAAV1-KLF4-shRNA (1.8×10 11 vg / 200μl / mouse) the other two groups were airway injection of rAAV1-control vector (control virus, rAAV1 packaging irrelevant sequence), and airway injection of normal saline (Saline). After continuing to smoke for 1 month, the rats in each group were anesthetized to measure hemodynamic indicators (same as in Example 2), the rats we...

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Abstract

The invention discloses an application of a recombinant adeno-associated virus of a target silent KLF4 gene, and primarily an application of a recombinant adeno-associated virus rAAV1-KLF4-shRNA of atarget silent KLF4 gene to preparation of medicines for preventing and treating pulmonary hypertension. According to the recombinant adeno-associated virus rAAV1-KLF4-shRNA of the target silent KLF4 gene, having GFP fluorescence labelling, disclosed by the invention, type 1 adeno-associated virus (AAV1) which can stably express target protein for a long term and is high in biological safety can beused as a transduction vector, the high tropism of AAV1 to vascular tissue is utilized, an administration mode of infusing medicines in air ducts is combined, and recombinant adeno-associated virusescan be stably expressed for a long term in lung vessels, so that the effects of enabling lung vessel KLF4 gene to be silent, restraining lung vessel remodeling and preventing and treating the pulmonary hypertension can be achieved.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and biomedicine, in particular to the application of a recombinant adeno-associated virus targeting silencing of the KLF4 gene in the prevention and treatment of pulmonary arterial hypertension. technical background [0002] Pulmonary hypertension (Pulmonary hypertension, PH) is a common life-threatening disease, characterized by persistent and significant elevated pulmonary artery pressure, which eventually leads to right ventricular failure and death. It is also a key pathological link in the development of many common diseases, such as chronic obstructive pulmonary disease, chronic thromboembolism, and connective tissue diseases. It is an independent risk factor that increases the incidence and reduces survival, and seriously affects the quality of life and prognosis of patients. It deserves our more attention and research. In recent years, although great progress has been made in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K9/00A61P9/12
Inventor 孙得胜刘虹延欧阳瑶
Owner AFFILIATED HOSPITAL OF ZUNYI UNIV
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