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Little interfered RNA preparation for internal preventing or curing respiratory system diseases and screening method thereof

A technology for respiratory diseases and screening methods, applied in the field of small interfering RNA preparations, can solve problems such as low cationic load

Inactive Publication Date: 2005-12-07
广州拓谱基因技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that the cationic load is relatively small

Method used

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  • Little interfered RNA preparation for internal preventing or curing respiratory system diseases and screening method thereof
  • Little interfered RNA preparation for internal preventing or curing respiratory system diseases and screening method thereof
  • Little interfered RNA preparation for internal preventing or curing respiratory system diseases and screening method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0069] Example 1: Preparation of cationic polymer / siRNA formulation. According to our research results at the cellular level, siRNA (see sequence listing) was synthesized by chemical methods (synthesis service provided by QIAGENE, Germany). Succinimide-based PEG3400 (PEG3400-N-hydroxy succinimide, purchased from Shearwater Polymers company) ( figure 2 ) was dissolved in 100mM HEPES (pH8) buffer solution, slowly added to PEI solution, kept stirring at room temperature for 5-9 hours, and the detected pH value was 9.5, left at room temperature for 4-6 days, freeze-dried the reaction product, and redissolved In 150mM NaCl (pH 7) solution prepared with 5mM HEPES, purified by gel column, dialyzed and ready for use. PEG-PEI and PEI were mixed at a ratio of 1:1 and dissolved in 5mM HEPES (pH8) buffer. The synthesized siRNA was also dissolved in 5mM HEPES (pH8) buffer. The two were mixed in a ratio of 2:1, and oscillated for 30 seconds.

Embodiment 2

[0070] Example 2: Preparation of cationic polypeptide / siRNA preparation.

[0071] Dissolve an appropriate amount of siRNA in RNase-free sterile water and mix gently. Due to the limited volume, a high concentration of siRNA can be used, generally 8-15 μg / μL for siRNA.

[0072] Take appropriate amount of siRNA and mix with cationic polypeptide (see sequence listing). SiRNA and cationic polypeptide were mixed at a ratio of 1:4-8, and incubated at room temperature for 30 minutes to form siRNA / cationic polypeptide complex. The prepared siRNA / cationic polypeptide complex can be used to introduce siRNA in vivo (including the respiratory system). This method is also applicable to in vivo introduction of DNA or DNA / siRNA complex.

Embodiment 3

[0073] Example 3: Preparation of nucleic acid protecting agent / siRNA preparation.

[0074] Dissolve an appropriate amount of siRNA (5-50 mg) in a solution of polyvinylpyrrolidine and glucose prepared by RNase-free sterile double-distilled water, and mix well at 4°C to make a complex of siRNA / nucleic acid protecting agent for use in Respiratory tract administration of siRNA.

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Abstract

Disclosed is a little-interfered RNA preparation for internal preventing or curing respiratory system diseases and screening method, which consists of leading highly performance siRNA preparation into the respiratory tracts of non-human Primates, inhibiting the reproduction and expression of the SARS coronavirus, screening siRNA with SARS disease preventing and treating actions. The invention provides the constituents of the siRNA preparation and the method for effectively inhibiting SARS viral infection, the preparation can be used for screening medicaments for preventing and treating respiratory tract infection diseases, such as asthma, COPD, RSV and ARS.

Description

technical field [0001] The invention relates to a small interfering RNA preparation for preventing or treating respiratory diseases in vivo and a screening method thereof. Background technique [0002] SARS is the first serious and easy-to-spread new infectious disease in the 21st century. Although the global SARS epidemic is currently under control, the rapid epidemic has had a serious impact on global health security, medical and health systems, people's livelihood, economic stability and growth. So far, people's understanding of this disease is still limited. On February 26, 2003, a Hong Kong resident in Hanoi, Vietnam, was admitted to the hospital due to unexplained fever and respiratory symptoms. In the following two weeks, similar infectious disease outbreaks occurred in Hong Kong, Toronto, Canada, and Singapore. . Through epidemiological investigation, it was found that the index cases from these countries and regions had all been to Hong Kong and lived on the same...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00
Inventor 李宝健陆阳唐清泉程度谢岳峰
Owner 广州拓谱基因技术有限公司