Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Multiple RNA polymerase III promoter expression constructs

An RNA polymerase and expression construct technology, which is applied in the field of multi-RNA polymerase III promoter expression constructs and can solve problems such as small differences

Active Publication Date: 2008-05-28
阿尔尼拉姆药物公司
View PDF40 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cells also use another family of promoters (transcribed by RNA polymerase I) to transcribe small structural RNAs, but the sequence of this family is less divergent than that of RNA polymerase III promoters

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Multiple RNA polymerase III promoter expression constructs
  • Multiple RNA polymerase III promoter expression constructs
  • Multiple RNA polymerase III promoter expression constructs

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0093] Pharmaceutical compositions can be prepared as described herein, for example comprising DNA plasmid constructs expressing 2, 3, 4, or more RNA polymerase III promoters under the control of 2, 3, 4, or more Related to, e.g., sequences from one or more genes of a target pathogen (e.g., a virus such as HBV, HCV, HIV, HPV, variola virus), or other nucleic acid sequences associated with another pathogen or human, veterinary and agricultural Sequences such as the human cell receptor sequence of anthrax virus, or other gene sequences associated with mammalian, vertebrate, or invertebrate diseases or conditions such as Huntington's disease or VEGF, or associated with macular degeneration or genetically engineered for treatment Short hairpin dsRNA molecules substantially homologous to avian influenza virus or West Nile virus sequences of human, equine or avian host cells). Expression constructs may include additional promoter sequences, such as the bacteriophage T7 promoter, whe...

Embodiment 1

[0130] Example 1: An expression construct based on tricistronic RNA polymerase III for producing shRNAs that reduce hepatitis B virus RNA production and replication

[0131] A series of plasmids was constructed to express three different shRNAs targeting hepatitis B virus under the control of three separate RNA polymerase III promoters. In the multiple cloning site of the vector, the U6 and 7SK promoter / shRNA cassettes were placed adjacent to each other, while a third promoter sequence was placed with the distant cloning site (adjacent to the kanamycin resistance gene) (Second copy of U6 promoter or H1 promoter). The 5' end of each shRNA element can be linked to each promoter's connected to the 3' end. Figure 4 The sequence of each promoter element is given. Figure 5Three shRNA sequences derived from conserved regions of HBV and placed into tricistronic vectors called 2791, 1907 and 1737 are shown. Each shRNA starts with a 21 bp HBV sequence, followed by a 9 base loop ele...

Embodiment 2

[0137] Example 2: Expression constructs based on four RNA polymerase III promoters for producing shRNAs that reduce hepatitis B virus RNA production and replication

[0138] Figure 9is a schematic diagram of vector pHB4 containing 4 polymerase III promoter-shRNA cassettes. Labels in the figure illustrate the promoter name, the associated shRNA (targeted to the HBV genome) and the direction of transcription of each cassette. Vectors were constructed using an iterative method of incrementally adding shRNA cassettes, as described above. The data in Figure 10 (a luciferase assay substantially similar to that used in Example 1) demonstrates that all four promoter / shRNA cassettes are provided with a vector and a detectable substrate (luciferin in Example 1) for silencing Enzyme fusion constructs, now including the shRNA-799 construct) are active on the intracellular target sequence. The table in Figure 10 shows three independent experiments with pHB4 and the positive control, wh...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

Expression constructs comprising at least two different RNA polymerase III promoters, wherein each promoter is operably linked to a nucleic acid sequence encoding an RNA effector molecule, are disclosed herein. Further provided are expression constructs comprising multiple polymerase III promoters operably linked to sequences encoding short hairpin RNA molecules, which may comprise single and / or multiple fingers. The provided constructs are useful for in vivo delivery of RNA molecules effective in gene silencing, including of viral genes including HBV and HCV.

Description

[0001] Cross-references to related applications [0002] This application claims priority to U.S. Provisional Application No. 60 / 603,622, filed August 23, 2004, and U.S. Provisional Application No. 60 / 629,942, filed November 22, 2004, which are hereby incorporated by reference in their entirety. into this application. technical field [0003] The general field of the invention is the use and compositions of novel recombinant DNA molecules having the ability to efficiently direct (in cells, tissues or organisms) the production of small, genetically engineered RNA molecules in large quantities. ability, said RNA molecules can be used to suppress genes involved in disease or abnormal cellular function. In particular, the present invention provides methods for the simultaneous production of multiple (2, 3, 4, 5 or more) different small RNA molecules from a single recombinant DNA molecule with a design suitable for human therapeutic applications. Background technique [0004] R...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/85A61K48/00C12N15/11A61P31/12C12N15/66
Inventor 凯瑟琳·J.·帕丘克丹尼尔·E.·麦卡鲁斯钱德拉塞卡尔·萨蒂什钱德拉恩马丁·D.·西格
Owner 阿尔尼拉姆药物公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com