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A primary micro RNA expression cassette

An expression cassette and primary technology, applied in the field of viral gene expression inhibition, can solve problems such as the inability to generate functional RNAi effectors

Inactive Publication Date: 2010-06-23
UNIVERSITY OF THE WITWATERSRAND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Convenient generation of functional RNAi effectors from PolII transcription is not currently possible

Method used

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  • A primary micro RNA expression cassette
  • A primary micro RNA expression cassette
  • A primary micro RNA expression cassette

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0279] Example 1: Design and Proliferation of Anti-HBV pri-miR Expression Plasmids

[0280] 1. Design

[0281] By replacing naturally occurring miR-31 (SEQ ID NOs: 1 / 107 / 133) and miR-122 (SEQ ID NOs: 2 / 108 / 134) to design pri-miR expression cassettes. The complete sequence encoding the anti-HBVpri miR sequence is given in SEQ ID NO: 3(135) and 4(136). The wild-type sequences of miRs were maintained as much as possible and the computer-aided prediction of transcript secondary structure [38] would not be significantly different from that of the corresponding wild-type miRs. The final expression cassette contained 51 nucleotides of wild-type sequence flanked by either end of the pre-miR (Zeng and Cullen, 2005). In order to facilitate the cloning of miR, Nhe I and Spe I restriction enzyme sites were introduced at its 5' and 3' ends, respectively. figure 1 A schematic diagram of the target genomic loci of HBV together with the pri-miR expression cassette is shown in . figure ...

Embodiment 2

[0314] Example 2: Intracellular expression of anti-HBV sequences from pTZ-57R / Tpri-miR-31 / 5 and pTZ-57R / Tpri-miR-122 / 5 in transfected cultured cells.

[0315] Cultured cells were transfected with plasmids encoding miR-31 / 5 and miR122 / 5.

[0316] HEK293 cells were propagated in DMEM supplemented with 10% FCS, penicillin (50 IU / ml) and streptomycin (50 μg / ml) (Gibco BRL, UK). One day before transfection, 1,500,000 HEK293 cells were seeded into a petri dish with a diameter of 10 em. Transfection was performed with 10 μg of shRNA- or miR-expressing plasmids using cationic liposomes (Invitrogen, CA, USA) following the supplier's instructions.

[0317] Northern blot analysis. HEK293 cells were harvested 4 days after transfection, and total RNA was extracted using Tri reagent (Sigma, MI, USA) according to the supplier's instructions. 20 μg of RNA was resolved in a urine-denaturing 12.5% ​​polyacrylamide gel and printed on a nylon membrane. Radiolabeled DNA oligonucleotides serve ...

Embodiment 3

[0319] Example 3: Anti-HBV Efficacy Test of miR Sequences in a Cell Culture Model of HBV Replication

[0320] target plasmid. pCH-9 / 3091 was described previously [42]. It contains HBV sequences larger than the length of the genome, which is similar to the HBV Al subgenotype consensus, and a 3.5 kb HBV pregenomic transcript is generated from its CMV promoter. The pCH firefly Luc vector was prepared by replacing the preS2 / SORF of pCH-9 / 3091 with firefly luciferase-encoding DNA following a procedure similar to that used to generate pCHGFP as described previously [43]. Briefly, the firefly luciferase sequence was amplified from psiCheck2 (Promega, WI, USA) by PCR. The primer combination for amplifying the coding region of the firefly luciferase sequence is:

[0321] 5' ACTGCTCGAGGATTGGGGACCCTGCGCTGAACATGGTGAGCAAGGGCG3' (forward; SEQ ID NO: 52);

[0322] and 5'ACGTTCTAGGTATACGGACCGTTACTTGTACAGCTC3' (reverse; SEQ ID NO: 53).

[0323] The forward primer contained a sequence comp...

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Abstract

This invention relates to inhibition of hepatitis gene expression. More specifically, the invention relates to a method of using RNA sequences to inhibit Hepatitis B and C Virus replication. Expression cassettes that include DNA sequences derived from endogenous micro RNAs (miRs) are used in the method and are transcribed by Pol Il promoters, and then processed to generate sequences that are specific to target hepatitis virus sequences (RNAi effecter sequences). The RNAi effecter sequences can target the selected hepatitis virus sequences resulting in gene silencing or transcriptional inhibition of the hepatitis virus gene. The expression cassettes may be delivered in vitro or in vivo to host cells. A pharmaceutical composition containing the expression cassettes is also claimed.

Description

Background of the invention [0001] The present invention relates to the inhibition of viral gene expression. In particular, the present invention relates to methods of inhibiting the replication of hepatitis B virus using RNA sequences. In this approach, expression constructs containing sequences from endogenous microRNAs (miRs) are used to generate sequence-specific silencing of HBV sequences. [0002] RNA interference (RNAi) is a conserved evolutionary biological response to double-stranded RNA that has been described in plants [1], invertebrates [24] and mammalian cells [5]. The function of RNAi is to directly inhibit the expression of genes with homologous sequences to endogenous or introduced RNAi effectors [6] and have no effect on genes with unrelated sequences [7,8]. RNAi is considered an ancient response pathway that plays a role in regulating the expression of protein-coding genes [8] and mediates resistance to endogenous parasitic and exogenous pathogenic nucleic ...

Claims

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Application Information

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IPC IPC(8): C12N15/11A61K31/713C12N15/113
CPCC12N2310/14C12N2320/50C12N15/111C12N2310/141C12N15/1131C12N2310/111
Inventor 阿巴思诺特·帕特里克阿卜杜拉·伊利塔努沙·奈杜马克·索尔·温伯格维多利亚·玛丽·朗肖
Owner UNIVERSITY OF THE WITWATERSRAND
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