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Compounds and methods for modulating expression of gccr

An antisense compound and a representative technology, applied in the field of compounds and methods for regulating PCSK9 expression, can solve problems such as reduced sequence specificity

Inactive Publication Date: 2009-07-22
IONIS PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RNAi is an antisense-mediated form of gene silencing involving the introduction of dsRNA, which results in a sequence-specific decrease in the levels of the targeted endogenous mRNA

Method used

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  • Compounds and methods for modulating expression of gccr
  • Compounds and methods for modulating expression of gccr
  • Compounds and methods for modulating expression of gccr

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[1173] Cell culture and short antisense compound treatment

[1174] The effect of short antisense compounds on target nucleic acid expression can be tested in any of a variety of cultured cell lines or primary cell lines. The cell lines can be obtained from publicly available sources, such as the American Type Culture Collection (Manassas, VA). The cells are cultured according to methods known to those of ordinary skill in the art.

[1175] When the cells reach the proper confluence, use The cells were treated with oligonucleotides as described. When the cells reach 65-75% confluence, they are treated with oligonucleotides. in Reduced serum medium (Invitrogen LifeTechnologies, Carlsbad, CA) combines oligonucleotides with (Invitrogen LifeTechnologies, Carlsbad, CA) mix to achieve the desired oligonucleotide concentration and 2.5 or 3μg / mL per 100nM oligonucleotide concentration. This transfection composition was incubated at room temperature for about 0.5 hours. For cells grown...

Embodiment 2

[1178] Example 2 Real-time quantitative PCR analysis of target mRNA levels

[1179] use The 7600, 7700 or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) performs real-time quantitative PCR to quantify the target mRNA level according to the manufacturer's instructions.

[1180] Before the quantitative PCR analysis, the GAPDH amplification reaction was used to evaluate the ability to be "multiplexed" of the primer-probe set specific to the tested target gene. After separation, reverse transcriptase (RT) reaction and real-time PCR are performed on the RNA sequence, and the two reactions are performed in the same well. RT and PCR reagents were obtained from Invitrogen Life Technologies (Carlsbad, CA). RT, real-time PCR are carried out in the same well, 20μL PCR cocktail (without MgCl 2 2.5x PCR buffer, 6.6mM MgCl 2 , DATP, dCTP, dCTP and dGTP each 375μM, forward and reverse primers each 375nM, 125nM probe, 4 units of RNase inhibitor, 1.25 units of Taq, 5 uni...

Embodiment 3

[1188] Example 3: Targeting ApoB nucleic acid and having 2'-MOE or methylene group (4'-CH 2 -O-2’) BNA modified short antisense compound

[1189] 6-week-old male Balb / c mice (Jackson Laboratory, Bar Harbor, ME) were injected intraperitoneally (i.p.) with an antisense compound targeting ApoB twice a week for three weeks. Antisense compound dosages include 2.4, 1.2, 0.6, 0.3 and 0.15 μmol / kg. For antisense compounds of 14 nucleotides in length, these doses are equal to approximately 12, 6, 3, 1.5, or 0.75 mg / kg, respectively. Shown in Table 25 are the sequences and motifs of the antisense compounds used in this study. The antisense compound is 20 or 14 nucleotides in length, and has a central "gap" region composed of 10 2'-deoxynucleotides, on both sides of which there are 2'-O-methoxyethyl Base (2'-MOE) wings or BNA modified "wings". For example, the 2-10-2 MOE gapmer motif (gapmer motif) represents such an antisense compound, which has a gap of 10 nucleotides and two nucleotides w...

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Abstract

The present disclosure describes short antisense compounds, including such compounds comprising chemically-modified high-affinity monomers 8-16 monomers in length. Certain such short antisense compound are useful for the reduction of target nucleic acids and / or proteins in cells, tissues, and animals with increased potency and improved therapeutic index. Thus, provided herein are short antisense compounds comprising high-affinity nucleotide modifications useful for reducing a target RNA in vivo. Such short antisense compounds are effective at lower doses than previously described antisense compounds, allowing for a reduction in toxicity and cost of treatment. In addition, the described short antisense compounds have greater potential for oral dosing.

Description

[0001] Sequence Listing [0002] The present invention is submitted together with the sequence table in electronic format. The sequence list provided is a file named CORE0061WO10SEQ.TXT generated on May 7, 2007, with a size of 700Kb. The sequence listing information in electronic format is incorporated herein by reference in its entirety. Background of the invention [0003] Nearly 40 years ago, it was first proposed to target gene sequences that cause diseases (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), and ten years later, antisense activity was demonstrated in cell culture (Zamecnik Et al., Proc. Natl. Acad. Sci. USA, 1978, 75, 280-284). One advantage of antisense technology in treating diseases or conditions formed by disease-causing genes is that it is a direct genetic method with the ability to regulate the expression of specific disease-causing genes. [0004] Generally, the mechanism of antisense technology is that the antisense compound hybridizes with the target n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/70C07H21/02C07H21/04C12Q1/68
Inventor 桑杰·巴诺特理查德·S·吉尔里罗伯特·麦凯布雷特·P·莫尼亚普尼特·P·塞思安德鲁·M·西科夫斯基埃里克·E·斯韦兹爱德华·万斯威茨
Owner IONIS PHARMA INC
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