Inhibition of Vegf-A secretion, angiogenesis and/or neovascularization by sina-mediated knockdown of Vegf-C and RhoA

A technology of VEGF-C and VEGF-A, applied in DNA/RNA fragments, cardiovascular system diseases, recombinant DNA technology, etc., can solve problems such as differences in signaling pathways, and achieve minimal side effects

Inactive Publication Date: 2011-12-07
RELIANCE LIFE SCI PVT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This may be due to differences in the signaling pathways used for angiogenesis and lymphangiogenesis

Method used

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  • Inhibition of Vegf-A secretion, angiogenesis and/or neovascularization by sina-mediated knockdown of Vegf-C and RhoA
  • Inhibition of Vegf-A secretion, angiogenesis and/or neovascularization by sina-mediated knockdown of Vegf-C and RhoA
  • Inhibition of Vegf-A secretion, angiogenesis and/or neovascularization by sina-mediated knockdown of Vegf-C and RhoA

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0174] 2. Preparation of siNA;

[0175] 3. Compound efficacy test;

[0176] 4. Comparative data for siRNAs having 19-30 nucleotides, for example 21, 22 and 27 nucleotides; and

[0177] 5. Efficacy evaluation in animal models.

[0178] In one example, the design of suitable siNAs includes the design of siRNAs with 21, 23 and 27 nucleotides for modulation of RhoA and VEGF-C, without chemical modifications. Accessible sites of target genes such as RhoA and VEGF-C were screened and siRNAs were synthesized considering the open reading frames (ORFs) of RhoA and VEGF-C.

[0179] Consider the following general requirements in siNA design:

[0180] i. Four or more A, T, G or U not in a row consecutively

[0181] ii Avoid the following sequences as they can elicit an interferon response.

[0182] A) 5'-UGUGU-3' and B) 5'-GUCCUUCAA-3'

[0183] iii. The first 200 bases were deleted from the start codon to avoid binding regulatory elements.

[0184] iv. Each siNA duplex was tested i...

Embodiment 1

[0205] Example 1: Design of 21, 22, 23 and 27mer siIRNAs for regulation of RhoA and VEGF-C gene expression

[0206] Identification of target sites:

[0207] Based on the literature: Henshel, A et al., "DEQOR: A web based tool for the design and quality control of siRNAs," (DEQOR: Internet-based tools for the design and quality control of siRNA) Nucleic Acids Res.2004; 32: Wl 13-W120; Ui-Tei, K, et al., "Guidelines for the selection of highly effective siRNA sequences formammalian and chick RNA interference," (for the selection guideline of the efficient siRNA sequences for the RNA interference of mammals and chicks ) Nucleic Acid Res.2004; 32(3):936-48; Sui, G., et al., "A DNA vector based RNAi technology to suppress gene expression in mammalian cells," (DNA vector based on RNAi technology in mammalian cells Suppression of gene expression in plasmacytoid dendritic cells through TLR7) Proc.Natl.Acad.Sci USA 2002;26(2):199-213; Kim, D.H., et al., "Synthetic dsRNA dicer-substrat...

Embodiment 2

[0226] Example 2: Preparation of siNA molecules

[0227] The siNA molecules were synthesized chemically using commercially available tools. Based on the type of protecting group bound at the 2′-carbon site of ribose, chemical methods are classified as follows:

[0228] 1.2'-tert-butyldimethylsilyl (TBDMS)

[0229] 2.2'-O-triisopropylsilyloxymethyl (TOM)

[0230] 3. 2′-Acetoxyethoxy Chemistry (ACE)

[0231] Cycling begins with the 3'-terminal nucleotide attached to a solid support material or bead. The second nucleotide binds to the 5-hydroxyl group of the first nucleotide. Capping effectively prevents the extension of wrong or short nucleotides. And then oxidize the phosphate bonds between nucleotides to the final P(V) state. Finally, the 5'-protecting group on the new nucleotide is removed and the extending oligonucleotide is ready for the addition of the next nucleotide. Once the nucleic acid molecule reaches the desired length, it is further deprotected, cleaved from...

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Abstract

The present invention relates to the use of short interfering nucleic acid molecules (siNA, such as siRNA) that modulate the expression of VEGF-C and/or RhoA involved in neovascular angiogenesis. In the present invention, inhibition of VEGF-C and/or RhoA gene expression results in decreased expression of VEGF-A, which is required for the initiation and persistence of angiogenesis. Furthermore, the present invention also relates to the inhibition of RhoA expression levels and VEGF-C expression levels to gain the benefit of downregulating two different targets required for angiogenesis. The present invention describes compounds, compositions and methods for the inhibition of neovascularization. In certain embodiments, the present invention relates to methods for inhibiting neovascularization, and compounds for treating ocular diseases such as age-related macular degeneration (AMD), diabetic retinopathy, glaucoma, and other neovascular diseases , such as VEGF-C siRNA and RhoAsiRNA.

Description

[0001] sequence listing [0002] This application contains a Sequence Listing that has been submitted via EFS-Web, which is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 18, 2009, is named RLS PCT037.txt and is 7,300 bytes in size. [0003] Cross references to related patent applications [0004] This application claims the benefit of Provisional Indian Application No. 2459 / MUM / 2008 filed November 21, 2008, which is hereby incorporated by reference in its entirety. field of invention [0005] The present invention relates to the use of short nucleic acid molecules, such as short interfering nucleic acid (siNA) molecules, to regulate gene and protein expression, including compounds, compositions and synergistic combinations of small nucleic acid molecules that modulate RhoA and / or VEGF-C gene expression. The compounds and methods of the invention are useful alone or in combination with other therapies in modulating Rho-A, VEGF-C and VE...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713A61P9/00
CPCC12N2310/14C12N15/113A61P27/02A61P27/06A61P35/00A61P9/00
Inventor 克里什那·阿迪帕利·穆拉里库马尔·巴拉特吉乐·莎伊拉迦
Owner RELIANCE LIFE SCI PVT
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