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Multiple target point small interference RNA cocktail agent for treating ophthalmic disease and preparing method thereof

An ophthalmic disease, small interference technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, gene therapy, etc., can solve the problem of no way to ensure siRNA and so on

Inactive Publication Date: 2008-06-18
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

[0028] Importantly, not every potential siRNA molecule can effectively inhibit the expression of the targeted gene. There is no way to ensure that the designed siRNA can inhibit the expression of the targeted gene. The efficacy of the siRNA molecule must be tested and analyzed for its inhibition of the targeted gene. Effect

Method used

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  • Multiple target point small interference RNA cocktail agent for treating ophthalmic disease and preparing method thereof
  • Multiple target point small interference RNA cocktail agent for treating ophthalmic disease and preparing method thereof
  • Multiple target point small interference RNA cocktail agent for treating ophthalmic disease and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0196] Example 1 Design of siRNA double-stranded molecules against VEGF, VEGF-R1, VEGF-R2

[0197] Six siRNA double-stranded molecules were designed for each gene, and a total of 18 siRNAs were chemically synthesized at Qiagen (Germantown, MD). All siRNAs have a dTdT overhang at the 3' end, and there are no other modifications. After screening in the cell model, two siRNAs with the best activity were selected from every six siRNAs, and the siRNAs were mixed in equal proportions. The siRNA sequence is as follows:

[0198] mVEGF-siRNA (a): (forward) 5'-GCCGUCCUGUGUGCCGCUGdtdt-3', (reverse)

[0199] 5'-CAGCGGCACACAGGACGGCdtdt-3'; mVEGF-siRNA(b): (forward)

[0200] 5'-CGAUGAAGCCCUGGAGUGCdtdt-3', (reverse)

[0201] 5'-GCACUCCAGGGCUUCAUCGdtdt-3'; mVEGFR1-siRNA(a): (forward)

[0202] 5'-GUUAAAAGUGCCUGAACUGdtdt-3', (reverse)

[0203] 5'-CAGUUCAGGCACUUUUAACdtdt-3'; mVEGFR1-siRNA(b): (forward)

[0204] 5'-GCAGGCCAGACUCUCUUUCdtdt-3', (reverse)

[0205] 5'-GAAAGAGAGUCUGGCCUGCdtdt-...

Embodiment 2

[0218] Example 2 Screening Candidate siRNA Double-Strand Molecules on Cell Model

[0219]Candidate mouse VEGF siRNA was screened using RAW264.7 gamma NO(-) cells (ATCC, CRL-2278), cell culture condition: RPMI containing 10% FBS. Candidate mouse VEGFR1 and VEGFR2 were screened using SVR cells (ATCC, CRL-2280), cell culture conditions: DMEM containing 5% FBS. The cells were planted in 6-well plates, and the transfection reagent was LipofectAmine (Invitrogen, Carlsbad, CA). After 24 hours or 48 hours, RNA was extracted, and RNA copy number changes were analyzed by RNA-specific PCR (RS-PCR).

Embodiment 3

[0220] Embodiment 3 prepares siRNA / polymer nanoparticle

[0221] A branched lysine-histidine polypeptide complex (HKP) was synthesized on a Ranin Voyager synthesizer (PTI, Tucson, AZ). The HKP polypeptide used here is PT73, and its structural characteristics are (R)K (R)-K(R)-(R)K(X), where R=KHHHKHHHNHHHNHHHN, X=C(O)NH2, K=lysine, H=histidine and N=Asperagine. PT73 and siRNA were respectively dissolved in water and mixed at a mass ratio of 4:1 to form nanoparticles with an average diameter of 150-200 nm. The targeted nano-conjugated polymer carrier RPP for systemic administration is an amphiphilic three-segment chimera, and its structural feature is RGD-PEG-PEI, wherein RGD is an adhesion protein whose sequence is H-ACRGDMFGCA-OH, PEG is polyethylene glycol, and PEI is polyethyleneimine. RPP and siRNA were dissolved in water respectively, and mixed according to the molar ratio of 2:1, which can self-assemble to form 120-150nm nanoparticles. The ζ-potential analysis showed t...

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Abstract

The invention discloses a multi-target small-interfering RNA cocktail preparation for treating ophthalmic diseases and the preparation method. The multi-target small-interfering-RNA cocktail preparation is composed of three or more than three small-interfering RNA aiming to three or more than three different genes and knocking down simultaneously a plurality of pathogenic genes; the multi-target small-interfering-RNA cocktail preparation is prepared by a plurality of small-interfering RNA in a certain proportion according to different diseases. The multi-target small-interfering-RNA cocktail preparation is a plurality of double-bond RNA molecules of different lengths from 19to 27nt, with blunt ends or overhanging ends; the RNA sequence in the multi-target small-interfering-RNA cocktail preparation has the homology to the gene targets of human, rat and other nonhuman primate; . The multi-target small-interfering-RNA cocktail preparation aiming to the following gene sequences: (1) virus-affection-related gene; (2) inflammation-arosing gene; (3) neovescular-related gene. The invention provides a novel treatment for a plurality of ophthalmic diseases, including retinopathy of prematurity, senile fundus macula lutea, retinopathy caused by senile diabetes, herpes simplex corneal stromal opacification and uveitis.

Description

technical field [0001] The invention relates to a multi-target small interfering RNA cocktail preparation for treating ophthalmic diseases and methods for its preparation, introduction and application. Background technique [0002] There are still no more effective therapeutic drugs for the treatment of infection, allergic reaction, angiogenesis and age-related ophthalmic diseases. Many eye diseases are caused by neovascularization or abnormal proliferation of blood vessels in the eye. In many serious eye diseases, neovascularization is an early pathological change that damages vision. Although there are new drugs for these diseases, neovascularization is still the most common cause of permanent blindness in European, American and Asian countries. main reason. Abnormal ocular neovascularization promoted by certain eye diseases can further damage vision. However, there are currently no effective treatments and drugs to treat ophthalmic diseases caused by abnormal ocular ne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K47/34A61K47/42A61P27/02A61P29/00C12N15/12C12N15/38C12N15/57A61K47/26
Inventor 陆阳梁晓玲程度
Owner 广州拓谱基因技术有限公司
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