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Medicament containing natriuretic peptide gene as well as preparation method and application thereof

A natriuretic peptide and drug technology, applied in the field of gene medicine for the treatment of glaucoma, can solve the problem, which can only be injected into the eye through subconjunctival and vitreous injection, natriuretic peptide has not been widely used, natriuretic peptide gene drug therapy Glaucoma has not yet been reported and other issues

Inactive Publication Date: 2012-05-23
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since natriuretic peptide can be hydrolyzed and cleared by neutral endopeptidase, repeated administration is required to maintain the therapeutic effect. However, due to the poor corneal permeability of natriuretic peptide, it can only be injected into the eye through subconjunctival or vitreous injection (the former However, repeated subconjunctival and intravitreal injections have no clinical practical significance, so natriuretic peptides have not been widely used in the treatment of glaucoma
[0006] In the prior art, the use of natriuretic peptide gene drugs to treat glaucoma has not yet been reported at home and abroad

Method used

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  • Medicament containing natriuretic peptide gene as well as preparation method and application thereof
  • Medicament containing natriuretic peptide gene as well as preparation method and application thereof
  • Medicament containing natriuretic peptide gene as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Amplification and cloning of embodiment 1 CNP gene

[0027] 1 material

[0028] 1.1 Plasmids and cell lines

[0029] 1) Lentivector expression systems (pPACKF1 TM Packaging Mix): Purchased from SBI Company in the United States, it is prepared by mixing three plasmids pCDF1-MCS2-EF1-copGFP, pFIV-34N and pVSV-G in a certain proportion.

[0030] 2) 293FT cell line: from Invitrogen Company of the United States, which is preserved in our laboratory.

[0031] 1.2 Tool enzymes and main reagents

[0032] 1) Reagent, Lipofectamine TM 2000 and trypsin (Tyrisin) for digestion: purchased from Invitrogen, USA

[0033] 2) TakaRa One Step RNA PCR Kit (AMV) and diethyl pyrocarbonate (diethypyrocarbonate, DEPC): purchased from Japan TakaRa Company

[0034] 3) Plasmid extraction kit PureYield TM Plasmid Midiprep System: purchased from Promega, USA

[0035] 4) Restriction endonuclease and T4DNA ligase: purchased from MBI Fermentas, USA

[0036] 5) DNA agarose gel electrophore...

Embodiment 2

[0094] Example 2 Construction of recombinant plasmids

[0095] 1 pCDF1-MCS2-EF1-copGFP empty vector and PCR product of CNP gene digested and recovered

[0096] The pCDF1-MCS2-EF1-copGFP empty vector and the CNP gene PCR product were double digested with restriction endonucleases BamH I and EcoR I, respectively. The double enzyme digestion reaction system is as follows, and the reaction condition is 37°C water bath for 4h.

[0097]

[0098] After the plasmid pCDF1-MCS2-EF1-copGFP was double digested, after 0.7% agarose gel electrophoresis for 30 minutes, the gel at the position of 6749bp was accurately cut, and then the gel was recovered. After double enzyme digestion of the PCR product, after 1.5% agarose gel electrophoresis for 30 minutes, the gel at the position of 410 bp was accurately cut, and then the gel was recovered.

[0099] The digested vector and PCR products were recovered using the gel recovery kit from BioBasic.

[0100] 2 Construction of pCDF1-MCS2-EF1-cop...

Embodiment 3

[0119] Example 3 Packaging of lentivirus

[0120] pPACKF1 mediated by liposome Lipofectamine2000 TM Packaging Plasmid Mix and expression plasmid (empty vector pCDF1-MCS2-EF1-copGFP or recombinant plasmid pCDF1-MCS2-EF1-copGFP-CNP) were co-transfected into 293FT cells to package the virus. Virus packaging flowchart ( Figure 4 ).

[0121] Virus harvest, concentration and titer detection

[0122] 48 and 72 hours after transfection of 293FT cells, the medium supernatant was harvested. Cell debris was removed by centrifugation. Then ultracentrifugation was carried out, and the precipitate was resuspended with PBS, that is, the concentrated lentivirus containing the recombinant plasmid was harvested (the pCDF1-MCS2-EF1-copGFP Vector connected to the CNP gene was packaged into the protein envelope of the virus) FIV-CNP and Lentivirus containing empty vector (packaging of pCDF1-MCS2-EF1-copGFP Vector not connected with CNP gene into viral protein coat) virus stock solution.

...

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Abstract

The invention provides a recombinant plasmid which contains a nucleotide sequence, such as SEQ ID NO.1. The invention also provides a medicament composition which contains the recombinant plasmid. The invention also provides a preparation method and application of the medicament composition. An approach of gene treatment is adopted, namely a natriuretic peptide gene lentivirus expression vector is guided into a rat vitreous cavity to observe that a target gene has high expression, positive effect of obviously reducing the intraocular pressure of a live animal, less adverse reaction and low cost, thereby creating a new idea for glaucoma treatment.

Description

technical field [0001] The invention relates to a drug containing natriuretic peptide gene, specifically a gene drug capable of lowering intraocular pressure and treating glaucoma, and a preparation method thereof. Background technique [0002] Glaucoma is an optic neuropathy that is one of the leading causes of blindness. According to statistics, about 7 million people around the world are blinded by this disease every year. Although the pathogenesis of glaucoma is not yet fully understood, elevated intraocular pressure (IOP) is one of the major risk factors. Therefore, lowering intraocular pressure has always been the main treatment for glaucoma. Timely and effective reduction of intraocular pressure can slow down the course of the disease and reduce the damage to visual function. The currently clinically used ocular hypotensive drugs are limited to small-molecule compounds that can affect enzyme activity, such as carbonic anhydrase inhibitors, or small-molecule drugs th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/861C12N15/16A61K48/00A61K9/08A61P27/06
Inventor 刘旭阳曹桂群闫乃红马嘉
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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