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Remedy for disease associated with apoptotic degeneration in ocular cell tissue with the use of SIV-PEDF vector

一种组织细胞、药物的技术，应用在治疗青光眼领域，能够解决尚无青光眼治疗、治疗方法不合适等问题

Active Publication Date: 2008-05-14
DNAVEC CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Considering that glaucoma is a progressive disease, it is not appropriate to treat glaucoma with administration methods that only expect temporary effects
On the other hand, retroviral vectors are generally stably integrated into the chromosomes of dividing cells, enabling long-term gene expression. So far, there is no research example of glaucoma treatment using retroviral vectors inserted with PEDF.

Method used

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  • Remedy for disease associated with apoptotic degeneration in ocular cell tissue with the use of SIV-PEDF vector
  • Remedy for disease associated with apoptotic degeneration in ocular cell tissue with the use of SIV-PEDF vector
  • Remedy for disease associated with apoptotic degeneration in ocular cell tissue with the use of SIV-PEDF vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Construction of VSV-G pseudotyped SIV vector

[0083] Vectors were constructed using the figure 1 The 4 plasmids (gene transfer vector, packaging vector, rev expression vector, VSV-G expression vector) shown in . Regarding the three types of gene transfer vectors, packaging vectors, and rev expression vectors, they were prepared by transforming the original vector plasmid (PCT / JP00 / 03955). As for the VSV-G expression vector, an unmodified original vector was used.

[0084] For plasmid preparation, various commercially available kits were used. Products from New England Biolabs were used as restriction enzymes, and QIAGEN kits (QIAquick PCR purification kit, QIAquick NucleotideRemoval kit, QIAquick Gel extraction kit, Plasmid Maxi kit) were used for plasmid DNA extraction, purification, and recovery. PCR uses TaKaRa's EX Taq enzyme, and the primers used are synthesized by SIGMA GENOSYS JAPAN. Dephosphorylation of DNA termini uses TaKaRa's alkaline phosphata...

Embodiment 2

[0102] Example 2 Functional evaluation of the SIV vector carrying cPPT, WPRE

[0103] In order to investigate the introduction effect of cPPT and WPRE, in addition to the vectors carrying both cPPT and WPRE, a vector carrying cPPT alone and a vector carrying WPRE alone were also produced, and compared with the original control. All gene transfer vectors used carried EGFP. The packaging carrier uses the original type (serial number: 27).

[0104] 2-1. Preparation of SIV vector

[0105] The cell line 293T cells from human fetal kidney cells were divided into about 1×10 per 15cm plastic culture dish 7 Inoculate (70-80% density on the next day) and culture in 20 ml of D-MEM medium (Gibco BRL) containing 10% fetal bovine serum for 24 hours. After 24 hours of cultivation, the medium was replaced with 10 ml of OPTI-MEM medium (Gibco BRL) and used as transfected cells for later use.

[0106] Dissolve 6 μg of gene transfer vector, 3 μg of packaging vector, and 1 μg of VSV-G express...

Embodiment 3

[0118] Example 3 Mass preparation and concentration of SIV vectors carrying therapeutic genes

[0119] Such as figure 1 The SIV vector was prepared as follows based on the four plasmids shown in the modified gene transfer vector, packaging vector, rev expression vector, and VSV-G expression vector. The vector carrying the PEDF therapeutic gene is produced in units of 20 15cm dishes.

[0120] According to each 15cm plastic Petri dish about 1×10 7 The 293T cells were inoculated (at a density of 70-80% on the next day), and cultured in 20 ml of D-MEM medium containing 10% fetal bovine serum for 24 hours. After 24 hours of cultivation, the medium was replaced with 10 ml of OPTI-MEM medium for transfection. Dissolve 10 μg of gene transfer vector, 5 μg of packaging vector, 2 μg of rev expression vector, and 2 μg of VSV-G expression vector in 1.5 ml of OPTI-MEM medium for each culture dish, and then add 40 ul of PLUS Reagent reagent (Invitrogen) for stirring , at room temperature...

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Abstract

The invention provides a novel method of treating a diseases associated with apoptotic degeneration in an ocular cell tissue by effectively administering pigment epithelium-derived factor (PEDF). Attentions are focused on PEDF as a novel means of preventing neural ganglion death which is the final pathological stage in glaucoma. Further, attentions are paid to an SIV vector as an effective method of delivering PEDF and an SIV-PEDF vector is constructed. When the SIV-PEDF vector is subretinally administered to an ischemic reperfusion model and an NMDA-induced model, it is observed that neural ganglion death is remarkably inhibited. Thus, it is found out that the SIV-PEDF vector is efficacious as a drug for treating a diseases associated with apoptotic degeneration in an ocular cell tissue such as glaucoma.

Description

technical field [0001] The invention relates to a method for treating glaucoma with a lentiviral vector carrying neurotrophic factors. technical background [0002] Glaucoma refers to a disease characterized by at least one of the characteristic changes in the optic nerve head and visual field. Usually, the optic nerve damage can be improved or further deterioration can be prevented by sufficiently reducing intraocular pressure. Regarding glaucoma, if it cannot get proper treatment, it will often lead to blindness. The blindness caused by glaucoma accounts for the second reason of acquired blindness in our country. [0003] Glaucoma can be divided into primary glaucoma (primary glaucoma), secondary glaucoma, and congenital glaucoma. Primary mixed glaucoma. In a broad sense, primary open-angle glaucoma includes primary open-angle glaucoma (narrow sense) and normal tension glaucoma. Normal tension glaucoma, regardless of intraocular pressure within the normal range (below 21...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K38/00A61P27/02C12N7/00C12N15/09C12R1/93A61K35/76A61K38/22A61P27/06A61P43/00C12N7/01C12N15/86
CPCC12N2810/6081C12N2740/15043C07K14/475A61K48/00A61K48/005C12N15/86C12N2740/15045A61P27/02A61P27/06A61P43/00A61K38/00
Inventor 宫崎胜德米满吉和池田康博居石克夫田畑寿晃饭田章博上田泰次长谷川护
Owner DNAVEC CORP
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