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Bone generation by gene therapy

A technology for osteoblasts and chondrocytes, applied in gene therapy, genetic engineering, osteoblasts, etc., can solve problems such as short duration and large amounts of recombinant proteins

Inactive Publication Date: 2013-07-03
TISSUEGENE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A major disadvantage of these approaches is the large amount of recombinant protein required to achieve therapeutic effect due to the short duration of action of the therapeutic protein in vivo

Method used

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  • Bone generation by gene therapy
  • Bone generation by gene therapy
  • Bone generation by gene therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Embodiment 1—Experimental steps of bone regeneration

[0139] The human BMP2 gene was cloned by PCR (polymerase chain reaction) using human fetal brain cDNA and two primers. The 5' primer was 5'-TCCCAGCGTGAAAAGAGAGACTGC-3' (SEQ ID NO: 1), and the 3' primer was 5'-TTTTGCTGTACTAGCGACACCCACAACC-3' (SEQ ID NO: 2). After utilizing the GC-rich PCR system (Roche), the TOPO TA cloning kit (Invitrogen) was used to clone into the pCRII-TOPO vector ( figure 1 A). For cloning into retroviral vectors, pCRIIbmp2DNA was cut with Sal I and the human BMP2 cDNA insert was ligated into pMTMLV with Sal I and Not I overhangs ( figure 1 B). The packaging cell line GP-293 cells (5x10 5 cells / p60 dish). GP-293 cells were transfected with pMTMLV or pMT-BMP2 using Fugene (Roche). 48 hours after transfection, neomycin was added to the medium for selection of neomycin-resistant cells. Screening lasted 10 days. 293MT and 293MTBMP2 cells (5x10 5 cells / p60 dish) was used for transfection of ...

Embodiment 2

[0140] Example 2—Injection of NIH3T3-BMP-2 cells into rabbits

[0141] New Zealand white rabbits weighing 2.0-2.5 kg were selected for animal studies. The tibia was exposed and a defect (2 cm long and 0.5 cm deep) was created with orthopedic instruments. After suturing, control NIH3T3-neo or NIH3T3-BMP-2 cells (2ml2x10 6 cells / ml) were injected into the defect site. Eight weeks after injection of the cells, radiological analysis and histological examination were performed.

Embodiment 3

[0142] Example 3 - weekly X-ray examination

[0143] New Zealand white rabbits weighing 2.0-2.5 kg were selected for animal studies. The tibia was exposed and a defect (2 cm long and 0.5 cm deep) was created with orthopedic instruments. After suturing, NIH3T3-BMP-2 cells (2ml2x10 6 cells / ml) were injected into the tibial defect site. Radiographic analysis was then performed at 1, 2, 3, 4, 5, 6 and 7 weeks after cell injection. Samples were harvested 7 weeks after injection and plotted. Histological examination was performed after harvest.

[0144] figure 2 A-2F show bone regeneration with NIH3T3-BMP-2 fibroblasts. figure 1 A and 1B show images of leg bones 8 weeks after injection of control NIH3T3 fibroblasts (A) and NIH3T3-BMP-2 cells (B). figure 2 C-2F shows radiographs of control (C&D) and experimental (E&F) leg bones before sacrifice of animals. Bone defects treated with cells expressing the BMP-2 protein healed 8 weeks after injection, whereas no bone regenerati...

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Abstract

The application discloses a method for making bone at a bone defect site for a person suffering from low bone mass which includes inserting a gene encoding a protein having bone regenerating function into a connective tissue cell operably linked to a promoter, and transplanting the mammalian cell into the bone defect site, and allowing the bone defect site to make the bone.

Description

[0001] The patent application of the present invention is an invention with the international application number PCT / US03 / 09718, the international application date is March 28, 2003, the application number entering the Chinese national phase is 03812002.X, and the name is "Osteogenesis through Gene Therapy" A divisional application of a patent application. technical field [0002] The present invention relates to a method of introducing at least one gene encoding a member of the transforming growth factor beta superfamily into at least one mammalian connective tissue cell for generating or regenerating bone, especially for repairing osteoporotic fractures or fused mammals The spine of an animal host. Background technique [0003] Homeostasis of living bone tissue is a dynamic process regulated by regulatory signals, such as hormones, growth and differentiation factors. Growth factors known to stimulate bone cell proliferation are bone morphogenic protein (BMP), transforming...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K38/18A61P19/08A61P19/10C07K14/495C07K14/51
CPCA61K38/1841A61K38/1875A61K48/00C07K14/495C07K14/51C12N2799/027A61L2430/02A61P19/00A61P19/08A61P19/10C12N15/867
Inventor S·U·宋李泳锡李宽熙卢文钟H·裴
Owner TISSUEGENE INC
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