Cytosine arabinoside and gland correlated virus composite preparations and uses thereof

A technology of cytarabine and compound preparation, applied in the field of biomedicine, can solve problems such as inability to replicate independently, and achieve the effects of improving the effect of gene therapy, increasing the positive rate and improving the effect

Inactive Publication Date: 2011-04-06
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although 80% of individuals in the population are reported to be seropositive for AAV, no clear association has been seen with either disease
Second, AAV is a defective virus that cannot replicate independently
No one has attempted to use Ara in gene therapy, especially for degenerative and genetic diseases, to improve AAV-mediated transduction efficiency, gene expression levels, and duration

Method used

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  • Cytosine arabinoside and gland correlated virus composite preparations and uses thereof
  • Cytosine arabinoside and gland correlated virus composite preparations and uses thereof
  • Cytosine arabinoside and gland correlated virus composite preparations and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Cytarabine Accelerates and Improves the Transduction Efficiency and Gene Expression Level of Retinal Cells by rAAV-GFP

[0065] 2×10 per hole 5 RPE (APRE-19, ATCC) cells were seeded in 6-well cell culture plates, and after 24 hours, the cells adhered to the wall, and 1×10 9 wxya 2 - GFP alone or in combination 0.0028 μg / ml (0.0028 μg), 0.014 μg / ml (0.014 μg), 0.028 μg / ml (0.028 μg), 0.14 μg / ml (0.14 μg), 0.28 μg / ml (0.28 μg), Cytarabine at concentrations of 0.56 μg / ml (0.56 μg), 1.4 μg / ml (1.4 μg), 2.8 μg / ml (2.8 μg), and 4.2 μg / ml (4.2 μg) was added to the cultured cells. Then observe and record the brightness of GFP fluorescence, cell morphology and lesion state of the infected cells with a fluorescent microscope every day. After 7 days, trypsinization was performed, the number of GFP-positive cells and the fluorescence intensity of GFP were detected by flow cytometry, and the expression level of GFP was detected by Western blot.

[0066] The above test...

Embodiment 2

[0068] Cytarabine Accelerates and Improves the Transduction Efficiency and Gene Expression Level of rAAV-GFP to Various Tumor Cells

[0069] 1×10 per well 5 Tumor cells (7721, 7901, NCIH446, NCIH460, A549) were seeded in 24-well cell culture plates, and after 24 hours, the cells adhered to the wall, and 1×10 9 wxya 2 - GFP was added to the cultured cells alone or simultaneously with cytarabine at a final concentration of 0.56 μg / ml (0.56 μg), 1.4 μg / ml (1.4 μg), 2.8 μg / ml (2.8 μg). Fluorescent microscope observation 24 hours after infection, 24 hours after infection, rAAV alone 2 -There was no obvious fluorescence in the five kinds of tumor cells infected with GFP; in the tumor cells combined with different concentrations of cytarabine, some cells in SMMC7721, SGC7901 and NCIH446 showed GFP fluorescence, the cell shape was good, and no rounding of cells was seen . After 48 hours, rAAV alone 2-GFP-infected SGC7901, NCIH446, and SMMC7721 cells showed a small amount of cells...

Embodiment 3

[0072] In vivo experiments have confirmed that cytarabine can significantly improve the transduction efficiency and gene expression level of adeno-associated virus to living retinal cells, and improve the therapeutic effect

[0073] Cytarabine increases the expression level of AAV-mediated genes in living retinal cells

[0074] Select 8-week-old Balb / c mice and inject AAV into the subretinal space after anesthesia 2 -Luc and AAV 2 -Luc+Ara-c each 2ul. Specific dose: left eye AAV 2 -GFP 2×10 9 (2ul) viral particles, right eye AAV 2 -GFP2×10 9 A virus particle combined with Ara-c 0.001μg. After 24, 48, 72 hours and 6 days, 9 days, 21 days, and 28 days of subretinal injection, the mice were anesthetized, and the expression of reporter gene Luc in the mouse retina was observed by small animal live imaging. The results showed that AAV alone 2 -When Luc was injected subretinally, the expression of Luc in the retina was not visible until the 6th day; however, when combined wi...

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Abstract

The invention belongs to the biologic medicine filed and relates to a novel gene therapy compound preparation and the use and application method thereof. When adeno-associated virus is used as gene delivery carrier, a certain dose of cytarabine is combined in use, and obviously increases transfer efficiency of normal cells and tumor cells by the adeno-associated virus, advances expression time offoreign gene mediated by adeno-associated virus, obviously increases positive rate and expression level, thereby improving the gene therapy effect employing adeno-associated virus as carrier, particularly the application in gene therapy for diseases, such as ocular fundus disease, genetic disease and tumor. In tumor gene therapy, the invention has functions of inhibiting tumor cell growth, inducing tumor cell apoptosis, and obviously increases the transfer efficiency and expression level of the gene mediated by adeno-associated virus.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a new compound preparation for gene therapy and its application and application method. Background technique [0002] Adeno-associated virus, referred to as AAV, belongs to the Parvoviridae family. It is a linear single-stranded DNA virus with an icosahedral capsid protein and a diameter of 20nm. It is the smallest animal virus. It can be divided into many different serotypes, among which AAV2 is the most commonly used one in gene therapy. AAV virus has the following characteristics: First, AAV has no obvious pathogenicity. It has been reported that although 80% of the individuals in the population were seropositive for AAV, there was no significant correlation between it and any disease. Second, AAV is a defective virus that cannot replicate independently. When there is no helper virus (such as adenovirus, herpes virus, etc.) infection, AAV is in a "latent" state. Third, site-specifi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K39/235A61K31/7068A61K9/08A61P35/00A61P19/02A61P1/16
Inventor 黄倩张圣海吴继红吴小兵董小岩李川源
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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