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Adeno-associated virus vector preparation and medicinal composition containing it and its use

A virus carrier and composition technology, applied in the field of adeno-associated virus carrier preparations, can solve the problems of virus stability and infectivity decline, virus particle aggregation, precipitation, etc.

Inactive Publication Date: 2007-12-26
NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the number of virus particles of adeno-associated virus vector preparations (AAV) that have entered clinical trials is usually 2-3 orders of magnitude higher than that of adeno-associated virus vector preparations (Adv), that is, the number of adeno-associated virus vectors (AAV) per unit volume is much higher than that of adeno-associated virus vector preparations (Adv). It is higher than the adenovirus vector (Adv), which leads to the aggregation and precipitation of virus particles during the preparation and concentration of high-concentration adeno-associated virus vector preparations (AAV), which leads to the decline of virus stability and infectivity. Therefore, how to improve It is of great significance to study the stability of high-concentration adeno-associated virus vector (AAV) preparations and to study its further application in the field of gene therapy

Method used

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  • Adeno-associated virus vector preparation and medicinal composition containing it and its use
  • Adeno-associated virus vector preparation and medicinal composition containing it and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1 vector construction

[0066] Firstly, the total RNA of human liver tissue was extracted, and human hNGFβcDNA was amplified by RT-PCR (mRNA purification and RT-PCR kit were purchased from Promega, USA). -3', the length of the amplified fragment is 1067bp, and it is connected with the plasmid pUC18 to obtain the recombinant cloning plasmid pUC18 / hNGFβ. DNA sequence determination confirms that its nucleotide sequence is consistent with the literature records. The hNGFβ gene was ligated with rAAV-2 universal vector plasmid pSNAV-1 (WuZJ, Wu XB, HOU YD, Chin J viral 2000; 16:1-6.) to obtain the recombinant vector plasmid pSNAV-1 / hNGFβ, and transfected with the plasmid BHK-21 cells were screened under the pressure of G418 (neomycin) to obtain the vector cell line SN carrying the vector plasmid pSNAV-1 / hNGFβ. With the participation of the rep and cap genes), the virus was packaged and finally purified (Wu XB, Dong XY, Wu ZJ, et al., Chin Sci Bull 2000; 45:2071-20...

Embodiment 2

[0070] Example 2 In vitro expression of rAAV-2 / hNGFβ and determination of biological activity

[0071] 1. In vitro expression

[0072] The ability of rAAV-2 / hNGFβ to transduce cells was determined by measuring the content and biological activity of hNGFβ in the cell supernatant after infecting BHK-21 cells. With 1640 medium (RPMI 1640 medium purchased from American GIBCO Company) containing 10% calf serum (the calf serum is a product of HyClone Company), at 37 ° C, 5% CO 2 BHK-21 cells were cultured under these conditions. BHK-21 cells by 4×10 5 Each well was connected to a six-well plate, and after 8 hours, the MOI was 2.5×10 4 with 1.0×10 6 Add recombinant rAAV-2 / hNGFβ virions. Cells were washed twice with fresh serum-free 1640 before adding virus particles. And adjust the ratio of virus to cells (MOI) with serum-free 1640, then make up the final volume to 1ml with serum-free 1640, add to the 6-well plate cells that have been washed and prepared; and at 37 ° C, 5% CO ...

Embodiment 3

[0076] Example 3 Factors Affecting Mannitol-Induced BBB Opening

[0077] 1. The influence of mannitol concentration: the suitable concentration of mannitol is 22-28% (w / v)

[0078] Wistar rats were used as experimental animals, with a body weight of 250g±20g, and both ♀♂ were used. (n=20, 5 in each group) 4 groups of rats were treated at 37°C with 0.12ml·s -1 , 2 minutes after inputting 20%, 22%, 25%, and 28% mannitol at a speed of 30 seconds, the gray levels of the left blue-stained areas of the EB group were 181.5±1.41, 115.5±1.32, and 81.1±1.21, respectively. and 80.25±1.46, and the percentages (%) of blue-stained areas of the left brain were 32.42±1.12, 49.85±1.70, 92.10±1.50, and 94.31±1.32, respectively. However, rats in the 28% mannitol group had bleeding points, and 28% mannitol was more likely to precipitate at room temperature, indicating that at 37°C, 0.12ml·s -1 , 30s given the above different concentrations of mannitol, it can be seen that the opening of the BB...

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Abstract

The present invention relates to a kind of adeno-associated virus vector preparation with gene carrying neurotrophic factor, and the preparation contains mannitol in 3-7 wt% to raise the stability of the preparation. The present invention also relates to medicine composition containing the said preparation and high permeability solution, which is selected from mannitol, arabinase, urea, fructose, lactamine and glycerin. The preparation and the medicine composition are used in preventing and treating central neurodegeneration, such as Alzheimer disease, Parkinson'e disease, etc. as well as in treating cerebral ischemic injury and cerebral traumatic injury, promoting the recovery of cerebral function after surgical operation, and preventing mental retardation of infant caused by anoxia damage.

Description

technical field [0001] The invention relates to an adeno-associated virus (adeno-associated virus, AAV) vector preparation carrying a neurotrophic factor gene, and the preparation contains 3-7% mannitol. In addition, it also relates to a pharmaceutical composition containing the preparation and a hypertonic solution, and the hypertonic solution induces the opening of the blood-brain barrier to allow the viral vector to enter the brain for infection and expression. The present invention also relates to the use of the preparation and / or pharmaceutical composition in the prevention and treatment of central neuron degenerative diseases such as Alzheimer's disease and Parkinson's disease, and the preparation and / or pharmaceutical composition It can also be used to reduce neuronal death and promote brain function recovery after cerebral ischemic injury, traumatic brain injury, and traumatic brain surgery; it can also be used to prevent brain-injured mental retardation or brain dyspl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K38/18A61P25/00A61P43/00
Inventor 王军志吴勇杰高凯时小燕吴小兵饶春明
Owner NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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