CN1242426A

CN1242426A - HSV-1 virus carrier system, method for building-up and quick guiding-in therewith

Info

Publication number: CN1242426A
Application number: CN98114171A
Authority: CN
Inventors: 任常山; 张朝东; 隋承光; 陈红
Original assignee: China Medical University
Current assignee: China Medical University
Priority date: 1998-07-22
Filing date: 1998-07-22
Publication date: 2000-01-26

Abstract

The present invention relates to a HSV-1 virus carrier system and its constitution and quick introduction method. It is characterized by cloning the complementary DNA (cDNA) functional fragment of exogenous target gene NGF gene onto the enzymic incision site of incision enzyme BamH1 of virus carrier, tranfecting the constituted plasmid and packaging so as to constitute the invented HSV-1 virus carrier system. By utilizing the characteristics of that the virus possesses the ecotropic property for nerve, the olfactory nerve is selected and used as introduction channel of said virus carrier, then the obtained high-titer HSV-1 virus carrier 0.1 ml (concentration 16 to the power 6 ptu/ml) is injected from patient's nasal cavity, and said carrier can be quickly come into the pathologic changed position of Alzheimer's disease so as to attain the goal of gene therapy of the Alzheimer's disease.

Description

HSV-1 virus carrier system and structure and quick introduction method

The present invention relates to genetic engineering C12N 15/00, the gene transfer technique and the structure of virus vector mediation.

Along with improving and development of Community health's security system, aging of population phenomenon is increasingly significant in China and even the whole world, and patient's quantity of suffering from AlzheimerShi disease (senile dementia) also constantly increases thereupon.This be a kind of be the big brain degenerative diseases of main clinical manifestation with the chronic progressive external dementia, up to now, still do not have any effective treatment way.Because this disease causes the patient can't take care of oneself, cause white elephant for family and society.Therefore, the AlzheimerShi disease has become the human a great problem of puzzlement.

In recent years, the develop rapidly of Protocols in Molecular Biology and modern medical service technology provides possibility for the gene therapy of AlzheimerShi disease.Therefore the same with other the difficult disease and the gene therapy of incurable disease, the gene therapy research of AlzheimerShi disease has become the focus that present countries in the world are paid close attention to.Neuropathological Study proves, the main pathological change of AlzheimerShi disease is the neuronal cell at brain Medial Temporal Lobe position, with advancing age, sex change, minimizing and atrophy constantly take place, and cause that therefrom these neuronal functions reduce or forfeiture, because these neurones are relevant with memory function, therefore, above-mentioned neuronic sex change and afunction thereof will cause dementia.These Neuropathological Study are the result also tell us, if carry out the gene therapy of AlzheimerShi disease, its key is how to improve and recover the sex change function of neurons.At present in the world, especially the gene therapy of U.S. AlzheimerShi disease, researchdevelopment is very fast, and set up the large-scale research (Geron of enterprise that is intended to the gene therapy technology practicability of AlzheimerShi disease, California, 1993), specialize in the molecular biology research of cell senescence, at the gene therapy of amyloidosis with somatomedin is the gene therapy research of goal gene, and what have also carries out the clinical experiment stage.But the treatment plan that these gene therapies are adopted all is to do operation of opening cranium to patient earlier, and underwent operative is opened carrier cell is directly injected at the cranium position in brain somatic gene therapy scheme then.This gene approach that imports does not still have wound property, and is difficult to accurate location, for the patient of AlzheimerShi disease, all must implement earlier operation of opening cranium, this point is to be difficult to accepting, and will keep result of treatment, need open cranium once more to the encephalic repeat administration, this just more difficult enforcement.So this unique treatment plan also is difficult to apply.Therefore, explore by peripheral nerve and adopt non-surgical way, to the new way of cortical area importing goal gene, significant for the gene therapy of implementing neuronal disease in the brain.

The purpose of this invention is to provide a kind ofly need not perform the operation, the gene therapy material of patient's no pain, its construction process and administering mode rapidly and efficiently, the structure and the quick introduction method of a kind of hsv-I type (HVS-1 virus) carrier system.The expression formula of HVS-1 virus carrier system:

It contains defective type hsv HSV ₁Promotor IE, external source goal gene nerve growth factor NGF, SV40 and HSV ₁Packing point as, the function fragment cDNA that it is characterized in that goal gene NGF forms the HSV-1 virus carrier system on the BamH1 restriction enzyme site.

Character: host's neurocyte is had high degree of specificity, particularly the neuronal cell after the mitotic division is had highly close preferendum.

The construction process of HVS-1 virus carrier system: the gene recombination technology that comprises the external source goal gene NGF that utilizes Bio-engineering Products, hsv-I type, restriction endonuclease, ligase enzyme, applied molecular biology, it is characterized in that complementation (cDNA) function fragment of exogenous target gene NGF (nerve growth factor) is cloned on the restriction enzyme site of restriction endonuclease BamH1 of virus vector, be built into the HSV-1 virus carrier system.The plasmid that builds is through after transforming, increasing, and with N,O-Diacetylmuramidase SDS cracking bacterium, phenol chloroform extraction DNA, ethanol sedimentation, TE dissolving DNA are by csCl-ethidium bromide gradient balance Gao Xinfa, 45000rpm18 hour, purifying DNA plasmid.The deletion mutation packing technique of applied molecular biology carries out transfection and the HSV-1 vector virus is packed the particle of above-mentioned structure.With HSV-1 17tsk helper virus vero cells infection, liposome DNA mixture is added to mixing in the serum-free medium gradually, cultivated three days for 31 ℃, obtain to have the vector virus of outer virionic membrane, forming the measuring virus titer with plaque is 10 ⁷Pfu/ml.

The HSV-1 virus carrier system is used for clinical quick introduction method, utilizes its height parent preferendum to neuronal cell, selects olifactory nerve as introduction channel, and with 0.1mlHSV-1, virus titer is 10 ⁶Pfu/ml, inject from nasal cavity, the diseased region (Medial Temporal Lobe positions such as the hippocampus of maincenter, amygdala) that this hsv that this is built into-I type carrier system just can enter the AlzheimerShi disease rapidly reduces function by external source goal gene product nerve growth factor NGF or the pathology neurone of forfeiture plays and composes the effect of living, thereby reaches the purpose to the treatment of AlzheimerShi ospc gene.

(Herpes simplex virus typeI HSV-1), has the advantages that to have a liking for neurocyte to herpes simplex virus I-type, can infect the neurocyte after the mitotic division, and move between neural axon and cynapse, long-term latent infection in neurone.Viral DNA is present in the nucleus with the double chain form of closed hoop, does not influence the function of cell.So characteristics according to HSV-1, the HSV-1 system of application build carries ngf gene and passes through olfactory neural pathway, being directed into Alzheimer nervous center diseased region fast and obtaining the effect of high reactivity, long-term high expression level, is determinative and the key problem in technology that successfully carries out therapy of tumor.The structure of virus carrier system of the present invention and quick introduction method at home and abroad end appear in the newspapers and lead, and are world initiative.

As everyone knows, the treatment of implementation efficient gene, one of its key also whether can gene therapy in the target cell that imports retroviral vector compare, it has obvious characteristic: (1) novel vector has high degree of specificity to host cell (neuronal cell), particularly the neuronal cell after the mitotic division is had highly close preferendum.(2) novel carriers has 100% importing efficient.(3) physiological characteristic of olifactory nerve just is connected with the temporal lobe position of brain inboard, utilize this virus vector, by the natural infection approach, after goal gene imported from specific peripheral nerve, can drive in the wrong direction for arriving is attached thereto in the maincenter cortical neuron that is, and long-term expression foreign gene therein.Therefore select this passage administration, import the location definitely, be easy to bring into play curative effect.

Claims

1, a kind of HSV-1 virus carrier system is used for the gene therapy of AlzheimerShi disease, it is characterized in that: hsv-I type (Herpes Simplex Virus Type I, HSV-1) carrier system expression formula:

It contains promotor IE, external source goal gene NGF, SV40, PolyA site and the HSV1 packing point as of defective type hsv HSV-1, the function fragment cDNA that it is characterized in that goal gene NGF forms the HSV-1 virus carrier system on the BamH1 restriction enzyme site.

2, a kind of construction process of HSV-1 virus carrier system, the gene recombination technology and the deletion mutation packing technique that comprise the goal gene ngf gene that utilizes Bio-engineering Products, defective virus, restriction endonuclease, ligase enzyme, applied molecular biology, it is characterized in that cDNA function fragment with exogenous target gene nerve growth factor gene is cloned on the restriction enzyme site of restriction endonuclease BamH1 of defective virus, is built into the HSV-1 virus carrier system; The plasmid that builds is through after transforming, increasing, and with N,O-Diacetylmuramidase SDS cracking bacterium, phenol chloroform extraction DNA, ethanol sedimentation, TE dissolving DNA are by csCl-ethidium bromide gradient equilibrium centrifugation method, 45000rpm18 hour, purifying DNA plasmid.

3, the construction process of HSV-1 virus carrier system according to claim 2, it is characterized in that the deletion mutation packing technique that adopted carries out transfection to the plasmid that makes up and vector virus is packed: with the HSV-1 17tsk helper virus vero cells infection of 1pfu/ml, absorption is 1 hour in 37 ℃, discard viral liquid, give a baby a bath on the third day after its birth time with the RPMI-1640 of serum-free, add the 2ml serum-free medium, preprepared liposome DNA mixture is added to mixing in the nutrient solution gradually, put in 31 ℃ and cultivated three days, the virus of results has the vector virus of outer virionic membrane for packing; Forming the measuring virus titer with plaque is 10 ⁷Pfu/ml.

4, a kind of HSV-1 virus carrier system of structure that makes is used for clinical quick introduction method, it is characterized in that utilizing virus that neurocyte is had the characteristic of close preferendum, selects the introduction channel of olifactory nerve as this virus vector; With the high titre HSV-1 virus vector 0.1ml that obtains, titre 10 ⁶Pfu/Ml, inject from patient's nasal cavity, this carrier can enter the diseased region of AlzheimerShi disease rapidly, by external source goal gene product NGF the effect that tax is lived is played in the reduction or the forfeiture of pathology neuronal function, thereby is reached the purpose to the treatment of AlzheimerShi ospc gene.