CN101338302B - Recombination herpes simplex virus for tumor gene therapy - Google Patents
Recombination herpes simplex virus for tumor gene therapy Download PDFInfo
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- CN101338302B CN101338302B CN2008100254337A CN200810025433A CN101338302B CN 101338302 B CN101338302 B CN 101338302B CN 2008100254337 A CN2008100254337 A CN 2008100254337A CN 200810025433 A CN200810025433 A CN 200810025433A CN 101338302 B CN101338302 B CN 101338302B
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Abstract
The invention relates to a gene treatment method which adopts a recombinant herpes simplex virus to treat knub, in particular to a defect ecombinant I type herpes simplex virus which lacks the repeated section (IR) which comprises the genes of ICP0, ICP4 and ICP34.5 in a fixed point way as well as the application thereof in treating the knub. The invention further relates to a drug compound whichcomprises the viruses. Compared with the existing recombinant herpes simplex virus, the recombinant virus of the invention has the characteristics of high stability and safety as well as good knub-restraining effect.
Description
Invention field
The present invention relates to a kind of employing recombinant herpes simplex virus and treat the genetic treatment of tumor method.Especially, relate to a kind of fixed point deletion one and comprise ICP0, the defective type reorganization I type herpes simplex virus of the repetition section (IR) of ICP4 and ICP34.5 gene, and the application in the treatment tumour.The invention further relates to the pharmaceutical composition that contains above-mentioned virus.
Technical background
Tumour is to influence human health even life-threatening common disease, frequently-occurring disease, tumor treatment is normally adopted operation, radiotherapy, chemotherapy or treatment by Chinese herbs scheme, but the curative effect of centering late tumor is not good enough.
From nineteen ninety French Anderson etc. adenine deaminase (ADA) gene has successfully been treated ADA defective disease, started the precedent of utilizing people's gene treatment disease, initial gene therapy only is used for the heredopathia (Anderson et al., Science, 1992) of genetic flaw.Along with the development of gene clone technology and the thinking of gene therapy are widened, people are applied to human body gene to tumor treatment again.
Along with going deep into of oncomolecularbiology research, find that tumorigenic molecular mechanism is extremely complicated, normally series jump, displacement, amplification and the overexpression because of proto-oncogene causes abnormal activation, or/and owing to sudden change, the disappearance of cancer suppressor gene causes due to the gene inactivation, thereby cause that the neoplasm of cell and apoptosis are out of control and carcinogenic.But present most of genetic treatment of tumor still is in initial stage, only has minority to enter the II/III clinical trial phase.
The subject matter that exists in the therapy of tumor is: the selected therapeutic genes of (1) oncotherapy is not often broken away from the gene therapy pattern of hereditary defect disease, therapeutic gene is single, it is unusual to be difficult to correct fully the several genes that tumorigenesis is relevant in the tumor tissue cell, so result of treatment is not ideal.(2) all there are some defectives in various degree in the carrier that is used for therapy of tumor at present: carry the plasmid vector of therapeutic gene, though easy to operate also very safe, transfection efficiency is low, and therapeutic gene can not obtain long-term stability and effectively expressing, and its curative effect is limited; Although the virus vector that carries therapeutic gene can overcome some defectives of plasmid vector, also there is weak link in various degree, early stage virus vector commonly used mostly is the adenovirus (AdV) and the retrovirus (RV) of replication defect type greatly.The problem of the maximum of this class carrier is that the diffustivity in tumour is very poor.Since the volume of virus and surface with electric charge, make it the ability extreme difference of physical diffusion in the tumour entity, if can not duplicate voluntarily in tumour, then can only rely on " bystander effect " to reach increases the effect of curative effect.Just because of above-mentioned reason, important breakthrough does not still take place in the clinical efficacy of genetic treatment of tumor at present, most of I clinical trial phase result only demonstrates lower clinical efficacy, need can synergy by treatment plan commonly used clinically such as combined radiotherapy, chemotherapy, operative treatment toward contact.
At the weak point of above-mentioned non-replicating virus, in recent years can the tumour internal specific duplicate and cause the tumour cell cracked to melt the knurl C-type virus C and become in the development trend aspect the oncotherapy of gene.The characteristics of melting the knurl C-type virus C be utilize virus vector in tumour cell optionally occurrence condition duplicate and the cracking tumour cell, the biology diffusion that can pass through again after virus discharges and constantly expansion melt the knurl effect.Therefore the copy choice oncolysis that splits the cancer virus carrier may seem more effective than the virus expression carrier that carries therapeutic gene in oncotherapy, has good prospects for application more.Virus only could massive duplication by the machine translator of cell and energy in sensitive cells, and some virus can be by the cracked cell and a large amount of release after assembling, and produces tangible cytopathic effect (CPE).Along with virusology and molecular biological further investigation and development, people have had more deep understanding to the molecular mechanism of tumorigenic molecule mechanism and virus replication and regulation and control, and then by the cancer virus that splits that is used for kill cancer cell has been set up in screening (as reovirus) and genetic modification (as adenovirus, simplexvirus) naturally, making it optionally to carry out conditionality in tumour cell duplicates, and then only special cracking is taken place tumour cell, but normal cell is not caused injury.
People I herpes simplex virus type (HSV-1) belongs to herpetoviridae, and herpes simplex virus belongs to, and is a kind of double-stranded DNA virus, eye, oral cavity and the skin of face mucous membrane of normal infected person, be a kind of therapy of tumor use always melt knurl C-type virus C carrier.
(this genom sequence NCBI sequence number is NC_001806 to the genomic dna of HSV-1, genome total length 152261bp) forms by covalently bound long segment (L) and short segment (S), two ends at each sections, contain inverted repeats, comprise terminal repeat (TR) and internal repeat (IR).Usually, in a single day HSV DNA enters in the nucleus, linear DNA just can be connected to form ring-type by the terminal repeat (TR) at two ends, viral genome in host cell nuclear is then expressed viral protein in the mode of cascade reaction, according to the difference of genetic expression sequential, the HSV gene be divided into extremely early stage (α), early stage (β) and late period (γ) gene.The α gene is infecting utmost point early transcription, its product is (as ICP4, ICP27) regulate β gene and some γ genetic transcriptions jointly, β albumen mainly participates in the viral nucleic acid metabolism, also be that virus replication is necessary simultaneously, the γ gene is divided into γ 1 again, γ 2, γ 1 gene (as γ 34.5) is infecting relative early expression, it is synthetic not rely on viral DNA, and γ 2 genes are only expressed late, the structural protein of main coding virus (Roizman B.Thefunction of herpes simplex virus genes:a primer for genetic engineering ofnovel vectors[J] .Proc Natl Acad Sci USA, 1996,93:11307-11312).
In the prior art, existing a plurality of research groups have made up the defective type herpes simplex virus vector that is used for oncotherapy on the basis of HSV-1.
Split cancer HSV-1 mutant strain (U.S. G207) (the R3616 mutant strain by HSV-1F is transformed) disappearance γ 34.5 genes (coding ICP34.5 albumen), and insert the LacZ marker gene in second BamHI site of ICP6 gene (coding viral nucleotide reductase enzyme), make the ICP6 gene that inactivation take place to insert.This virus virulence is extremely low, reproducible is not bred in normal cells such as human neure, therefore do not damage the human normal cell, but can in the glioblastoma cell that propagation is enlivened, breed, because of this oncocyte enlivening owing to propagation, the virus that can be this defective provides enough exogenous ribonucleotide reductases, remedied virus replication defective and can be in born of the same parents propagation in a large number, finally cause the cracking of glioblastoma cell, and discharge a large amount of viruses, and biological diffusion takes place in cell towards periphery, splits cancer effect (Toshihiro M, Samuel D R and constantly enlarge, Takahito Y, et al.Attenuatedmultimutated herpes simplex virus-1 for the treatment of malignantgliomas[J] .Nat Med, 1995,1:938-940).
Britain has also made up 1716 strains of HSV-1 γ 34.5 deletion mutantions that are different from U.S. G207, its the same present clinical experiment of carrying out with G207, the two difference of 1716 strains and G207 is, the G207 of the U.S. is transformed by the R3616 mutant strain of HSV-1F, the dual-gene disappearance that wherein contains ICP6 and γ 34.5 genes, and inserted the LacZ gene at the ICP6 section, and Britain 1716 is to be transformed by the HSV-1-17 strain, the 1716th, only cause γ 34.5 genetically deficients, ICP6 is disappearance not, does not also carry any foreign gene, and its toxicity is a little more than G207, the result who is used for the I clinical trial phase of malignant glioma shows that using dosage is up to 3 * 10
9During Pfu, still do not have obvious toxic and side effects and (Rampling R occurs, Cruickshank G, Papanastassiou V, et al.Toxicity evaluation of replication-competent herpes simplex virus (ICP 34.5null mutant 1716) in patients with recurrent malignant glioma[J] .GeneTher, 2000,7:859-866; With Markert J M, Medlock M D, Rabkin S D, et a1.Conditionally replicating herpes simplex virus mutant, G207 for thetreatment of malignant glioma:results of a phase I trial[J] .Gene Ther2000,7:867-874).
Also made up in China in recent years and both be different from G207 and also be different from 1716 Brainwel and split cancer defective virus (CN 1397642 A), be used for the treatment of the I clinical trial phase of human glioma.Preliminary experiment is the result show, Brainwel is the same with G207 and 1716 to be safe in to the human glioma treatment.Brainwel is transformed by the HSV-1F strain, it is positioned at two copies of γ 34.5 genes and all partly removes and inactivation, also partly removed and inserted the fusion gene of the inhibition tumor vessel formation of Endostatin and Angiostatin simultaneously at the ICP6 constant gene segment C, more be better than G207 at the BamHI point of contact.
But, the recombinant herpes simplex virus that is used for therapy of tumor for the above-mentioned first-generation that has entered the clinical experiment stage in the prior art is discovered, no matter be G207 or Brainwel strain or 1716 strain recombinant viruses, all there is following defective in it: owing to there is repetition section (the Internal Repeat of an about 15kb among the wild-type HSV-1, IR), comprise ICP0 in this repetition section, the duplicated gene sequence of ICP4 and ICP34.5 gene, thereby said gene is deposited and is all had two copies (referring to Fig. 1) in wild-type virus, this causes and the reorganization phenomenon might occur in virus replication, thereby the inconsistent situation of gene phenotype occurs.This reorganization phenomenon can have a strong impact on the quality stability of virus formulation and have potential security hidden danger. and in order to overcome the defective of above-mentioned recombinant herpes simplex virus of the prior art in therapy of tumor, the inventor has made up a kind of brand-new absence type recombinant herpes simplex virus carrier and has been used for genetic treatment of tumor.
Summary of the invention
In order to overcome the above-mentioned defective that the existing first-generation is used for the recombinant herpes simplex virus of gene therapy, the inventor has made up a kind of new herpes simplex virus carrier that is used for gene therapy, described carrier is that a fixed point disappearance one comprises ICP0, ICP4, defective type reorganization I type herpes simplex virus with the repetition section (IR) of ICP34.5 gene, have safe, Stability Analysis of Structures and oncolytic good effectiveness, can improve present herpes simplex virus gene therapy process safe problem greatly.
Hereinafter, the inventor is constructed new herpes simplex virus carrier called after HEPAWEL, and it is described in further detail.
The viral genome structure of HEPAWEL:
HEPAWEL virus be with I herpes simplex virus type (HSV-1) F strain be framework construction melt knurl C-type virus C carrier.This viral genetic characteristics is to repeat section (Internal Repeat, IR) DNA of about 15KB in having removed in the viral genome.This section includes the ICP0 of HSV-1 virus, and ICP4 and ICP34.5 gene are removed this section, makes recombinant virus HEPAWEL only include each copy of above-mentioned three genes (Fig. 1 is the gene structure figure of recombinant virus HEPAWEL).Among the figure in the genome of wild-type HSV-1 a-b and A-B and c and C be two reverse iterons, and U
LAnd U
SThen be long non-repeated segments and short non-repeated segments, comprise the coding region of other genes of HSV-1 virus respectively.
The viral biology characteristic of HEPAWEL:
Virus genomic fundamental research shows to HSV based on prior art, and the IR fragment is to (Poffenberger, 1983 of duplicating not necessarily of HSV-1 virus; Meignier, 1988).The genome of removing the recombinant virus behind this section is shorter than the summary of wild-type virus.Particularly, in wild virus, because the existence of the tumor-necrosis factor glycoproteins of a-b and A-B and c and C, occurring inevitably recombinating and causing progeny virus when duplicating is to include the U with different directions
LAnd U
SThe mixing of the virus of the gene phenotype of section.After the tumor-necrosis factor glycoproteins of having removed IR, reorganization no longer appears in virus when duplicating, present stable genome structure.The genome structure of aforementioned stable helps to improve the security of HEPAWEL in therapy of tumor.
The characteristics of HEPAWEL of the present invention are:
At first the most important biological property of HEPAWEL is 3 entrained main utmost point early gene ICP0 of wild-type virus, ICP4 and ICP34.5 respectively have been removed a copy in HEPAWEL, thereby reduced viral toxicity significantly, thereby it is safer to be applied to the human body gene treatment.
Secondly, the recombinant virus of IR disappearance can be used for tumor treatment as melting tumor virus, shown in specific embodiment result subsequently, no matter still is the external good tumor killing effect that all has in vivo.Also preserve the ICP6 gene of wild-type after the IR genetically deficient of HEPAWEL, melt the knurl effect, make the efficient of virus improve greatly and do not influence security thereby improved.
Once more, remove the segmental recombinant virus structure of IR owing to removed the interior repetition section of genome, so structure is more stable.After the mouse intracranial inoculation, get back to cell in vitro and cultivate, still keep original genome structure after so going down to posterity for 9 times repeatedly, proved its virus genomic high stability.
Once more, IR disappearance recombinant virus is used for gene therapy and is better than being used at present clinical herpes simplex virus carrier.IR disappearance recombinant virus of the present invention is to belong to the herpes simplex virus that the s-generation is used for therapy of tumor.Be used for first-generation oncotherapy, that all entered the clinical experiment stage that is used for the malignant glioma treatment at present the earliest clinically and for example melt the knurl herpes simplex virus, the G207 of the U.S., 1716 of Britain, its common feature is two copies all having removed viral ICP34.5 gene.IR of the present invention disappearance virus (HEPAWEL) is melted tumor virus with the single blister of the above-mentioned first-generation and is compared, and maximum difference has been only to remove a copy of ICP34.5 gene.Compare with first-generation virus such as G207, IR disappearance virus of the present invention (HEPAWEL) is also preserved the ICP6 gene of wild-type.Remove ICP34.5 and ICP6 gene fully and make the toxicity of HSV virus reduce greatly, but its replication of while also is lower than virus (McAuliffe, 2000 of wild-type significantly; Bennett, 2002).Thereby can infer in theory, HEPAWEL of the present invention compares with first-generation virus to have and better melts the knurl effect.
At last, owing to the HEPAWEL recombinant viral vector does not adopt tk gene GFP (green fluorescent protein) to carry out selected marker when the recombination to construct, the tk gene that therefore makes up back HEPAWEL viral genome itself does not change.And in the adorned recombinant virus of those genomes tk gene, because behind the tk genetic modification, low-level tk expresses and can influence this virus susceptibility .Acyclovir and the Ganciclovir of gancyclovir (GCV) and acyclovir (ACV) put into practice for many years clinically, can control herpesvirus infection effectively, in case thereby clinical gene therapy is out of control, said medicine can effectively suppress viral growth, thereby interrupt infecting, for the individuality that carries out gene therapy provides further safety precaution.Therefore when clinical treatment, control the diffusion of virus if desired and when using above-mentioned antiviral, the safety and effectiveness of HEPAWEL will improve greatly.Therefore from the safety perspective of herpes simplex virus, HEPAWEL is more responsive to GCV, thereby security is higher.
Particularly, the present invention relates to the recombinant herpes simplex virus of a kind of called after HEPAWEL, it is characterized in that its specificity has lacked the sequence of virus genomic the 117071st to the 131534th base pair of wild-type HSV-1, thereby make the ICP0 that includes only a copy in the recombinant virus, ICP4 and ICP34.5 gene.
The invention still further relates to a kind of method that makes up recombinant herpes simplex virus, it is characterized in that, section is repeated in the inside that specificity has lacked in the wild-type HSV-1 viral genome, thereby makes the ICP0 that includes only a copy in the recombinant virus, ICP4 and ICP34.5 gene.
The invention still further relates to a kind of method of structure recombinant herpes simplex virus as indicated above, it is characterized in that:
At first, with 2554bp fragment between the EcorRI of SalI to the 117071 base pairs of the 114517th base pair of wild-type HSV-1 virus, and the 1986bp fragment is cloned into respectively in the SstI of pbluescriptKS and the XhoI multiple clone site and is built into the pKSdelIR plasmid between the XhoI of EcoRI to the 133520 base pairs of the 131534th base pair;
On two HSV fragment intermediary NotI point of contacts of plasmid pKSdelIR, insert the GFP expression cassette, be built into plasmid pKSdelIRGFP;
With plasmid pKSdelIRGFP and HSV-1 viral DNA, on the Vero cell of cotransfection, pick out and express the viral plaque of the proteic green of GFP;
Obtain recombinant virus HepaGreen through 3 purifying; Again with the Vero cell of the viral DNA cotransfection of plasmid pKSdelIR and recombinant virus HepaGreen;
Pick out colourless viral plaque from the viral plaque of green, purified and be accredited as the HEPAWEL that has lacked IR.
The invention still further relates to a kind of is the recombinant virus that carries other allogenic genes of skeleton with reorganization herpes simplex virus as indicated above, and it comprises the gene that can cause apoptosis of tumor cells, can suppress the gene of vasculogenesis, and the gene of energy immune stimulating activity.
The present invention relates to a kind of recombinant herpes simplex virus as indicated above and be used for the treatment of application in the medicine of tumour in preparation.
The invention still further relates to a kind of medicine that is used for the treatment of tumour, it is characterized in that containing recombinant herpes simplex virus mentioned above as activeconstituents.
The invention still further relates to a kind of recombinant herpes simplex virus as indicated above and be used for the treatment of application in the medicine of tumour in preparation, it is characterized in that can be by various route of administration in the oncotherapy, include but not limited to the systemic vein injection, medication in local tumor intravascular administration and the tumour.
The invention still further relates to a kind of recombinant herpes simplex virus as indicated above and be used for the treatment of application in the medicine of tumour in preparation, it is characterized in that in the oncotherapy can with the combining of various other treatment means, include but not limited to chemical medicinal treatment, radiotherapy, immunity and other biological preparation therapy in part or the knurl.
Description of drawings
Fig. 1 is the gene mapping of the defective virus HEPAWEL of wild-type HSV-1 and the present invention's structure;
Fig. 2 is that the gene mapping of HEPAWEL of the present invention and existing several defective viruss compares;
Fig. 3 is a HEPAWEL gene recombination synoptic diagram of the present invention;
Fig. 4 is for identifying the restriction map spectrum of treatment pKSdelIR;
Fig. 5 is the fluorescence photo of recombinant virus HepaGreen;
Fig. 6 is the restriction map of recombinant virus HEPAWEL;
Fig. 7 is that the virus replication experimental result of HEPAWEL of the present invention compares;
Fig. 8 is the susceptibility experimental result of HEPAWEL of the present invention to GCV;
Embodiment
Except that specifying, plasmid, carrier, enzyme and primer used in the specific embodiments of the present invention are all available from U.S. Gibco company.
(1) structure of HEPAWEL
Embodiment 1HSV-1 nucleic acid extraction
Get wild-type HSV-1 virus F strain (buy the company in Gibco, and preserve in this chamber, its genom sequence and concrete gene structure are seen NCBI, and its NCBI sequence number is NC 001806) and under the condition of MOI=1, infect the Vero cell of cultivating.After 24-36 hour, when treating that big portion cell presents cytopathy (CPE), collecting cell also changes the centrifugal removal cell culture fluid of 15ml centrifuge tube over to.Adding the 0.3ml cell pyrolysis liquid (contains: 100Mm NaCl, 10Mm Tris.HCl pH8.25MmEDTA pH8 and 0.5%SDS) cracking cells infected and handled 1 hour with RNaseA (10mg/ml) 65 ℃, above-mentioned lysate is used isopyknic phenol/chloroform (1: 1) mixed solution extracting again with isopyknic phenol earlier, the aqueous phase solution that contains DNA adds the 7.5M Ammoniom-Acetate of 1/2 volume and the raw spirit of 2 times of volumes after transferring to new centrifuge tube, leave standstill the centrifugal viral DNA precipitation that obtains after 10 minutes, dissolve the back in 4 ℃ of preservations with the 20-100ulTris/EDTA damping fluid.
The segmental disappearance of embodiment 2IR
As shown in Figure 3, with 2554bp fragment between the EcorRI of SalI to the 117071 base pairs of the 114517th base pair in the genome, and the 1986bp fragment is cloned into respectively among the SstI and XhoI multiple clone site of pbluescriptKS between the XhoI of EcoRI to the 133520 base pairs of the 131534th base pair.Be built into the pKSdelIR plasmid.
On two HSV fragment intermediary NotI point of contacts of plasmid pKSdelIR, insert the GFP expression cassette, be built into plasmid pKSdelIRGFP.
With plasmid pKSdelIRGFP and HSV-1 viral DNA, on the Vero cell of cotransfection, pick out and express the viral plaque of the proteic green of GFP.Obtain recombinant virus HepaGreen through 3 purifying.Again with the Vero cell of the viral DNA cotransfection of plasmid pKSdelIR and recombinant virus HepaGreen.Pick out colourless viral plaque from the viral plaque of green, purified and be accredited as the HEPAWEL that has lacked IR.
The evaluation of embodiment 3 recombinant viruses
As Fig. 4,5 and shown in Figure 6.Observe the green fluorescence and the enzyme of recombinant virus through microscopically and cut detection validation, obtain recombinant virus, and with its preservation.The result shows through gene order-checking, and recombinant virus HEPAWEL compares with wild-type HIV-1 sequence, has lacked the sequence between the 117071st base pair to the 131534 base pairs.
(2) replicability of HEPAWEL and safety experiment
The virus replication of embodiment 4HEPAWEL
HEPAWEL virus behind the purifying at first on the Vero cell of cultivating with the titration of malicious spot assay method, to determine viral effective concentration, the HEPAWEL of known titre and other contrast virus strain infect the individual layer Vero cell of cultivating with MOI=0.01 respectively on 6 hole culture dish, metainfective cell every 24 hours results once, every kind of virus is gathered in the crops each two hole at every turn, till 5 days, the cell of results is centrifugal removal cell residue after freeze thawing three times, the supernatant liquor that contains virus is used the titration of Vero cell once more, and two porocytes of getting results on the same day contain the concentration of the mean value of virus as this day virus.
Experimental result as shown in Figure 7, experimental result illustrate HEPAWEL of the present invention still kept with existing clinical experiment in the G207 virus replication ability suitable used with the F strain.
Embodiment 5HEPAWEL is to the susceptibility experiment of GCV
HEPAWEL, G207 and Brainwel strain virus infect the Vero cell of normal growth in containing the DMEM nutrient solution of 10% serum respectively with the titre of MOI=0.01. grow not adding or be added with in the nutrient solution of GCV of different concns respectively, at back 48 hours harvested cells of infection and carry out malicious spot titration then.
Concrete experimental result as shown in Figure 8.
Because gancyclovir (GCV) has put into practice for many years clinically, be used for the treatment of herpesvirus infection, thereby in case clinical gene therapy is out of control, it can effectively suppress viral growth, thereby interrupt infecting, for the individuality that carries out gene therapy provides further safety precaution.This experimental result shows that HEPAWEL has kept the susceptibility of wild-type virus HSV F strain to GCV fully, and its safety and effectiveness is much higher than recombinant virus G207.
(3) result of the inhibition test of HEPAWEL
The external inhibition test of embodiment 6HEPAWEL
Test method is as follows: with logarithmic phase cell 3 * 10
4It is individual that (the HepG2 poor growth suitably strengthens the cell inoculation amount, as 3 * 10
5Individual) move in 96 orifice plates 200 μ l/ holes, 5%CO
2Cultivate 24h for 37 ℃, cover at the bottle end about 70% so that cell grows to.The careful suction removed substratum, and (the HEPAWEL 50 μ l/ holes of 0.0001-10 are contrast with the EPAWEL of 65 ℃ of 10min deactivations, and every titre is all established two multiple holes to add the different vaccination titre.5%CO
237 ℃ hatch 0.5h after, add the substratum 150 μ l/ holes contain 2% calf serum again, each hole OD value when measuring 96h with mtt assay.(during 92h) adds MTT liquid (5mg/ml) 10 μ l before measuring, and adds cytolysate 100 μ l behind the continuation cultivation 4h and spends the night, and after the dissolving to be crystallized, the 570nm wavelength is measured every hole absorbancy (OD) on enzyme-linked immunosorbent assay instrument.Inhibitory rate of cell growth=(control group OD average-experimental group OD average)/control group OD average * 100%.
With the corresponding computation program " IC50 calculating " of inserting of inhibiting rate of above calculating, calculate the half amount of suppression (pfu value) of HEPAWEL to different cells with virus titer (pfu) value.
Partial results:
1. to people's cancer of the stomach BGC-823 cell strain, IC50=1745.9pfu;
2. to the cell lung cancer NCI-H460 of National People's Congress cell strain, IC50=2334.4pfu;
3. to everybody chronic marrow-derived leukocythemia K562 cell strain, IC50=953.7pfu;
4. to human hepatoma HepG2 cell's strain, IC50=3532.7pfu;
5. to people's liver cancer SMMC-7721 cell strain, IC50=823.5pfu;
Above Multiplicity of Infection (MOI refers to the ratio of virus inoculation amount and inoculating cell) is about 10
-2--10
-3
Above-mentioned experiment in vitro presentation of results, HEPAWEL of the present invention has a good tumor killing effect external, can very effective inhibition tumor growth, have the good curing prospect.
Press down the knurl experiment in the body of embodiment 7HEPAWEL
Concrete experimental technique is as follows: the S180 in the vegetative period of taking the logarithm, H22 cell 2 * 10
6Individual, every mouse 200 μ l are inoculated in under the right armpit of sex Kunming mouse, and tumor growth to volume was about 60-100mm in about fourth, fifth day
3When (length is measured with vernier callipers, and volume calculates by 0.5 * major diameter * minor axis 2),, give HEPAWEL by multi-point injection in the knurl or intravenous injection respectively with the animal random packet.Fortnight is put to death mouse, cuts open to get tumour and weigh, and calculates tumor control rate.Tumor control rate=(control group tumor weight average-experimental group tumor weight average)/control group tumor weight average * 100%.
2 * 106 in QGY-7701 cell, every mouse 200 μ l are inoculated under the right armpit of male nude mouse, about the 20th day tumor growth to volume is about 60-100mm3, and (length is measured with vernier callipers, volume calculates by 0.5 * major diameter * minor axis 2) time, with the animal random packet, give HEPAWEL by multi-point injection in the knurl.Value when this test-results is the 35th day.
Tumor control rate=(control group tumor weight average-experimental group tumor weight average)/control group tumor weight average * 100%.
1.S180 knurl strain:
Multi-point injection in the knurl is administered once 1 * 10 every other day totally 3 times
7Pfu/mouse, tumour inhibiting rate 70.9%;
2.H22 knurl strain: multi-point injection in the knurl is administered once 3 times every other day totally.
A.1 * 10
7Pfu/mouse, tumour inhibiting rate 60.4%;
B.5 * 10
4Pfu/mouse, tumour inhibiting rate 42.9%;
C.1 * 10
4Pfu/mouse, tumour inhibiting rate 31.4%;
3.H22 knurl strain: multi-point injection in the knurl.
A. be administered once 1 * 10 in per 3 days totally 3 times
7Pfu/mouse, tumour inhibiting rate 53.3%;
B. be administered once 1 * 10 in per 7 days totally 2 times
7Pfu/mouse, tumour inhibiting rate 44.4.0%;
4.H22 knurl strain: tail vein injection is administered once 3 times every other day totally.
A.5 * 10
7Pfu/mouse, tumour inhibiting rate 68.0%;
B.5 * 10
4Pfu/mouse, tumour inhibiting rate 30.7%;
C.1 * 10
4Pfu/mouse, tumour inhibiting rate 24.5%;
5.H22 knurl strain: tail vein injection.
A. be administered once 1 * 10 in per 3 days totally 3 times
7Pfu/mouse, tumour inhibiting rate 26.0%;
B. be administered once 1 * 10 in per 7 days totally 2 times
7Pfu/mouse, tumour inhibiting rate 22.5%;
6. human hepatocellular QGY-7701
The inoculation nude mice, multi-point injection in the knurl is administered once 1 * 10 every other day totally three times
7Pfu/mouse, tumour inhibiting rate 79.3%;
Experimental result explanation in the above-mentioned body, HEPAWEL recombinant virus of the present invention also has good tumor killing effect in vivo fully.
(4) result of the toxicity test of HEPAWEL
Cavy is an abdominal injection, simulates interventional therapy in the clinical liver; Rabbit is intravenous injection, simulation clinical vein dosage regimen.The administration cycle: three months, administration was 1 time weekly.Immunotoxicity test, stimulation, allergy and hemolytic test experimental result show that HEPAWEL is very low to the toxicity of cavy and rabbit.It is the very high recombinant virus of a kind of security.
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Claims (8)
1. the recombinant herpes simplex virus of a called after HEPAWEL, it is characterized in that its specificity has lacked the sequence of the 117071st to the 131534th base pair of wild-type HSV-1 virus F pnca gene group, thereby make the ICP0 that includes only a copy in the recombinant virus, ICP4 and ICP34.5 gene.
2. a method that makes up recombinant herpes simplex virus is characterized in that, section is repeated in the inside that specificity has lacked in the wild-type HSV-1 virus F pnca gene group, thereby makes the ICP0 that includes only a copy in the recombinant virus, ICP4 and ICP34.5 gene.
3. the method for structure recombinant herpes simplex virus as claimed in claim 2 is characterized in that:
At first, with 2554bp fragment between the EcorRI of SalI to the 117071 base pairs of the 114517th base pair of wild-type HSV-1 virus F pnca gene group, and the 1986bp fragment is cloned into respectively in the SstI of same pbluescriptKS and the XhoI multiple clone site and is built into the pKSdelIR plasmid between the XhoI of EcoRI to the 133520 base pairs of the 131534th base pair;
On two HSV fragment intermediary NotI point of contacts of plasmid pKSdelIR, insert the GFP expression cassette, be built into plasmid pKSdelIRGFP;
With plasmid pKSdelIRGFP and HSV-1 viral DNA, on the Vero cell of cotransfection, pick out and express the viral plaque of the proteic green of GFP;
Obtain recombinant virus HepaGreen through 3 purifying; Again with the Vero cell of the viral DNA cotransfection of plasmid pKSdelIR and recombinant virus HepaGreen;
Pick out colourless viral plaque from the viral plaque of green, purified and be accredited as the HEPAWEL that has lacked IR.
4. one kind is the recombinant virus that carries other allogenic genes of skeleton with reorganization herpes simplex virus as claimed in claim 1, wherein said other allogenic genes comprise the gene that can cause apoptosis of tumor cells, the gene that can suppress vasculogenesis, and the gene of energy immune stimulating activity.
5. the described recombinant herpes simplex virus of claim 1 is used for the treatment of application in the medicine of tumour in preparation, and described tumour is cancer of the stomach, lung cancer, chronic marrow-derived leukocythemia or liver cancer.
6. a medicine that is used for the treatment of cancer of the stomach, lung cancer, chronic marrow-derived leukocythemia or liver cancer is characterized in that described medicine contains the described recombinant herpes simplex virus of claim 1 as activeconstituents.
7. the described recombinant herpes simplex virus of claim 5 is used for the treatment of application in the medicine of tumour in preparation, it is characterized in that described medicine can be by various route of administration administrations in oncotherapy.
8. the described recombinant herpes simplex virus of claim 7 is used for the treatment of application in the medicine of tumour in preparation, it is characterized in that described route of administration is the systemic vein injection, administration in local tumor intravascular administration and the tumour.
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