CN103330723A - Applications of genetically engineered respiratory syncytial virus ([delta]NS1 RSV) in medicines treating cancer - Google Patents

Abstract

The invention relates to applications of a genetically engineered respiratory syncytial virus ([delta]NS1 RSV) in medicines treating cancer. The genetically engineered respiratory syncytial virus can be used for medicines treating lung cancer and breast cancer, and can be used for medicines treating liver cancer, skin cancer, prostatic cancer, ovarian cancer, rectal cancer, pancreas cancer, laryngocarcinoma and other cancer. The genetically engineered respiratory syncytial virus is advantageous in that the virus is used for medicines treating cancer; and the virus kills only cancer cells and does not impair normal cell in a human body.

Description

The purposes of genetic engineering respiratory syncytial virus (△ NS1 RSV) in the treatment cancer drug

Technical field

The present invention relates to respiratory syncytial virus (RSV), specifically the purposes of a kind of gene NS1 deficiency respiratory syncytial virus (△ NS1 RSV) in the treatment cancer drug.

Background technology

The World Health Organization's publish data shows that the year two thousand twenty whole world cancer morbidity will be than present increase by 50%, and the newly-increased cancer patient's number in the whole world will reach 1,500 ten thousand people/every year.The demand of cancer therapy drug will further increase, and development good effect, safe PTS will become hot fields and the main direction of new drug research.

At present, the prevalence of cancer rises year by year, and lacks effective treatment means at present.The main Therapeutic Method of malignant tumor still is traditional chemotherapy, radiotherapy and excision.But chemotherapy exists drug resistance and the tangible poisonous side effect of medicine problem that can't avoid, and clinical therapeutic efficacy is very undesirable.And there is serious toxic and side effects equally in radiotherapy, and some human organ is responsive especially to radiotherapy, and the incompatible radiotherapy of selecting is treated.Only less than 30% the operative treatment that is applicable to, and postoperative has recurrence more and shifts, and causes mortality high in the tumor patient.Therefore need a kind of new medicine of exploitation or new therapy badly.The oncolytic virus medicine receives industry personage concern day by day with the tumor effect of killing of its uniqueness.

Oncolytic virus (oncolytic virus) is the tumor-killing type virus that a class has replication capacity, and the report that occurs oncolytic virus in the world the earliest is because of found at that time that cervical cancer patient tumor after infecting rabies virus disappeared thereupon.

1991, people such as Martuza found that transgenic HSV has certain curative effect in the glioblastoma treatment, just receive publicity day by day thereby adopt virus to carry out oncolytic therapy.The principle of oncolytic virus treatment tumor is to carry out genetic modification by some virus that nature is existed to make special virus, and this virus can optionally infect cancerous cell and final destroyed tumor cell.

Avian pneumo-encephalitis virus (newcastle disease virus, NDV), herpes simplex virus-1(herpes simplex virus-1, HSV-1), reovirus (reovirus), adenovirus (adenovirus) etc. be used to be transformed into oncolytic virus because having the natural tumor characteristic of having a liking for, they can infect cancerous cell and finally destroy it.Because these oncolytic viruses do not injure organism normal cell, seldom occur by toxic and side effects in theory.But the wild type strain of these oncolytic viruses has stronger pathogenic originality, it is pathogenic though these viruses have reduced through genetic modifications, under certain condition, do not get rid of the probability that reverts to the wild type strain but enter behind the human body oncolytic virus after these genetic modifications, thereby unavoidably there is potential safety hazard in these viral clinical practices.Moreover these viral antigenic types are stronger, and virus can stimulate body to produce stronger neutralizing antibody after entering body, and the follow-up virus of the same race that enters in the body reduces the oncolytic effect thereby this antibody can neutralize.In addition, the wild type of these oncolytic viruses has stronger pathogenic, and relevant Susceptible population mostly inoculated relevant vaccine.Therefore the neutrality antibody that produces among the crowd may carry throughout one's life, thereby certainly will influence the actual effect that adopts these oncolytic virus treatment cancers among this crowd patient.Therefore it is extremely urgent to seek new oncolytic virus pathogenic low, that antigenicity is weak.

Respiratory syncytial virus (RSV) is a kind of RNA viruses, belongs to Paramyxoviridae.The wild-type RSV nucleotides sequence is classified single strand RNA as, and its gene order is followed successively by 3 '-NS1-NS2-N-P-M-SH-G-F-M2-L-5 '.Its N, L and P gene code core protein, M gene code stromatin, G gene code glycoprotein, gene F encoding fusion protein, SH gene code SH albumen, the M2 gene has two overlapping genes M2-1 and M2-2, the corresponding protein of encoding respectively, these albumen are referred to as structural protein.In addition, RSV also contains NS1, NS2 two genes encode respectively viral non-structural protein NS 1 and NS2.The RSV gene of people, cattle and other animals has similar nucleotide sequence.People's wild-type RSV virus is main to infect neonate and 6 months with interior baby, and adult and older child also can infect this virus, but infection rate is not high.The patient mainly shows as upper respiratory tract infection, but only needs anti symptom treatment mostly or need not special treatment and can recover.Body is to the immunity imperfection of rsv infection simultaneously, and patient is repeated infection in a short time, and the indication body is difficult to produce effective neutralizing antibody, and this makes RSV might become desirable oncolytic virus candidate.

Present each big pharmaceuticals of the U.S. very good oncolytic virus medicine future market all.Though the oncolytic virus product of U.S. Biovex company still is in the primary stage of clinical anticancer test, Biovex company (wherein 4.25 hundred million is cash) was then purchased with 1,000,000,000 dollars in 2011 by U.S. Amgen company.Show this PTS especially in the critical role in anticancer field in future.The anticancer biological medicine of oncolytic virus of new generation that development has China's independent intellectual property right is very necessary.

Invention Inner holds

The objective of the invention is to have anticancer function when applying biological science and technology means make its forfeiture pathogenecity after with the respiratory syncytial virus genetic modification: a selectivity kill cancer cell, do not kill and wound the human normal cell.

The purposes of genetic engineering respiratory syncytial virus of the present invention (Δ NS1 RSV) in the treatment cancer, this virus not only can be applied to treat pulmonary carcinoma, breast carcinoma, can also be used for the treatment of hepatocarcinoma, skin carcinoma, carcinoma of prostate, ovarian cancer, cervical cancer, rectal cancer, cancer of pancreas, laryngeal carcinoma and other cancers.

Described genetic engineering respiratory syncytial virus (Δ NS1 RSV) refers to NS1 gene function disappearance RSV.

Described this respiratory syncytial virus (Δ NS1 RSV) virus can derive from the RSV of people RSV A, B hypotype and other Strain and cattle and other animals.

The RSV of described NS1 gene function disappearance makes it afunction or NS1 gene front and back end and introduces other nucleotide sequences to suppress NS1 gene transcription or translation or to use antisense technology to make virus N S1 gene inactivation at gene level for removing the part or all of gene of NS1 or introduce other nucleotide sequences in the NS1 gene.

Described genetic engineering modified, can obtain NS1 deficiency RSV with the NS1 gene in the reverse genetic technology deletion respiratory syncytial virus; Or by technology such as gene insertion, gene mutation, gene sealing or enzyme action, make the loss of function of NS1 gene.

The purposes advantage of genetic engineering respiratory syncytial virus of the present invention (Δ NS1 RSV) in the treatment cancer comprises: 1) anticancer spectrum is wide: can effectively kill and wound pulmonary carcinoma, breast carcinoma, hepatocarcinoma, skin carcinoma, carcinoma of prostate, ovarian cancer, cervical cancer, rectal cancer, cancer of pancreas, laryngeal carcinoma and other cancer cell; 2) toxic and side effects is low: virus is not damaged the human normal cell; 3) bright in producing neutralizing antibody, make virus to reuse; 4) can kill and wound drug resistance cancerous cell (comprising multidrug resistant); 5) has unique anticancer mechanism: a) viral direct killing effect; B) cause cancer cell-apoptosis by mitochondrion mechanism in the born of the same parents; C) PT-PORE mechanism changes on the mitochondrial membrane, and passage continues exploitation, causes calcium ion outflow in the mitochondrion, and then causes mitochondrial swelling to break; D) the mitochondrion transmembrane potential reduces; E) Caspase 9-dependency/dependent/non-dependent double mechanism; F) this oncolytic virus can kill and wound P53 antioncogene feminine gender and positive cancer cell.

Description of drawings

Fig. 1 shows that causing the Hep-2 cancerous cell with the people RSV behind the antisense oligonucleotide sealing NS1 gene produces apoptosis.

The let others have a look at CPE of the prostate gland cancer cell LN of △ NS1 RSV CAP of Fig. 2.

Fig. 3 shows the zoopery of testing the anti-carcinoma of prostate LN CAP of △ NS1 RSV that lets others have a look at.

Fig. 4 △ NS1 RSV that lets others have a look at causes hepatocarcinoma HepG2 and produces CPE.

The let others have a look at zoopery of △ NS1 RSV antagonism hepatocarcinoma of Fig. 5.

Fig. 6 is the lethal effect table of the tumor cell of △ NS1 RSV.

The specific embodiment:

The specific meanings of used abbreviation is among the present invention:

RSV: respiratory syncytial virus

ATCC: American Type Culture Collection

Δ NS1 RSV:NS1 gene defection type respiratory syncytial virus

Hep-2: laryngeal cancer cell strain

LN CAP: prostate gland cancer cell strain

HepG2: hepatoma cell strain

CPE: cytopathic effect

The present invention has following marked feature:

The present invention utilizes reverse genetic technology deletion people RSV NS1 gene order to be included as 122 to 630 base sequences on the viral RNA: the respiratory syncytial virus that is removed the NS1 gene is called NS1 gene defection type respiratory syncytial virus (△ NS1 RSV), and △ NS1 RSV is by obtaining with a plurality of plasmid co-transfection Hep-2 cells that carry totivirus cDNA, virus N-gene, M2-1 gene, P gene and the L gene etc. of removing the NS1 gene respectively.

△ NS1 RSV also can prepare by other ways: for example insert one section meaningless nucleotide sequence and make its NS1 albumen of can not encoding out in the NS1 gene order of wild-type strain RSV A2 strain; Or with restriction endonuclease cutting inactivation NS1 gene, make the NS1 afunction; Or use antisensenucleic acids sealing NS1 gene, thereby generate the virus of NS1 functional defect type.

△ NS1 RSV is through behind the Hep-2 cell amplification, carries out purification and makes viral concentrated solution through high speed centrifugation or through separating chromatographic column, is kept in-80 ℃ of low temperature or the liquid nitrogen.Also can be prepared into viral freeze-dried powder preparation by Freeze Drying Technique.

△ NS1 RSV has a marked feature: the selective killing cancerous cell, and to not influence of normal cell.Under the experiment in vitro condition, △ NS1 RSV with the DMEM cell culture fluid be diluted to MOI=10 respectively infected person hepatoma carcinoma cell, melanoma cell, prostate gland cancer cell, ovarian cancer cell, cervical cancer cell, rectum cancer cell, pancreatic cancer cell, laryngeal cancer cell with and corresponding normal cell, 37 ℃ of cultured cells are microscopically observation of cell pathological changes two days later, the result shows that △ NS1 RSV has tangible lethal effect to above-described cancerous cell, under the mirror showed cell become justify, come off, pathological changes such as necrosis, and the normal cell form is not seen obvious change.

For checking △ NS1 RSV tumor effect extremely in vivo, choose hepatoma carcinoma cell, skin cancer cell, prostate gland cancer cell, ovarian cancer cell, cervical cancer cell, rectum cancer cell, cancer of pancreas or laryngeal carcinoma arbitrarily, and be inoculated into the nude mice hind leg left and right sides.Treat that the cancerous cell tumor grows up to diameter 4cm, respectively intratumor injection or intravenous injection △ NS1 RSV and wild-type RSV.I.e. side nude mice hind leg injection △ NS1 RSV, opposite side injection wild-type RSV in contrast.The dry powder that this △ NS1 RSV can make for the viral liquid that concentrates or lyophilization.Its injection titre is 1X10 ¹-1X10 ¹⁰⁰Behind the locally injected into tumor, observe and measure the size of tumor continuously and find that after the week, the experimental side gross tumor volume is 30% of control sides, showing △ NS1 RSV has and significantly presses down tumor effect, with experimental mouse euthanasia and peel off tumor, weighing tumor body weight.

The present invention has an other marked feature: RSV and is not limited to specific RSV Strain.Any virus, as long as it contains the gene of similar NS1, the generation that this albumen can antagonism host I-type IFN just might make it this and becomes oncolytic virus by deleting or lack this gene.

Its candidate's virus can be all Strain and other genotypic a plurality of Strain of people RSV A, B hypotype including, but not limited to specific RSV Strain, and the RSV of cattle and other animals.

Cell strain and virus: LN CAP, HepG2, Hep-2 cell and respiratory syncytial virus (RSV) A2 strain derive from ATCC (American Type Culture Collection).

The antisense oligonucleotide plasmid construction:

The sequence of siRNA is:

siNS1: 5’-GGCAGCAATTCATTGAGTATGCTTCTGGAAATAAGCATACTCAATGAATTGCTGCCTTTTTG-3’;

siNS1a: 5’-GTGTGCCCTGATAACAATATTCAAGAGATATTGTTATCAGGGCACACTTTTTTG-3’;

siE7: 5’-GAAAACGATGAAATAGATGTTCAAGAGACATCTATTTCATCGTTTTCTTTTTT-3’;

siPB2: 5’-GGCTATATTCAATATGGAAAGAACTCGAGTTTTGTTCTTTCCATATTGAATATAGCCTTTTTG-3’;

siUR: 5’-GGTCACGATCAGAATACTTCGCTCGAGCGAAGTATTCTGATCGTGACCCTTTTTTG-3’。

SiRNA oligonucleotide sequence is inserted into the corresponding site of plasmid psMWZ-1, generates the siRNA of respiratory syncytial virus strain (RSV) NS1 gene; The siRNA of virus HPV18 gene E7, and the siRNA of A type influenza virus gene PB2 and pUR is in contrast.Its antisense oligonucleotide carrier cloning process is seen document 1.

The structure of NS1 deficiency respiratory syncytial virus (RSV):

The extraction of viral nucleic acid and the preparation of cDNA chain thereof: the Hep-2 cell that has infected HRSV A2 in the 6 porocyte culture plates is scraped and centrifugal collection supernatant, adopt QIAamp Viral RNA Mini kit (QIAGEN) to extract the HRSV viral RNA.Get viral RNA 1 μ g and add random primer (random hexamers, Invitrogen) 125 μ g, moisturizing to 20 μ l.Response system is placed on the PCR instrument, arrange 70 ℃ 10 minutes, transposition is 25 ℃ then, maintains 4 ℃ at last.Press the product description gradation and in above-mentioned response system, add 5 x First Strand Buffer, DDT, dNTP (10 mM each) (Invitrogen), SuperRase inhibitor (Ambion), SuperScript II (Invitrogen) and do not have RNA water to cumulative volume 40 μ l circulates at the PCR instrument at last.The PCR program arrange 25 ℃ 10 minutes, 37 ℃ 45 minutes, 42 ℃ 45 minutes, 70 ℃ 15 minutes, 30-45 the circulation after, product is deposited stand-by for 4 ℃.

The structure of viral gene expression carrier: carrier for expression of eukaryon pVAX1 (Invitrogen) is respectively applied to express virus N-gene, M2 gene, P gene and L gene.RT-PCR is used for the preparation of viral related gene, and template is seen above-mentioned viral cDNA.The amplimer of its corresponding viral gene is respectively as follows: the virus N-gene primer is: forward:5 '-TAGGATCCATGGGGCAAATA-3 ', reverse:5 '-AATGTCCGAATTCTTATTAACTCAA-3 '; Pfu enzyme (QIAGEN) with calibration function adds the Taq enzyme and is used for pcr amplification, and reaction buffer adopts Roche Expand buffer system.Amplification back fragment is through BamHI and EcoRI double digestion, and purification reclaims this fragment and inserts the pVAX1 carrier for the corresponding restriction enzyme site of clone's usefulness, and the capable conversion of connexon and picking recon carry out gene sequencing to be identified.The M2 gene primer is: forward:5 '-GGGCAAGCTTATGAAAACTGG-3 ' (containing enzyme Hind III sequence), reverse:5 '-CGCGCTCGAGTTAAATAACTATC-3 ' (containing enzyme Xho I sequence).The P gene primer is: forward:5 '-GGAAGCTTATGGGGCAAATA-3 ' (containing Hind III restriction enzyme site), reverse:5 '-CGTTTGGGCCCCTATATTATA-3 ' (containing Apa I restriction enzyme site).The L gene primer is: forward:5 '-AACTGCAGAGTTGTGGGACA-3 ' (containing Pst I restriction enzyme site), reverse:5 '-GCGATGGGCCCTTAATAACTA-3 ' (containing Apa I restriction enzyme site).Use the correlated virus gene outcome of above-mentioned primer amplification gained and insert the pVAX1 carrier respectively for the corresponding enzyme action position of clone's usefulness, order-checking is identified through recon equally;

The structure of viral gene defective vector: have virus leader sequence and NS1 in order to make up, clone's of NS2 and N gene, we adopt RT-PCR method (seeing said method).Primer sequence is: forward:5 '-AGCTGCAGACGCGAAAAA-3 ', reverse:5 '-CCCCGGATCCTTATTAACTCAA-3 '.Its product is examined through gene sequencing through inserting site corresponding in the PUC18 cloning vehicle again and produce the sub-pUCR4 of clone with reclaiming behind restriction endonuclease Pst I and the Bam HI double digestion again.In order to remove virus N S1 gene, we adopt the PCR method that virus N S2 gene is directly connected to the 45th base place, NS1 gene start codon upstream equally.Be template to clone sub-pUCR4, primer adopts: forward:5 '-pAAAATGGGGCAAATAAATCA-3 ', reverse:5 '-pAGTGGTACTTATCAAATTCTTATTTGC-3 '.Transformation gene engineering bacterium DH5 α after amplified production self the connection cyclisation, picking clone is done the deficiency plasmid pUCR Δ 3 that the gene sequencing checking has obtained lacking NS1 gene and upstream portion sequence thereof.Be similarly and obtained having viral gene P, M, SH, clone's of G and F, primer is selected for use: forward:5 '-AAGGATCCATGGGGCAAAT-3 ', reverse:5 '-CGCCGGGGTACCTTATATAACTATAA-3 '.Its PCR product is examined through gene sequencing through inserting restriction enzyme site corresponding in the PUC18 cloning vehicle again and produce the sub-pUCR5 of clone with reclaiming behind restriction endonuclease Bam HI and the Kpn I double digestion again.For the primer of cloning viral M2 and L two genes is: forward:5 '-AGGAAGGTACCATGAAAACTGG-3 ', reverse:5 '-GGAGGGGTCGACTTAATAACTATAATTG-3 '.Its PCR product is examined with gene order surveying method equally through inserting restriction enzyme site corresponding in the PUC19 cloning vehicle again and produce the sub-pUCR2 of clone with reclaiming behind restriction endonuclease Kpn I and the Sal I double digestion.Last gradation is with respective segments under the enzyme action: as downcutting targeting sequencing and virus N S2 with Pst I and the two enzymes of Bam HI from cloning sub-pUCR Δ 3, recovery inserts that corresponding restriction enzyme site becomes new recon pTR12A among the carrier pTR12 again after the N genetic fragment; Remove the viral gene P that contains among the sub-pUCR5 of clone with Bam HI and Kpn I double digestion, M, SH, the big fragment of G and F, recovery is inserted the corresponding site of carrier pTR12A that has been inserted with viral gene NS2 and N again and is made it to become new support pTR12AB.The two enzymes of the same Kpn I of employing and Sal I downcut viral M2 and L two genetic fragments from pUCR2, reclaim to insert to have among the expression vector pTR12AB of T7 promoter and fourth type hepatovirus Ribozyme sequence again.This have except the NS1 gene whole viral genes and 44 gene orders of upstream from start codon thereof expression vector we be referred to as pTR-L2NS1;

The generation of defective virus: in order to produce genetic flaw HRSV virus, we with the NS1 gene with and 44 base sequences in upstream lack as collaborative each the corresponding virus protein N of 1 μ g of expression vector pTR-L2NS1 1 μ g, M2, the expression plasmid cotransfection Hep-2 cell of P and L, cell culture are collected supernatant and are infected new Hep-2 cell after 6 to 8 days.Continue to observe infection cell and collect the new Hep-2 cell of supernatant infection, produce cytopathic effect (CPE) until cell.The defective virion that adopts traditional plaque-forming assay to come purification to obtain again detects each viral gene with RT-PCR at last, and NS1 albumen is screened with Western blot.

The △ NS1 RSV of results makes viral concentrated solution through separation and purification, is kept in-80 ℃ or the liquid nitrogen, also can be prepared into viral lyophilized powder by Freeze Drying Technique.Carry out hepatocarcinoma with this virus concentrated solution or viral lyophilized powder, skin carcinoma, carcinoma of prostate, ovarian cancer, cervical cancer, rectal cancer, laryngeal carcinoma, the pharmacodynamic experiment of cancer such as cancer of pancreas and other cancers.

Embodiment 1: cause people's laryngeal cancer cell apoptosis with the RSV NS1 gene that infects among the antisense oligonucleotide closing cell in the laryngeal cancer cell

Get 1 * 10 ⁶Individual Hep-2 laryngeal cancer cell is inoculated on the 6 porocyte culture plates 37 ° of C and is cultured to 70%-80% and merges, respectively with the antisense oligonucleotide siNS1(of 4 μ g sealing people RSV NS1 gene), siE7(sealing people HPV E7 gene), siPB2(sealing human influenza virus PB2 gene) and the siUR(random sequence contrast) transfection Hep-2 cell (Lipofectamine, Life technologies), test group infected person wild-type RSV (MOI=2) more separately after 24 hours.RSV after the sealing of NS1 gene can optionally cause the Hep-2 cell and produce apoptosis as shown in Figure 1, and other contrast antisense oligonucleotides siE7, siPB2 and siUR group Hep-2 cell no obvious apoptosis except producing spontaneous apoptosis produce.Confirm that no matter what method to remove the NS1 function by the RSV that loses the NS1 function all can kill and wound cancerous cell.

Embodiment 2: the lethal effect of the carcinoma of prostate of Δ NS1 RSV

The external killing prostate cancerous cell of Δ NS1 RSV: this experiment prostate gland cancer cell strain LN CAP cell and normal prostatic epithelial cell derive from ATCC.Get 1 * 10 ⁶Individual LN CAP cell 37 ° of C on 6 orifice plates are cultured to 70%-80% and merge, equivalent inoculation Δ NS1 RSV, wild-type RSV and viral dilution agent (MOI=5).Δ NS1 RSV optionally kills and wounds LN CAP cell as shown in Figure 2, and wild-type RSV and viral dilution liquid then do not have lethal effect to this cell.Repeat this experiment in an other strain prostate gland cancer cell PC3, Δ NS1 RSV has tumor effect extremely, and wild-type RSV and viral dilution liquid level are not then killed the tumor effect.

Δ NS1 RSV induces the apoptotic experiment of LN CAP: LN CAP cell infects wild-type RSV and Δ NS1 RSV (MOI=5) respectively.Detected in conjunction with testing with Annexin V by transfect cell.The result shows: Δ NS1 RSV induces LN CAP apoptosis.

The tumor inhibition effect of the Human Prostate Cancer Cells strain of Δ NS1 RSV LN CAP mice with tumor:

Set up the BALB/c nude mice subcutaneous transplantation tumor model of Human Prostate Cancer Cells strain LN CAP, be filtered into the good animal of tumor and be divided into 3 groups of (wild-type RSV, Δ NS1 RSV at random, matched group), with normal saline in contrast, Δ NS1 RSV virus liquid is as test sample, 5 every group.Concrete grouping and dosage are as follows:

Route of administration: tail vein injection

Administration frequency and time limit: test sample once a day, equal 4 weeks of successive administration.

Evaluation of result:

General clinical observation

Every day at the upper and lower noon is respectively carried out 1 general clinical observation in the process of the test.

The mensuration of body weight and tumor volume

In process of the test, should be from grouping, administration (comprise last administration and euthanasia the same day) 2 times weekly, weigh and with vernier caliper measurement and record tumor major diameter, minor axis, calculate gross tumor volume, draw tumor growth curve according to gross tumor volume, and the difference of tumor growth curve between each group relatively.

Calculate gross tumor volume according to following formula:

V=1/2 * major diameter * minor axis 2

Its experiment the results are shown in Figure 3.

Embodiment 3: Δ NS1 RSV virus is to the lethal effect of hepatocarcinoma HepG2

Hepatoma carcinoma cell HepG2 and normal liver cell are all from ATCC.Get 1 * 10 ⁶Individual above-mentioned cell culture is cultured to the 70%-80% cell fusion at 37 ° of C on 6 orifice plates, infect wild-type RSV, Δ NS1 RSV (MOI=5) and viral dilution agent respectively.Microscopically observation of cell pathological changes.The result shows: Δ NS1 RSV virus has tangible lethal effect to hepatoma carcinoma cell, and normal liver cell is not had toxic and side effects.Wild-type RSV and viral dilution agent all do not have toxic and side effects (Fig. 4) to hepatoma carcinoma cell and normal liver cell.

The tumor inhibition effect of the human liver cancer cell HepG2 of Δ NS1 RSV mice with tumor:

Set up the BALB/c nude mice subcutaneous transplantation tumor model of human liver cancer cell HepG2, be filtered into the good animal of tumor and be divided into 3 groups (matched group, Δ NS1 RSV, wild-type RSV), 5 every group at random.As negative control, Δ NS1 RSV virus liquid obtains lyophilized powder after lyophilization with normal saline, with this lyophilized powder as test sample.Concrete grouping and dosage are as follows:

Route of administration: tail vein injection

Administration frequency and time limit: test sample is once a day.

Evaluation of result:

General clinical observation

Every day at the upper and lower noon is respectively carried out 1 general clinical observation in the process of the test.

The mensuration of body weight and tumor volume

In process of the test, should be from grouping, administration (comprise last administration and euthanasia the same day) 2 times weekly, weigh and with vernier caliper measurement and record tumor major diameter, minor axis, calculate gross tumor volume, calculate gross tumor volume according to following formula: V=1/2 * major diameter * minor axis ²

Carry out therapeutic evaluation according to gross tumor volume

In the process of the test, when tumor load surpasses 10% of body weight, the average tumor diameter surpasses 20mm, or tumor generation ulcer, necrosis or infect, to animal enforcement euthanasia.

As shown in Figure 5: Δ NS1 RSV lyophilized powder is 1 * 10 to the inhibiting onset dosage of the tumor growth of human liver cancer cell HepG2 mice with tumor ⁸PFU/, Δ NS1 RSV lyophilized powder has tangible tumor growth inhibitory action, and wild-type RSV does not have tumor-inhibiting action.

Embodiment 4: Δ NS1RSV virus is to the lethal effect of rectum cancer cell

Rectal cancer HCT116 cell, rectal cancer SW-620 cell and normal rectum cell are all from ATCC.Press the ATCC condition of culture, get 1 * 10 ⁶Individual above-mentioned cell culture is cultured to the 70%-80% cell fusion at 37 ° of C on 6 orifice plates, infect wild-type RSV, Δ NS1 RSV (MOI=5) and viral dilution agent respectively.Microscopically observation of cell pathological changes.Experimental result shows that the rectum cancer cell of Δ NS1 RSV has tangible lethal effect, and normal rectum cell is not had toxic and side effects.Wild-type RSV and viral dilution agent all do not have toxic and side effects to rectum cancer cell and normal rectum cell.

Embodiment 5: the specific skin cancer cell of killing and wounding of Δ NS1 RSV

Get 1 * 10 ⁶Individual skin cancer cell strain 888, skin carcinoma WM-115 cell are cultivated respectively on 6 orifice plates and are cultured to the 70%-80% cell fusion at 37 ° of C.Cell infects wild-type RSV, Δ NS1 RSV (MOI=5) and viral dilution agent respectively.Δ NS1 RSV induces the necrosis of skin cancer cell 888 and skin carcinoma WM-115 cell specifically, and wild-type virus and the agent of contrast viral dilution are to 888 skin cancer cell and the not influence of skin carcinoma WM-115 cell.This experiment is in other skin cancer cell, for example: repeat among the SK-MEL-3 and 624, obtained the same experimental result.

Apoptosis is through detecting with Annexin V combined techniques, and experimental result shows that Δ NS1RSV has induced the apoptosis of skin cancer cell 888, and wild-type RSV virus and viral dilution agent are to not influence of cell.This experimental result is the immuning hybridization experiment confirm.

Example 6: the lethal effect of the ovarian cancer of Δ NS1 RSV, cervical cancer, laryngeal carcinoma, cancer of pancreas

Human ovarian cancer C-13 cell, cervical cancer Hela cell, laryngeal carcinoma 1059, cancer of pancreas AR42J derive from ATCC, get 1 * 10 respectively ⁶Individual above-mentioned cell culture when 37 ° of C are cultured to cell 70-80% fusion, infect Δ NS1 RSV and wild-type RSV (MOI=5) respectively and detects cytopathy on 6 orifice plates.The result shows: Δ NS1 RSV can kill and wound above-mentioned cancerous cell effectively, and wild-type RSV is not then killed the tumor effect.

The embodiment of the invention is including, but not limited to above-mentioned example.

The research of Δ NS1 RSV oncolysis mechanism

Studies show that different cancer therapy drugs generally has different anticancer mechanism, but relate to the different cell signal path of blocking-up more.Research report p53 albumen can participate in the apoptosis that RSV induces.Whether the inventor is essential by NS1 RSV cell death inducing for research p53 gene, H1299 lung carcinoma cell with NS1 rsv infection p53 deficiency, the result shows that NS1 RSV can induce H1299 cell generation apoptosis equally, shows that NS1 RSV cancer cell specific induction of apoptosis does not need p53 to participate in.

Cell generation apoptosis also can be induced by the mitochondrion approach.For detecting the NS1 rsv infection to the influence of cell mitochondrial transmembrane potential (Δ Ψ m) value, this experiment has detected mitochondrion transmembrane potential (the Δ Ψ m) value behind the NS1 rsv infection A549 cancerous cell (MOI=5).The result shows that NS1 albumen can stop the decline of mitochondrion transmembrane potential (Δ Ψ m) value.Further confirmed this result under the ultramicroscope: with respect to matched group, the A549 cell mitochondrial electron density that Δ NS1 RSV infects descends, and swelling appears in mitochondrion.The present invention shows that Δ NS1 RSV can pass through mitochondrion approach inducing cell generation apoptosis.

Studies show that in the past, the generation of the IFN-beta of the host cell of NS1 has antagonism.And IFN-beta has the effect that suppresses viral growth.This also is that wild-type RSV can infect eupnea road cell, the reason that induces an illness.After NS1 gene function disappearance, RSV no longer suppresses the generation of host cell IFN-beta, host cell can generate the IFN-beta of higher concentration, thereby the propagation that suppresses RSV virus, therefore the RSV of NS1 afunction can not breed at human normal cell line, can not make human normal cell line generation pathological change.

Studies show that IFN-beta is virus inhibitory factor still not, also is a stronger tumor-inhibiting factor.One of mechanism of cell carcinogenesis is obstacle to have occurred because the IFN-beta of these cells generates, thereby can not suppress these cells effectively to the transformation of cancerous cell, so IFN-beta is one of drug candidate of oncotherapy clinically at present.

When the rsv infection cancerous cell of NS1 afunction, owing to can not produce enough IFN-beta behind the interferon system imperfection of cancerous cell own and the viral infection, thereby the RSV of NS1 afunction can breed in cancerous cell, thereby causes cancer cell death.

Our work further confirms a hypothesis: need only the gene inactivation with the similar NS1 gene function in the viral gene, just can make up the oncolytic virus that makes new advances.

Described genetic engineering modified, can use the gene NS1 in the reverse genetic technology deletion respiratory syncytial virus, and obtain NS1 deficiency RSV; Or by technology such as gene insertion, gene mutation, gene sealing or enzyme action, make the loss of function of NS1 gene.

Described by technology such as gene insertion, gene mutation, gene sealing or enzyme action, make the loss of function of NS1 gene,, above-mentioned technology is prior art in the genetic engineering, use these technology and make NS1 gene loss of function in the respiratory syncytial virus, no longer one by one tired stating.

List of references:

1.Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene.Zhang W, Yang H, Kong X, Mohapatra S, San Juan-Vergara H, Hellermann G, Behera S, Singam R, Lockey RF, Mohapatra SS.

Nat Med. 2005 Jan;11(1):56-62. Epub 2004 Dec 26. Erratum in: Nat Med. 2005 Feb;11(2):233.

Claims (5)

1. a genetic engineering respiratory syncytial virus (Δ NS1 RSV) is in the purposes for the treatment of in the cancer drug, can be applied to treat pulmonary carcinoma, breast carcinoma, it is characterized in that: can also be applied to treat hepatocarcinoma, skin carcinoma, carcinoma of prostate, ovarian cancer, cervical cancer, rectal cancer, cancer of pancreas, laryngeal carcinoma and other cancers.

2. genetic engineering respiratory syncytial virus according to claim 1 (Δ NS1 RSV) is characterized in that in the purposes for the treatment of in the cancer drug: described genetic engineering respiratory syncytial virus (Δ NS1 RSV) refers to NS1 gene function disappearance RSV.

3. genetic engineering respiratory syncytial virus according to claim 1 (Δ NS1 RSV) is in the purposes for the treatment of in the cancer drug, it is characterized in that: this respiratory syncytial virus (Δ NS1 RSV) comes from people RSV A, B hypotype and other a plurality of genotype, and the RSV strain of cattle and other animals.

4. genetic engineering respiratory syncytial virus according to claim 2 (Δ NS1 RSV) is in the purposes for the treatment of in the cancer drug, it is characterized in that: described NS1 gene function disappearance RSV, for removing the part or all of gene of NS1, or in the NS1 gene, introduce nucleotide sequence, make it afunction, or NS1 gene front and back end introducing nucleotide sequence, suppress NS1 gene transcription or translation, or use antisense gene to make the NS1 inactivation.

5. genetic engineering respiratory syncytial virus according to claim 2 (Δ NS1 RSV) is in the purposes for the treatment of in the cancer drug, it is characterized in that: described genetic engineering modified, can use the gene NS1 in the cdna reverse strategy deletion respiratory syncytial virus, and obtain NS1 deficiency RSV; Or make the loss of function of NS1 gene by technology such as gene insertion, gene mutation, gene sealing or enzyme action.

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