CN101538556B - Method for replicating adenovirus by way of selection and mutual compensation - Google Patents
Method for replicating adenovirus by way of selection and mutual compensation Download PDFInfo
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Abstract
The invention provides a method for replicating adenovirus by means of selection and mutual compensation, belonging to the field of biomedicine and relating to a method for replicating replication-defective adenovirus by means of selection and compensation. The method is characterized in that by mutual compensation, two replication-defective adenoviruses are provided with one early gene which is not contained in the other one, wherein the early gene of one replication-defective adenovirus is controlled by a tumor specific promoter, the other replication-defective adenovirus is provided with the early gene and the tumor therapy gene which are not contained in the former replication-defective adenovirus, and the adenoviruses can be selectively compensated and replicated in tumour cells by simultaneously adopting the two replication-defective adenoviruses. The method can selectively kill the tumour cells, without affecting normal cells. The invention can improve the safety, the targeting and the effectiveness of the operation in which adenoviruses carry target genes to carry out gene therapy on tumors.
Description
Technical field
The invention belongs to biomedical sector.Relate to the foundation of two kinds of replication-defective adenoviral selectivity, complementary ground clone method in tumour cell.
Background technology
Malignant tumour is one of serious disease that threatens human health, is the main cause of death in the whole world.World Health Organization report, global cancer mortality number in 2007 reach 7,900,000 (account for all death tolls 13%), estimate that the cancer mortality number will continue increase, and the year two thousand thirty will have 1,200 ten thousand people to die from cancer.The cause of death of the resident for the third time sample survey result that China Ministry of Health announces in April, 2008 shows that malignant tumour is China urban population cause of the death first place (accounting for dead sum 25.0%), is second of the people in the countryside cause of the death (accounting for dead sum 21.0%).
Up to the present, main treatment means as malignant tumour, though operation, chemotherapy and radiation have obtained certain result of treatment, but still exist significant limitation, as the strike, chemicotherapy of operation to the toxic side effect of systems such as liver kidney, hematopoiesis, immunity, the finiteness of late malignant tumour curative effect etc.In recent years to the research of malignant tumour mechanism prompting, the cell growth that the sudden change inactivation of oncogene active, cancer suppressor gene, cell cycle control gene change etc. cause is out of control, to cancerate be the main mechanism that malignant tumour takes place.At tumorigenic molecular genetics background, the genetic treatment of tumor technology develops rapidly thereupon, becomes the forefront of current oncotherapy research, for brand-new approach has been opened up in oncotherapy.
One of key of gene therapy is to select suitable carriers that the external source goal gene is imported target cell safely and efficiently, and foreign gene is expressed in target cell or specifically by requirement regulating and expressing necessarily.Carrier commonly used at present is the harmless virus through transforming, as retrovirus, adenovirus, adeno-associated virus, hsv etc.The ideal carrier should possess low toxicity, efficient, these several essential condition of large vol.Human 2 types and 5 type adenovirus are used to therapy of tumor more, because they have following characteristics: 1. host range is wide, and the epithelial cell of having a liking for is arranged, and human most tumour is the epithelial cell source; 2. pathogenic low to the people; 3. in propagation and non-proliferative cell, all can infect and expressing gene; 4. unconformability does not have the mutagenicity of insertion in karyomit(e); 5. can express a plurality of genes simultaneously; 6. can effectively increase, the titre height, and can carry out the heavy dose preparation.
Replication-defective adenoviral is one of gene therapy carrier commonly used, owing to lost copy function, this carrier has higher biological safety, but there is significantly deficiency in conventional effect.Behind the replication-defective adenoviral cells infected owing to do not have a replication, goal gene can not be imported in all or the most of target cell, add the scavenging(action) of body immune system, the time of expression alien gene is shorter, need be in therapeutic process repeatedly, in a large number, topical application, and this may cause body intensive sensitization by activate immunity anamnestic response.
In recent years, people utilize the difference of some biological character between tumour cell and the normal cell, have made up tumour and have selected replication type adenovirus.This adenovirus carrier can make the external source goal gene optionally efficiently duplicate final kill tumor cell in tumour cell, reduces the required viral dosage of treatment simultaneously, reduces to normal cytotoxicity with to host's immune response.
The essential gene of research and utilization tumor-specific promoters (Telomerase TERT promotor) control virus replication is arranged at present, as E1a gene etc., these genes are only expressed in tumour cell, and in normal cell, do not express, thereby virus is only duplicated in tumour cell.
It is that genetic treatment of tumor has been opened up new approaches that this tumour is selected replication type adenovirus, does not have the TERT activity although must be noted that human body overwhelming majority normal cells, and sexual cell and hemopoietic stem cell have been proved and have telomerase activation.This replication type adenovirus may be in time multiplexed cell systems such as hemopoietic stem cells, thereby critical functions such as human body hematopoiesis are affected.For fear of the defective of above-mentioned copy choice virus, improve the safe and effective property of treatment, we are necessary to explore new therapeutic strategy.
Summary of the invention
The advantage that the objective of the invention is comprehensive above-mentioned replication-defective adenoviral and selection replication type adenovirus, further avoided their defective, by making up the replication-defective adenoviral that two kinds of selectivity, complementations are duplicated, set up a kind of novel, high selectivity, efficiently, safer therapy of tumor method.
Characteristics of the present invention are that two kinds of replication-defective adenovirals lack a kind of early gene that has nothing in common with each other respectively, as E1a and E4, the replication-defective adenoviral that wherein lacks the E4 gene contains the E1a gene of TERT promotor control, and the another kind of replication-defective virus that lacks the E1a gene has E4 gene and oncotherapy gene.When the two separately existed in normal cell and the tumour cell, they are for want of required albumen of adenoviral replication and reproducible not all.As the two being mixed the postoperative infection tumour cell, thereby they can provide the expressing protein complementation of the other side's missing gene to duplicate within it mutually, behind the virus replication, and the tumour cell around infecting again, and complementation is duplicated once more.Because the E1a gene is to be controlled by the TERT promotor, this complementation is duplicated and the Circulation process of infection again can constantly be carried out in having the active tumour cell of TERT, stop at tumor border at last, and in not having the active normal cell of TERT, this complementation is duplicated no longer and is taken place.Like this, can make each tumour cell in the complete infected tumor of the adenovirus that carries the oncotherapy gene tissue, reach best knurl effect extremely, and don't can have influence on normal cell.
Because two kinds of viruses must just may have the characteristics of replicability in same cell, even a small amount of like this virus spills from tumour, enter tissues such as infecting marrow by circulation of blood, the possibility of virus replication also reduces greatly, also just greatly reduced attack simultaneously, selected the security of replication type adenovirus that large increase has been arranged normal plasma cell.
Have TERT promotor control E1a gene and lack the replication-defective adenoviral of E4 gene, can unite use with the multiple replication-defective adenoviral that carries Antioncogene and E1a genetically deficient existing now or that constantly occur in the future, can well overcome the shortcoming of this genoid treatment, improve the effect of this class therapy of tumor.
The present invention has the following advantages: 1. pair tumour cell high selectivity; 2. higher security; 3. the ability of carrying the external source goal gene is higher; 4. can continue to use and verify in the past that effective replication-defective adenoviral, various scheme combination made treatment versatile and flexible.
Realize that step of the present invention comprises: 1. use molecule clone technology, separate TERT promotor and E1a gene; 2. make up complementary, replication-defective adenoviral; 3. packing, amplification, purified virus; 4. virus infection and replication are identified.
Description of drawings
Figure 1A d-TERTp-E1a Δ E4 and Ad-GFP Infection in Vitro tumour cell (Hela cell) situation.Infect next day, observe every Kong Junyou under the fluorescent microscope and seldom measure the green fluorescence expression, illustrate that every Kong Jun has cell by virus infection (A, B, C).Infected the back the 3rd day, Geminivirus infected group fluorescence strengthens (F), and single virus group fluorescence have no significant change (D, E).Replication-defective virus Ad-TERTp-E1a Δ E4 and Ad-GFP can infected tumor's cells, and the two could duplicate in tumour cell when using simultaneously in complementation.
Fig. 2 Ad-TERTp-E1a Δ E4 and Ad-GFP Infection in Vitro normal cell (MRC-5 cell) situation.Infect next day, observe every Kong Junyou under the fluorescent microscope and seldom measure the green fluorescence expression, illustrate that every Kong Jun has cell by virus infection (A, B, C).Infected the back the 3rd day, single virus and Geminivirus group fluorescence have no significant change (D, E, F).Though replication-defective virus Ad-TERTp-E1a Δ E4 and Ad-GFP can infect normal cell, no matter still be separately that common application is not all duplicated in normal cell.
Fig. 3 Ad-TERTp-E1a Δ E4 and Ad-GFP Infection in Vitro tumour cell (Hela cell) situation.200MOI virus infected cell (A, D, G), observing every Kong Jun under the fluorescent microscope has egfp expression, illustrates that every Kong Jun has cell by virus infection.Infected the back the 3rd day, and got half cell pyrolysis liquid respectively and add (B, E, H) in the Hela cell of another hole, very strong fluorescent signal appears in the Geminivirus infected group, and the virion (H) that has produced permissive cell is described.Continuation is infected next porocyte (C, F, I) with half cell pyrolysis liquid, and the Geminivirus infected group is the virus (I) of reproducible generation permissive cell still.
Fig. 4 Ad-TERTp-E1a Δ E4 and Ad-GFP Infection in Vitro normal cell (MRC-5 cell) situation.200MOI virus infected cell (A, D, G), observing every Kong Jun under the fluorescent microscope has egfp expression, illustrates that every Kong Jun has cell by virus infection.Infected the back the 3rd day, and got half cell pyrolysis liquid respectively and add (B, E, H) in the MRC-5 cell of another hole, only the Geminivirus group has very weak fluorescent signal (H).Continuation is infected next porocyte (C, F, I) with half cell pyrolysis liquid, and three groups are not all had fluorescent signal.In normal cell,, can not duplicate and produce the virus that infection ability is arranged even two kinds of viruses are used simultaneously.
Fig. 5 Ad-TERTp-E1a Δ E4 and the Ad-GFP immunohistochemical methods GFP dyeing situation in vivo tumor.The single infection group and viral infection group (a) of Ad-GFP, virus are only expressed the GFP albumen (the pale brown look dyeing in the cell of a figure top) that carries in the tumour cell of infective virus, and can not infect other cells (a figure bottom) again.(b, c), virus can be duplicated in the SKOV3 cell that has infected and be continued to infect other tumour cells, until infecting all tumour cells (all having a large amount of pale brown look dyeing in b, the c figure cell) for Ad-TERTp-E1a Δ E4 and Ad-GFP Geminivirus infected group.B is the high power lens picture, shows GFP protein expression in the tumour cell, and c is the low power lens picture, shows that nearly all tumour cell is all by virus infection and express GFP albumen.
Embodiment
Embodiment 1
Utilize molecule clone technology to separate tumour-specific TERT promotor and E1a gene.
The present invention passes through round pcr, be template with human cervical carcinoma Hela cell and wild-type adenovirus Ad5DNA respectively, amplification TERT promotor and E1a gene order are inserted among the shuttle plasmid pAdtrack, construct the pAdtrack-TERTp-E1a plasmid of being expressed by TERT promotor control E1a.
The primer sequence of amplification TERT promotor:
Upstream primer 5 '-CAATGATATCTTCCCAGGGCCTCCACATCATG-3 '
Downstream primer 5 '-ATAGTTTAGCGGCCGCACGCAGCGCTGCCTGAAACTC-3 '
The primer sequence of amplification E1a gene:
Upstream primer 5 '-ACCGGGACTGAAAATGAGAC-3 '
Downstream primer 5 '-TTAAGCATAATCTGGAACATCATATGGATATGGCCTGGGGCGTTTAC-3 '
Embodiment 2
Make up the replication-defective virus Ad-TERTp-E1a Δ E4 by TERT promotor control E1a genetic expression of disappearance E4 gene.
The present invention utilizes the AdEasy adenovirus system to make up replication-defective adenoviral, and implementation step is:
1. skeleton plasmid pAdEasy-2 (not being with the E4 gene) electricity transforms the BJ5183 bacterium, the microbiotic plate screening is chosen the amplification of clone back, extracting plasmid, and enzyme is cut the integrity of checking pAdEasy-2, select correct clone and be the BJ5183-Ad-2 bacterium, with its preparation electroreception attitude cell.
2. utilize shuttle plasmid pAdtrack (carrying green fluorescence protein gene) and TERT promotor and E1a fragment construction recombination plasmid pAdtrack-TERT p-E1a.
3.pAdtrack-TERTp-E1a electricity transforms BJ5183-Ad-2, the microbiotic plate screening is chosen clonal expansion, extracting plasmid, and enzyme is cut detection, selects correct clone and is pAdEasy-2-TERTp-E1a.
4. transfection, packing, amplification and purified virus Ad-TERTp-E1a Δ E4.
Plasmid pAdEasy-2-TERTp-E1a is behind restriction enzyme PacI linearization for enzyme restriction, with liposome lipofectamine 2000
TMThe transfection packing cell.
The 911E4 cell is expressed E4 albumen under 0.4mg/ml G418 (Geneticin), 0.2mg/ml Totomycin (Hygromycin B), 1 μ g/ml doxycycline (Doxycyclin) acting in conjunction; 2V6.11 cell is expressed E4 albumen under 1 μ g/ml pine sterone (Ponasterone A) effect.Express the proteic clone of E4 for these two kinds and all can be used for Ad-TERTp-E1a Δ E4 virus packing and amplification.Thereby utilize the fluorescence microscope luciferase expression to judge virus packing, amplification situation.With cesium chloride density gradient centrifugation and dialysis method purified virus.The virus titer of purifying about 10
7Fu/ μ l.
Embodiment 3
The selectivity complementation is duplicated the infection of adenovirus in cultured cell in vitro and is duplicated (one)
The replication-defective adenoviral commonly used of gene therapy at present is generally E1 genetically deficient and has inserted therapeutic gene in this site, and this virus has the E4 gene.With the replication-defective adenoviral Ad-GFP that has green fluorescent protein GFP gene is example, with virus of A d-TERTp-E1a Δ E4 and its co-infected cultured cell in vitro of our structure.
Inoculate human cervical carcinoma Hela cell, normal human embryonic lung fibroblast MRC-5 in six porocyte culture plates, every hole 5 * 10
5Individual cell.In Hela, MRC-5 cell, add virus respectively: promptly singly add Ad-TERTp-E1a Δ E4 group by following three groupings; Singly add the Ad-GFP group; Add Geminivirus Ad-TERTp-E1a Δ E4 and Ad-GFP group.Calculate institute by each cell infection 10MOI and add virus quantity, the single virus group of every kind of virus quantity of Geminivirus group reduces by half to keep the total virus amount identical.Observe every Kong Junyou next day under the fluorescent microscope and seldom measure the green fluorescence expression, illustrate that every Kong Jun has cell by virus infection.Observed 5 days continuously, find that the Hela cell adds in two groups of single virus, fluorescence does not have considerable change, strengthens gradually and add Geminivirus group fluorescence.Two of MRC-5 cell add that fluorescence has no significant change in single virus group and the Geminivirus group.This result shows that replication-defective adenoviral Ad-TERTp-E1a Δ E4 and Ad-GFP can infected tumor's cell and normal cells, but no matter separately still common application all in normal cell, do not duplicate; Have only two kinds of viruses infected tumor's cell simultaneously, could in tumour cell, complementation duplicate.(Fig. 1, Fig. 2)
Embodiment 4
The selectivity complementation is duplicated the infection of adenovirus in cultured cell in vitro and is duplicated (two)
Inoculate human cervical carcinoma Hela cell, normal human embryonic lung fibroblast MRC-5 in six porocyte culture plates, every hole 5 * 10
5Individual cell.In Hela, MRC-5 cell, add virus by each cell infection 200MOI virus: singly add Ad-TERTp-E1a Δ E4 group by following grouping; Singly add the Ad-GFP group; Add Geminivirus Ad-TERTp-E1a Δ E4 and Ad-GFP group, the single virus group of every kind of virus quantity of Geminivirus group reduces by half to keep the total virus amount identical.Observing every Kong Jun next day under the fluorescent microscope has a large amount of green fluorescences to express, and illustrates that every Kong Jun has cell by virus infection.Infect after 3 days, substratum is removed in every hole, and PBS washes cell 2 times, uses the tryptic digestion collecting cell, and freezing-thawing and cracking is also centrifugal, gets in the cell of the new uninfecting virus of cultivating of half cracking supernatant liquor adding.Per 3 days repeat this experiment, get half cell pyrolysis liquid and infect the new cultured cells in next hole.The purpose of this checking is the virion that whether contains the continued cells infected that newly duplicates generation in the checking cell pyrolysis liquid, infect new cell owing to only get half cell pyrolysis liquid at every turn, if newly do not duplicate the virion of the continued cells infected of generation, green fluorescence is expressed and will inevitably be reduced until disappearance gradually, otherwise the expression of fluorescin will continue to exist.
Thereby the result shows single virus infection and can not duplicate the lasting virus that infection ability is arranged that produces in tumour cell and normal cell; Geminivirus Ad-TERTp-E1a Δ E4 and Ad-GFP in normal cell not reproducible produce the virus that infection ability is arranged, and these two kinds viruses can be duplicated in complementation in tumour cell, lasting generation has the virus of infection ability, constantly infected tumor's cell.(Fig. 3, Fig. 4)
Embodiment 5
The selectivity complementation is duplicated the infection of adenovirus in vivo tumor and is duplicated.
With logarithmic phase human cervical carcinoma Hela cell digestion, counting, use the PBS suspension cell, by every nude mice injection 5 * 10
5It is subcutaneous that individual cell, cumulative volume are that 50 μ l are injected in the nude mice back leg.When tumour was grown to the about 0.5cm of diameter, the ovarian cancer SKOV3 cell in the vegetative period of taking the logarithm set up three groups separately and uses virus infection: 1, singly adds Ad-TERTp-E1a Δ E4; 2, singly add Ad-GFP; 3, infect two kinds of viruses of Ad-TERTp-E1a Δ E4 and Ad-GFP simultaneously.The infective virus amount is all calculated by each cell 100MOI, and the single virus group of every kind of virus quantity of Geminivirus group reduces by half to keep the total virus amount identical.Infect next day, fluorescent microscope is observed down and is seen all have a large amount of green fluorescences to express, and proves and infects successfully.The collecting cell counting will infect viral SKOV3 injection cell in nude mice Hela knurl body, every nude mice injection 1 * 10
6Individual cell, every group of 5 mouse.When treating the tumour continued growth, put to death nude mice, pathologic sampling, section to the about 1cm of diameter.In the section of the tumour that three groups of different virus infect, do the immunohistochemical experiment of DAB colour developing respectively, resist with one to be the anti-GFP of rabbit.The result shows: the tumour of single virus infection, and its GFP only distributes in the part tumor tissues, and the Geminivirus group can find in whole tumour that GFP distributes.Our experimental result shows two virus-free duplicating of single virus group, and the Geminivirus group is duplicated and infection conditions again, has occurred the GFP protein expression in nearly all tumour cell.(Fig. 5)
Our invention has shown that the selectivity complementation duplicates reasonableness and feasibility that adenovirus is used for oncotherapy.Replication-defective adenoviral Ad-GFP can be for loading other replication-defective adenovirals of various goal gene, with Ad-TERTp-E1a Δ E4 flexible combination, for efficient, the safe targeted therapy of tumour proposes new direction.
SEQUENCE?LISTING
＜110〉West China No.2 Hospital, Sichuan University
＜120〉method of replicating adenovirus by way of selection and mutual compensation
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Claims (1)
1. the composition that contains the replication-defective adenoviral that two kinds of complementations duplicate, it is characterized in that described two kinds of replication-defective adenovirals lack respectively different with duplicate relevant early gene, simultaneously can provide the missing gene expressed proteins mutually again, thereby can complementation duplicate;
Wherein, described a kind of replication-defective adenoviral carries the replication-defective adenoviral of oncotherapy gene simultaneously for lacking a kind of early genes; Be E1 genetically deficient and inserted the replication-defective adenoviral Ad-GFP that has the E4 gene of therapeutic gene in this site;
Wherein, described another kind of replication-defective adenoviral is regulated and control by tumor-specific promoters for it duplicates necessary a kind of early gene, described tumor-specific promoters is being controlled the early genes that the former lacks, and lacks the replication-defective adenoviral of another kind of early gene simultaneously; This replication-defective adenoviral is the replication-defective virus Ad-TERTp-E1a Δ E4 by TERT promotor control E1a genetic expression of disappearance E4 gene:
Ad-TERTp-E1a Δ E4 is prepared by following method: amplification TERT promotor and E1a gene order, be inserted among the shuttle plasmid pAdtrack, and construct the pAdtrack-TERTp-E1a plasmid of expressing by TERT promotor control E1a;
The primer sequence of described amplification TERT promotor:
Upstream primer 5 '-CAATGATATCTTCCCAGGGCCTCCACATCATG-3 ',
Downstream primer 5 '-ATAGTTTAGCGGCCGCACGCAGCGCTGCCTGAAACTC-3 ';
The primer sequence of described amplification E1a gene:
Upstream primer 5 '-ACCGGGACTGAAAATGAGAC-3 '
Downstream primer 5 '-TTAAGCATAATCTGGAACATCATATGGATATGGCCTGGGGCGTTTAC-3 ';
Utilize the AdEasy adenovirus system to construct the replication-defective virus Ad-TERTp-E1a Δ E4 by TERT promotor control E1a genetic expression of E4 gene.
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Citations (1)
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| US20080032283A1 (en) * | 2004-09-29 | 2008-02-07 | Oncolys Biopharma Inc. | Telomelysin/gfp-expressing recombinant virus |
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| US20080032283A1 (en) * | 2004-09-29 | 2008-02-07 | Oncolys Biopharma Inc. | Telomelysin/gfp-expressing recombinant virus |
Non-Patent Citations (2)
| Title |
|---|
| Woo，et al..Antiangiogenic Gene Therapy for Breast Cancer Using Targeted Recombinant Adenovirus.《storming media》.2003,20. * |
| 叶震敏.生物类基因治疗载体的研究新进展.《临床肿瘤学杂志》.2006,第11卷(第3期),230-234. * |
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