CN1294268C - Recombinant adenovirus vector capable of being duplicated and spread specifcally inside tumor cell - Google Patents
Recombinant adenovirus vector capable of being duplicated and spread specifcally inside tumor cell Download PDFInfo
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Abstract
The present invention relates to ' recombinant adenovirus vectors which are capable of being duplicated and spread specifically in tumor cells', and provides a method and a composite for treating tumor diseases based on a virus treating method. The present invention constructs a genetic engineering recombinant adenovirus which comprises a 5 type adenovirus E1a gene. Research shows that the viruses can be duplicated specifically in the tumor cells, and the E1a gene can promote the duplication of the recombinant adenoviruses in the tumor cells and the spread of the recombinant adenoviruses between the tumor cells. Test results show that the recombinant adenovirus can be used in the viral treatment and the gene treatment of a tumor.
Description
Invention field:
The present invention relates to gene therapy and viral therapy field, be specifically related to utilize the E1a gene to promote specific the duplicating and spread in tumour cell of a kind of special recombinant adenoviral vector (Ad.IR).
Background of invention:
The gene therapy of malignant tumour requires virus vector specificly efficiently to enter tumour cell, the express therapeutic gene, or specificly in tumour cell, duplicate, and then the kill tumor cell.
At present the common vector as gene therapy has retroviral vector, adenovirus carrier and gland relevant viral vector, wherein as therapy of tumor comparatively commonly used be adenovirus carrier.
Adenovirus is a kind of double-stranded DNA virus, and its genome length is about 36kb, is divided into early transcription district and late transcription district.Find that at present adenovirus hominis has 47 serotypes, adheres to A-F6 subgenus separately, wherein to be that present people study the most detailed a kind of for 5 type adenovirus of C subgenus.
Adenovirus is deleted the resulting adenovirus carrier in E1 district and E3 district often be called as first-generation adenovirus carrier, it is that 8kb is with interior foreign gene that this carrier can insert and express length, this adenovirus carrier not reproducible itself produces progeny virus, and adenovirus carrier DNA also unconformability goes in the genome of host cell.
Adenovirus carrier can infect the broad variety cell, comprises cell stationary phase.Adenovirus carrier is large scale culturing and obtain the viral pure product of high titre easier.These characteristics make adenovirus as therapy of tumor carrier commonly used.
Adenovirus is as the carrier of therapy of tumor, and it is crucial improving its efficiency of infection and specificity to tumour cell.
Report a kind of novel adenovirus carrier (Ad.IR) (SteinwaerderDS.et.al.Nature medicine.2001Feb is arranged; 7 (2): 240-243) be inverted tumor-necrosis factor glycoproteins with inserting two sections in the adenovirus carrier, can make adenovirus that orientable homologous recombination takes place in reproduction process, produce the gene structure of a brachymemma, making insertion be inverted foreign gene reverse in the tumor-necrosis factor glycoproteins can express by forward.And duplicating of this adenovirus carrier only occurs in the quick growing tumors cell.So, be reversed the foreign gene that is inserted in the adenovirus carrier and promptly can specificly be expressed in the tumour cell, if foreign gene is to have anti-personnel therapeutic gene, then its tumour cell of expressing therein will be killed.
For the therapy of tumor carrier, require on the one hand it can be at the specifically inside tumor cell expression alien gene; On the other hand, wish that also it can duplicate, produce progeny virus at specifically inside tumor cell and infect not infected tumour cell on every side, improves the fragmentation effect to tumour cell.Simultaneously, require it in normal cell, not duplicate as far as possible, lower and kill and wound Normocellular.For reaching this two purposes, the Ad.IR adenovirus carrier combined with 5 type adenovirus E 1 a genes will produce an ideal results.
5 type adenovirus E 1 a genes are positioned at the viral genome left end, have 84% conservative property is arranged.E1a two the main albumen of encoding, one have 243 amino acid (12S, 243R), one have 289 amino acid (13S, 289R).E1a-12S and E1a-13S albumen impel virus replication by the expression that activates virogene.(Shenk，T.1996.Adenoviridea，P.2111-2148。) E1a-12S and E1a-13S albumen is initial two albumen of expressing of adenovirus, have the function that activates adenovirus other gene, particularly E2 district and E4 district gene, and the function in these two districts is duplicating of relevant adenovirus.
E1a direct activation cytogene, the inducing cell dna replication dna, with cell cycle regulating protein matter pRb, pRb associated protein (P107/P130) or P300 interact.
E1a combines with pRb family protein molecule, discharges the transcription factor of E2F family, causes in the host cell some relative dna synthetic genes by positive regulation, makes the stationary phase cell enter the S phase.These and some other in S phase activated cytokine, produced a suitable viral DNA synthetic environment.In normal cell, E1a inducing cell cycle negative regulation causes the proteic accumulation of P53, stimulates P53 mediation G1 phase cell to stagnate (el-Deiry, W.S.et.al., 1993.Cell.75:817-25; Xiong, Y.et.al.1993.Nature, 366:701-4) or enter apoptotic pathways.In addition, the E1a inductive is transferred to die and also can be passed through not take place according to the P53 approach.(Teodoro，J.G.et.al.1995.Oncogene.11：467-74)
E1a gene past attempts is considered to an oncogene, can make animal embryo cell generation immortalization, and can interact with the oncogene of other viruses or cell.
In experimentation on animals, E1a can not bring out canceration separately, and needs to concur with other gene such as E1b.5 type adenovirus itself do not have carinogenicity, and other type has the adenovirus of the carinogenicity E1a gene independent with it not have necessary relation.Over past ten years, a lot of experiment confirms, 5 type adenovirus E 1 a genes not only do not have carinogenicity to human body cell, and in fact the effect that suppresses tumor growth can be arranged.The tumor inhibition effect of E1a may be relevant to the regulation and control of several genes with E1a.Mainly show the following aspects:
1. particularly the neck oral squamous cell carcinomas is closely related for overexpression of neu gene and human malignant tumor, and also prognosis and the resistance with tumour is relevant.And evidence show, the E1a gene can specific inhibition neu expression of gene at transcriptional level, can suppress as characteristics relevant such as adhesion, invasion and attack with metastases, the E1a gene also can suppress the canceration characteristic that the oncogene inductive comprises cell polymorphism etc., also can improve susceptibility (YuDH, the et.al.Molecular basis of oncology.1995:131-162 of the breast cancer cell of high expression level neu gene to chemotherapeutics; UenoNT, et.al.Proc AACR, 1998,39:360 (Abstract))
2. E1a produces antitumor action by improving the horizontal cell death inducing of cell P53.
3. E1a can improve cell to chemotherapeutic and the apoptotic susceptibility of radiation institute inductive such as 5 FU 5 fluorouracil, cis-platinums.
4. E1a can express at host cell surface, and killing and wounding, removing of the cell of raising body pair cell expression Ela gene reaches antitumous effect.
As therapy of tumor, existing issue is to improve its expression in tumour cell specifically, the Ad.IR recombinant adenoviral vector has this feature, and being applied to clinical prospect, the Ad.IR carrier is to improve it to the infection rate of tumour cell or it can be duplicated specifically effectively in tumour cell, and diffusion infect around the tumour cell of infected thing not, improve killing-efficiency to tumour cell.
The E1a gene applies in the clinical trial in recent years as Antioncogene.The inventor is through research trial, the E1a gene is inserted the Ad.IR carrier, construct a kind of new therapy of tumor adenovirus carrier M003, purpose provides a kind of adenovirus carrier and can duplicate in the specific tumour cell, express the E1a gene, thus the kill tumor cell of differential high efficient.
Adenovirus carrier provided by the present invention can be based on the duplicating and recombinating at specifically inside tumor cell of this carrier in the principle of tumour internal specific expression alien gene.
Adenovirus carrier provided by the present invention can promote its diffusion in tumour.For this tumour-specific carrier, the diffusion of vector virus helps inducing apoptosis of tumour cell, and enhanced virus discharges and infect other not infected tumour cell on every side from infected tumour cell, thereby strengthens the fragmentation effect to tumour cell.
The present invention also provides the pharmaceutical composition that contains this tumour-specific vector virus and pharmaceutically acceptable carrier.
The present invention also provides this tumour-specific carrier to be used for the treatment of purposes in the tumors pharmaceutical combination in preparation.
Summary of the invention:
The present invention relates to a kind of recombinant adenoviral vector, it has deleted E1 and/or E3 district gene, have the external source of inserting with 3 '-5 ' direction and insert gene adenovirus E 1 a gene, and insert the gene both sides in external source and have a pair of inversion multiple dna sequence dna, be inverted between the repeating sequences at two these same virus genomic these homologous recombination can take place.
Recombinant adenoviral vector of the present invention preferably includes one section adenovirus left side of following assembly: a. and is inverted terminal repeat; B. one section adenovirus packaging sequence is positioned at the 3 ' end that terminal repeat is inverted in the left side; C. one section promoter sequence is positioned at 3 ' of adenovirus packaging sequence and holds; D. a pair of inversion tumor-necrosis factor glycoproteins is inverted the 3 ' end that tumor-necrosis factor glycoproteins is positioned at promoter sequence for first section, and second section tumor-necrosis factor glycoproteins is positioned at 5 ' end of exogenous DNA array; E. one section exogenous DNA array is adenovirus E 1 a gene, oppositely is connected in first section 3 ' end of being inverted tumor-necrosis factor glycoproteins with 3 '-5 ' direction; F. at least one adenoviral gene is positioned at second section 3 ' end of being inverted tumor-necrosis factor glycoproteins; G. terminal repeat is inverted on the adenovirus right side, is positioned at 3 ' end of adenoviral gene.
For above-mentioned adenovirus carrier, preferably, behind described first section inversion tumor-necrosis factor glycoproteins 3 ' end, have one section bidirectional transcription termination site sequence, can block to transcribe and carry out.This bidirectional transcription termination site sequence preference is two-way SV40 poly adenosine sequence.
The invention still further relates to a kind of method that produces new adenovirus carrier by the homologous recombination between the virus vector of the present invention, wherein introduce at least 2 viruses of the present invention in the tumour cell in vitro, can recombinate between these two viruses, produce new recombinant adenoviral vector, this new recombinant adenoviral vector contains the adenovirus E 1 a gene that can express.
The invention still further relates to a kind of pharmaceutical composition, but it contains the above-mentioned recombinant adenoviral vector and the available pharmaceutical carrier of kill tumor cell.
The invention still further relates to a kind of composition, it comprises a kind of recombinant adenoviral vector with above-mentioned adenovirus characteristic, and a kind of chemotherapy compound.
The invention still further relates to a kind of composition, it comprises a kind of recombinant adenoviral vector with above-mentioned adenovirus characteristic, and a kind of immunosuppressive compounds.
The invention still further relates to above-mentioned recombinant adenoviral vector and be used for the treatment of purposes in the pharmaceutical composition of tumour in preparation.
Description of drawings:
1.M003 and M005 recombinant adenovirus gene structure
2.Ad.IR the recombinant adenoviral vector homologous recombination produces new recombinant adenovirus
3.E1a gene promotes duplicating of M003 recombinant adenovirus
4.E1a gene promotes duplicating of M003 recombinant adenovirus at different time
5.E1a gene promotes the lethal effect of M003 recombinant adenovirus to tumour cell
6.E1a gene promotes the diffusion of adenovirus carrier between tumour cell
Detailed Description Of The Invention:
Constructed recombinant adenoviral vector is 5 type adenopathies through transforming among the present invention Poison. The genome of 5 type adenovirus contains 35935 nucleotides. 5 type adenovirus are orders A kind of virus of front research. Mainly cause people's breathing on the epidemiology The road infects, and obvious self-healing property is arranged. It is existing several that adenovirus is applied to human body as vaccine The history in 10 years takes place from finding no transformation of human normal cell and carcinogenic phenomenon.
Make among the recombinant adenoviral vector that the present invention is constructed such as Fig. 1 the M003 that shows and M005. In M003, the promoter region of adenovirus vector E1a is deleted, adds RSV Copying of viral promotors (RSVp) the whole adenovirus vector of control. The RSVp promoter The downstream is one and expresses assembly. 5 '-3 ' direction is successively by alkaline phosphatase gene (AP), Internal ribosome entry site gene (IRES), E1a gene and poly adp gene (polyA) form, and be 3 '-5 ' direction and be inverted in the adenoviral gene group this table Reaching the assembly both sides is one of research usefulness with being inverted repetitive sequence (IR) .M005 respectively Individual contrast virus, basic structure and M003 are in full accord, are E1a gene wherein Direction is just opposite in M003 with it, is 5 '-3 ' direction. Theoretically, work as disease When poison copies in tumour cell, can be inverted at two heavy between two viral genome Recombinate between the complex sequences (Fig. 2), the RSV promoter can be replicated and Opposite direction connection Downstream to expressing assembly produces a little expression unit, in this unit, E1a gene and AP gene that originally 3 '-5 ' direction was inserted in M003 virus can To obtain expression. And the E1a gene that 5 '-3 ' direction is inserted in M005 virus is not Can express, it is multiple in the growth of Ad.IR viral vectors so just can to observe the E1a gene Role in the process processed.
Advantage of the present invention:
The invention solves two problems in the field of tumor gene therapy:
(1) how at the expressing gene of specifically inside tumor cell, by using Ad.IR Recombinant adenoviral vector can be at the specifically inside tumor cell expression alien gene, as The fruit specifically expressing some cytotoxicity foreign genes or with apoptosis-related outside The source gene will provide to the treatment of kinds of tumors a kind of effective means.
(2) how to promote the antitumous effect of gene therapy vectors of tumor. Present tumour The low problem of killing-efficiency is infected in the common existence of gene therapy vector. Make among the present invention With recombinant adenoviral vector, utilize 5 type adenovirus E 1 a genes, promote virus to carry Body copying and the diffusion between tumour cell in tumour cell. Simultaneously, E1a base Because also bringing into play antitumor action.
Definition
Unless different definition is arranged in addition, all technology and scientific terminology contains as used herein Common understand the same of those skilled in the art in justice and the affiliated field of the present invention. Although Similar or identical any method any and described here and material all can be used for this Bright enforcement or test. Only preferred method and material are described at this. Be this Invention is existing to be defined with regard to following term:
" foreign gene " this term refers to insert the dna sequence dna of any protein of interest of coding in the used adenovirus carrier among the present invention as used herein.The upper limit of inserting foreign gene length depends on the packing limit of adenovirus carrier.The albumen that any institute of the foreign gene codified that inserts needs can have medicinal or further feature.Foreign gene can get with the ordinary method preparation, as synthetic, and is got by natural origin extraction, clone etc.
" homology " this term refers to one section Nucleotide as used herein, and it wherein has more than 70% regional identical with the other end nucleotide sequence at least, the homologous recombination of can uniting between them.
These two terms of " tumour-specific genetic expression " and " tumour-specific " are meant the expression of adenovirus carrier in tumour cell as used herein.Though this carrier also has expression in normal cell, expression level is very low, makes carrier duplicate and packs and can't effectively carry out, to such an extent as to low like this copy package level can be ignored.
" first-generation adenovirus carrier " or " first-generation recombinant adenoviral vector " this term is meant any adenovirus carrier as used herein, has deleted E1 and/or E3 district gene, causes not reproducible of adenovirus carrier.
The structure of example I recombinant adenovirus M003 and M005
1.M003 the structure of virus
The structure of M003, M005 two recombinant adenovirus has below been described.They all are to have the 5 type adenovirus carriers of being inverted tumor-necrosis factor glycoproteins, and wherein the M003 recombinant adenovirus contains the E1a of function gene, and M005 contains non-functional E1a gene.
Unless otherwise indicated, the technology used in the present invention all is this area routine techniquess, and as molecule clone technology, microbiological technique, cytobiology technology, these technology all are fully explained in the literature.As " molecular cloning laboratory manual " second edition (1989) of Sambrook etc. etc.
The structure of plasmid vector: the method with point mutation constructs an AgeI restriction enzyme site before pXC1 plasmid E1a gene, with AgeI and HpaI restriction enzyme the E1a gene is cut down, and reclaims gene fragment.With BstXI and BsrGI restriction endonuclease cutting pIRES2-EGFP plasmid, reclaim the plasmid fragment that contains the IRES gene, reclaim fragment with two and connect, constitute a new plasmid pIRES.E1a.
With EcoRI and NsiI restriction endonuclease cutting plasmid pLAPSN, reclaim the dna fragmentation that contains the AP gene.With EcoRI and PstI restriction endonuclease cutting plasmid pIRES.E1a, reclaim big fragment, be connected with the AP gene fragment, constitute plasmid pAP.IRES.E1a.
Cut pAP.IRES.E1a with FspI, reclaim 3.6kb length fragment, this is for expressing assembly AP.IRES.E1a.With AvrII and B1pI cutting plasmid pAd.IR.BG, reclaim big fragment, AP.IRES.E1a is connected with the expression assembly, constitutes plasmid pM003.Adopt the calcium phosphate precipitation method with plasmid pM003 and plasmid pBHG10 cotransfection 293 cells, cell surface is spread 0.5% agar, include 1XMEM, 7% foetal calf serum places 37 ℃, 5%CO
2Incubator is cultivated, and after about 9 days, plaque appears in cell under the culture dish agar layer, and the picking plaque increases, and extracts recombinant dna, carries out the DNA restriction analysis, determines correct recombinant adenovirus poison strain.
2.M005 the structure of virus
The step of the construction process of M005 recombinant adenovirus and M003 virus is in full accord, just when the first step is cloned the E1a gene is oppositely linked to each other with the IRES gene, causes the E1a gene not express.
M003 and M005 recombinant adenovirus belong to the adenovirus carrier (Ad.IR) that activates the generation reorganization by duplicating, as shown in Figure 2, two viruses of the same race are being inverted generation reorganization between the tumor-necrosis factor glycoproteins (IR), produce the expression unit of a weak point, the promotor in left side is replicated and is connected to the right side that tumor-necrosis factor glycoproteins is inverted on the right side, make the oppositely insertion can not expressed exogenous gene (being the E1a gene) here, expressed.Because this carrier homologous recombination only occurs in the tumour cell,, external source also only occurs in the tumour cell so inserting the specific expressed of gene.
Embodiment 2
The E1a gene promotes M003 virus to duplicate intracellular
M003 and M005 recombinant adenovirus and wild contrast virus are infected the 293PMT cell respectively, preparation M003 and the M005 virus that methylates, culture condition is 37 ℃, DMEM nutrient solution, 10% foetal calf serum, 5%CO
2Purified virus adopts cesium chloride density gradient ultracentrifugation, two times centrifugal purifying.
Methylate virus and contrast virus of M003 and M005 is infected the SKHepI cell respectively, extract total DNA in the cell, cut with restriction endonuclease HindIII+XbaI enzyme and spend the night the separation of 0.8% agarose electrophoresis in metainfective different time points.With the hybridization of adenoviral gene fragment radioactive probe, do nucleic acid seal stain (Southern Blot) analysis.The result as can be seen from Fig. 3, expresses the M003 virus of E1a gene as shown in Figures 3 and 4, and its levels of replication and wild-type adenovirus are suitable, and M005 virus and contrast non-replicating adenovirus carrier do not have generation significantly to duplicate phenomenon.Result from different time points, the duplicate situation of wild-type adenovirus in the SKHep1 cell is, 24 hours viral replication in behind infective virus strengthen gradually, M003 recombinant adenovirus being replicated in metainfective 36 hours and reaching the highest gradually in the SKHep1 cell, and the M005 recombinant adenovirus does not effectively duplicate in the SKHep1 cell.These results show, the M003 viral genome of expressing the E1a gene is finished homologous recombination in cell after, the E1a gene is by effective expression, have quickened viral genome and have duplicated in that SKHep1 is intracellular.
The E1a gene promotes the fragmentation effect of M003 virus to tumour cell
With M003 virus, non-replicating adenovirus carrier and wild-type adenovirus are with different multiplicity of infection (MOI=3,10,30,100,300,1000) infect SKHep1 cell (not supporting the non-replicating adenovirus carrier to duplicate), LOVO cell (human large intestine cancer cell strain), HeLa cell (people official's neck JEG-3), with 293 cells (human embryonic kidney cell that 5 type adenovirus transform supports the non-replicating adenovirus carrier to duplicate), above cell is incubated at respectively in 96 orifice plates, culture condition is the DMEM substratum, 10% foetal calf serum, 37 ℃, 5%CO
2After 48 hours,, carry out violet staining, the results are shown in Figure 5 cell fixation.As can be seen from Fig. 5, for the SKHep1 cell, tumour cell, M003 virus has close lethality with wild-type adenovirus, but not the replication type adenovirus carrier is to the SKHep1 cell, the LOVO cell, the lethality of HeLa cell has been compared very big difference with M003 virus and wild-type adenovirus, shows that the E1a expression of gene has promoted the fragmentation effect of M003 recombinant adenovirus to tumour cell.
Embodiment 4
The E1a gene promotes in the test in animal body
The diffusion of M003 recombinant adenovirus between tumour cell
By B17 mouse portal vein inoculation people official neck cancer cells HeLa cell, after three weeks, form metastasis model in the human tumor cells liver in the Mouse Liver.
M003 and M005 recombinant adenovirus are incubated in 293 cells, and culture condition is the DMEM substratum, 10% foetal calf serum, 37 ℃, 5%CO
2Use the cesium chloride density gradient centrifugation purified virus, 35,000rpm two times centrifugal purified virus contains 1mM MgCl with rare being assigned in of virus at last
2, 10mM Tris, pH7.5 is in the solution of 10% glycerine.
Inject M003 and M005 recombinant adenovirus respectively by the tail vein of tumor model mouse in the liver, two days later injection once more.Kill mouse after three days,, carry out alkaline phosphatase staining, the results are shown in Figure 6 hepatic tissue section.
As seen from Figure 6, the all specific alkaline phosphatase gene of in tumour cell, expressing of M003 and M005 recombinant adenovirus, M003 virus is owing to E1a expression of gene, prolongation in time, the tumour cell dyeing of expressing alkaline phosphatase gene is a bunch shape, shows the diffusion of virus between tumour cell.This explanation E1a gene has promoted to duplicate adenovirus carrier (Ad.IR) specific expression and the diffusion between tumour cell in tumour cell that activates homologous recombination.
Although the present invention for the ease of clearly understanding by way of example mode describe in detail in some aspects, it is feasible obviously carrying out certain variation and modification within the scope of the claims.
Claims (9)
1. recombinant adenoviral vector, it has deleted E1 and/or E3 district gene, have the external source of inserting with 3 '-5 ' direction and insert gene adenovirus E 1 a gene, and insert the gene both sides in external source and have a pair of inversion multiple dna sequence dna, be inverted between the repeating sequences at two these same virus genomic these homologous recombination can take place.
2. the recombinant adenoviral vector described in the claim 1, this recombinant adenoviral vector comprises following assembly:
A. terminal repeat is inverted in one section adenovirus left side;
B. one section adenovirus packaging sequence is positioned at the 3 ' end that terminal repeat is inverted in the left side;
C. one section promoter sequence is positioned at 3 ' of adenovirus packaging sequence and holds;
D. a pair of inversion tumor-necrosis factor glycoproteins is inverted the 3 ' end that tumor-necrosis factor glycoproteins is positioned at promoter sequence for first section, and second section tumor-necrosis factor glycoproteins is positioned at 5 ' end of exogenous DNA array;
E. one section exogenous DNA array is adenovirus E 1 a gene, oppositely is connected in first section 3 ' end of being inverted tumor-necrosis factor glycoproteins with 3 '-5 ' direction;
F. at least one adenoviral gene is positioned at second section 3 ' end of being inverted tumor-necrosis factor glycoproteins;
G. terminal repeat is inverted on the adenovirus right side, is positioned at 3 ' end of adenoviral gene.
3. the adenovirus carrier described in the claim 2 wherein has one section bidirectional transcription termination site sequence behind described first section inversion tumor-necrosis factor glycoproteins 3 ' end, can block to transcribe and carry out.
4. the adenovirus carrier described in the claim 3, wherein said bidirectional transcription termination site sequence is two-way SV40 poly adenosine sequence.
5. method that produces new adenovirus carrier by the homologous recombination between the virus vector in the claim 1, introduce the virus at least 2 claims 1 in the tumour cell in vitro, can recombinate between these two viruses, produce new recombinant adenoviral vector, this new recombinant adenoviral vector contains the adenovirus E 1 a gene that can express.
6. pharmaceutical composition, but recombinant adenoviral vector any among the claim 1-4 of kill tumor cell and available pharmaceutical carrier contained.
7. composition, it comprises a kind ofly having the recombinant adenoviral vector that is selected from any one adenovirus characteristic among the claim 1-4, and a kind of chemotherapy compound.
8. composition, it comprises a kind ofly having the recombinant adenoviral vector that is selected from any one adenovirus characteristic among the claim 1-4, and a kind of immunosuppressive compounds.
9. any described recombinant adenoviral vector is used for the treatment of purposes in the pharmaceutical composition of tumour in preparation among the claim 1-4.
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| CN1453361A (en) * | 2003-05-30 | 2003-11-05 | 中国科学院上海生命科学研究院 | Construction method of virus as anticancer targeting adjustable gene |
| US9289477B2 (en) | 2011-04-29 | 2016-03-22 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers to reduce cytotoxic T lymphocyte responses |
| CA2888193A1 (en) | 2012-10-17 | 2014-04-24 | Eyal Breitbart | Treatment methods using adenovirus |
| EA201592106A3 (en) | 2013-05-03 | 2016-08-31 | Селекта Байосайенсиз, Инк. | LOCAL ACCOMPANYING INTRODUCTION OF TOLEROGENOUS SYNTHETIC NANOSATORS TO REDUCE HYPERSENSITIVITY TYPE I AND HYPERSENSITIVITY TYPE IV |
| WO2016037163A1 (en) * | 2014-09-07 | 2016-03-10 | Selecta Biosciences, Inc. | Methods and compositions for attenuating gene therapy anti-viral transfer vector immune responses |
| BR112019018748A2 (en) | 2017-03-11 | 2020-04-07 | Selecta Biosciences Inc | methods and compositions related to combined treatment with anti-inflammatories and synthetic nanocarriers comprising an immunosuppressant |
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