CN1865449A - Virus strains - Google Patents
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Abstract
The present invention provides a herpes virus with improved oncolytic properties which comprises a gene encoding an immunomodulatory cytokine and which lacks a functional ICP34.5 gene and a functional ICP47 encoding gene.
Description
The application is to be invention and created name the dividing an application for the Chinese patent application of " virus strain " (national applications number be No.01806750.6, international application no is PCT/GB01/00229) in January 22 calendar year 2001 the applying date.
Technical field
The present invention relates to for example non-zoo virus strain of simplexvirus (such as HSV), compare with the zoo virus strain, described non-zoo virus strain has improved oncolytic ability and/or gene transmission capacity.
Background technology
Propose or confirm, in many cases, virus has effectiveness in the various application of biotechnology and medical field.This is because virus has the unique ability that enters cell with high-level efficiency.This adds viral gene expression and duplicates and/or the expression of heterologous genes of inserting in such application.Thereby, the gene (perhaps virogene or other gene) that virus can or will can be used for for example gene therapy or vaccine development is delivered in the cell and at cell inner expression, and perhaps they can be used for the effect selective killing cancer cell for example by the lytic replication or the gene that transmits.
Proposed hsv (HSV) and both can be used as gene delivery vector in neural system and other system, can be used for oncolytic ruling by law again and treat cancer.Yet, in these two kinds of application, must make virus become defective type, it is pathogenic that it is no longer had, but make it still can enter cell and carry out required function.Thereby, for utilizing HSV to make nontoxic gene be passed to target cell, obviously in most of the cases, must stop the immediate early gene expression of virus or its expression is reduced to minimum.Cancer (this therapy also can comprise the gene that transmits the described treatment effect of enhancing) is treated in ruling by law for oncolytic, many sudden changes of HSV have been identified, these sudden changes of HSV still allow virus at culture or enliven in the splitted cell (for example tumour in) in vivo and duplicate, and still stop its effectively duplicating in healthy tissues.Such sudden change comprises the encoding gene that destroys ICP34.5, ICP6 and thymidine kinase.Up to now, in these mutated viruses, the virus with ICP34.5 sudden change or ICP34.5 sudden change and for example ICP6 sudden change has shown the most favourable safe distribution plan.Shown the virus that only lacks ICP34.5 external in many tumor cell types, duplicate and mouse brain tumour the artificial induction in copy choice, and do not injure surrounding tissue.Early studies in man shows that also they are safe in the mankind.
Yet though be hopeful various viruses (comprising HSV) are used for gene transmission/treat or are used for oncolytic ruling by law treating cancer, the virus strain that kept many years is used in the overwhelming majority's work in this work always in tissue culture cells.Only needing virus to enter in the application of cell with the transmission gene, this can prove unchallenged, because keep also needing virus to enter cell in cell cultures, is dissimilar or the cell of species though compare with the possible target cell of carrier usually.Yet in the application of other characteristic of needs, application experiment chamber virus strain can not make virus have whole potential in the concrete application of need utilization.
HSV has unique ability in the present virus of just developing as carrier, and this is because the natural infection neurone that is evolved into of HSV is also hidden hereinto.HSV also is evolved into from infection site (usually in periphery) and is transported to the pericaryon that is usually located at the spinal ganglion along neural with high-level efficiency.Do not need this ability in cell culture, because this ability needs the characteristic of the special evolution of HSV, further adapting to grows in culture may cause the most effective aixs cylinder transportcapacity of forfeiture.If keep the aixs cylinder transportation characterization with maximum efficiency always, the hsv vector that then is used for gene is passed to central nervous system or peripheral nervous system may show maximum efficiency.At this, inoculation then can allow most effectively gene to be passed to the peripheral neurons cell space in the peripheral part position, then may allow most effectively gene to be passed to the position of a plurality of connections in intracranial inoculation.May not allow based on the carrier of laboratory HSV strain at present this incident takes place with possible maximum efficiency.In fact, because HSV is strong along the ability of nerve transportation, therefore between the conservative characteristic of needs and the characteristic that may in culture, keep, may there be king-sized difference.
HSV and other virus (for example adenovirus or rheovirus) also have potential effectiveness aspect the oncolytic ruling by law treatment cancer.Yet, be used for these purpose viruses and before kept at culture widely always developing.Because oncolytic ruling by law is treated cancer and need relatively slowly effectively duplicated in the human tumor cells of growth usually, therefore expection adapts to the zoo virus strain to grow in specific culturing cell, may reduce the efficient of lytic replication in may the suitableeest generation human tumor cells or the efficient of infected person tumour cell.
Summary of the invention
The invention provides the chance of the improved virus of ability in exploitation gene transmission and/or the tumoricidal body of cracking performance.At this,, make up the virus strain that is applicable to these purposes based on the up-to-date clinical separation strain of suitable virus rather than the laboratory strain of the previous continuous passage of using always.Therefore, potentiality of the present invention provide the virus with following characteristic: the ability of infected person cell is improved, is improved and (with regard to HSV with regard to) is transported to the ability improvement of pericaryon from inoculation position along nerve in this class time multiplexed cell system/cracked ability in the body.The present invention utilizes HSV to illustrate, but is equally applicable to just developing at present as carrier and/or is used for other virus that the oncolytic method is destroyed cancer cell.
We are verified, compare with HSV1 strain 17+ (standard laboratory strain), and two kinds of HSV1 clinical separation strains (strain JS1 and BL1) have strengthened duplicating in some human tumor cell line.
We make clinical separation strain JS1 disappearance ICP34.5, and then compare with the HSV1 strain 17+ (standard laboratory strain) that wherein also lacks ICP34.5, compare the potential that duplicates in the human tumor cells type.This strain (JS1/ICP34.5-) is a kind of modified strain that derives from clinical separation strain, and is a kind of modified non-laboratory strain of the present invention therefore.
Compare with the HSV1 ICP34.5 disappearance strain 17+ (being the standard laboratory virus strain) with identical modification, the JS1 with ICP34.5 disappearance demonstrates growth enhancing in some tested person's tumour cell.Yet, to compare with the laboratory strain that derives from strain 17+, JS1/ICP34.5-virus strengthens the cell killing ability in all tested tumor cell lines.
Therefore, can observe the non-zoo virus strain of application and strengthen the anti-tumor capacity of this viroid, and in all tested so far tumor cell lines, be confirmed.This will be applicable to the cancer therapy of human patients.
If these viruses are used for transmitting the gene with anti-tumor activity subsequently, then also can expect further enhanced activity.Such gene comprises that coding prodrug activator, tumor suppressor protein or short apoptosis factor (pro-apoptotic factors) or immunity irritate proteic gene.
For this purpose, we have produced the ICP34.5 absence type clinical separation strain of the HSV1 of expressing human GMCSF.This virus is used for strengthening after the injection anti-tumor immune response in tumour.
The present invention also provides the virus of the present invention of carrying one or more heterologous genes.The term heterologous gene is included in non-existent any gene in the described viral genome.Described heterologous gene can be any allelic variation body of wild type gene, and perhaps it can be a mutator gene.Preferably heterologous gene effectively is connected with the control sequence that allows the inherent cell inner expression of described heterologous gene body.Therefore, virus of the present invention can be used to will allow in the cell of its expression being passed in one or more heterologous gene bodies.For the oncolytic virus therapy, the albumen of the tumor destruction characteristic that the common coding of such gene can enhanced virus.These genes can encode himself have Cytotoxic albumen, the prodrug activator or can encode and can irritate/strengthen the albumen of anti-tumor immune response of can encoding.For utilizing HSV that gene is passed to peripheral nervous system, described one or more heterologous genes can be encoded to change pain stimulation is replied or the polypeptide of relieve chronic pain, for example can completely cut off for example albumen or Substance P or other neuropeptide of nerve growth factor, other regulating pain neurotrophic factor or neurotrophic factor sample molecule.Described one or more heterologous genes also can be encoded can stimulate injured nerve regrowth in the degenerative disease or prevent the neural further polypeptide of sex change.In central nervous system, heterologous gene can comprise those have potential benefit in neurodegenerative disease (for example Parkinson's disease or presenile dementia) gene, and can comprise usually that in this class disease coding can strengthen the neurotrophic factor of all the other cytoactives and/or the gene of enzyme.In all cases, a kind of virus can be carried one or more heterologous genes.
Therefore, the invention provides:
Use modified non-laboratory oncolytic virus strain and produce the medicine that cancer is treated in oncolytic ruling by law;
Use modified, no replication, as to comprise a kind of heterologous gene non-zoo virus strain and produce the medicine that is used for described gene is passed to the curee;
A kind ofly determine that a kind of gene is whether to the phenotype relevant with peripheral nervous disease or to the influential method of the cell of the peripheral nervous system relevant with peripheral nervous disease, described method comprises:
(i) simplexvirus of the present invention that will comprise a kind of heterologous gene, no replication is inoculated in the peripheral nerve; With
(ii) monitor the influence of the phenotype of described disease or described expression of gene, determine thus whether described gene has and described disease related effect described cell;
A kind ofly determine that a kind of gene is whether to the phenotype relevant with central nervous system disease or to the influential method of the cell of the central nervous system relevant with central nervous system disease, described method comprises:
(i) simplexvirus of the present invention that will not have a replication is inoculated in the cell of central nervous system; With
(ii) monitor the influence of the phenotype of described disease or described expression of gene, determine thus whether described gene is influential to described cell or described phenotype described cell;
Whether a kind of definite a kind of gene is encoded and is caused a disease and infect or the relevant antigenic method of cancer, described method comprises with the virus infection dendritic cell of the present invention or the scavenger cell that comprise a kind of heterologous gene of coding for antigens, no replication, and monitor the antigen presentation of polypeptide product of described gene or the phenotype of described expression of gene effect or described pathogenic infection or cancer, determine whether the encode antigen relevant and himself whether have treatment potential or the target of therapeutic interference of described gene thus with described infection or cancer;
A kind ofly measure non-zoo virus strain for the adaptive method that is modified into modified as defined herein strain, described method comprises:
(i) randomly from the host, separate non-zoo virus strain;
Described non-zoo virus strain (ii) is provided;
(iii) estimate the characteristic of described virus aspect one or more desired characteristics; And randomly,
(iv) select for the virus strain with ideal behavior of modifying usefulness;
A kind ofly measure non-zoo virus strain for the adaptive method that is modified into the modified oncolytic strain of the present invention, described method comprises:
(i) randomly from the host, separate non-zoo virus strain;
(ii) estimate the growth of described virus in the tumour cell of one or more types; And randomly
(iii) select for the virus strain with high growth rates or cell killing ability of modifying usefulness;
A kind ofly measure non-zoo virus strain for the adaptive method that is modified into modified oncolytic strain, described method comprises:
(i) provide a kind of non-zoo virus strain, randomly it is selected with the method that limits just now;
(ii) modify described strain, make it become the oncolytic strain; With
(iii) estimate the ability of described modified non-laboratory oncolytic strain killing tumor cell; And randomly
(iv) option table reveals the strain of high tumor cytotoxicity ability, is used for further modification; And randomly
(v) further modify;
A kind ofly determine the whether method of the anti-tumour effect of enhanced virus of a kind of gene, described method comprises:
(i) provide a kind of the present invention modified non-laboratory oncolytic strain;
(ii) described gene is inserted in the described virus as heterologous gene; With
(iii) compare, estimate the ability of described modified non-laboratory oncolytic strain killing tumor cell with the ability of the precursor strain that provides in the step (i);
A kind of method that produces modified non-laboratory oncolytic virus strain, described method comprises:
(i) from the host, separate a kind of non-zoo virus strain;
(ii) optional definite to its adaptability of modifying as defined above; With
(iii) it is modified, make it become oncolytic virus; And randomly
(iv) further modify;
A kind of method that produces modified non-zoo virus strain, described method comprises:
(i) provide a kind of non-zoo virus strain;
(ii) it is modified, make its virus that becomes no replication and
(iii) insert a kind of heterologous gene;
A kind of non-laboratory oncolytic virus strain as defined herein, modified;
A kind of as defined herein, that comprise a kind of heterologous gene, modified non-zoo virus strain;
Application is accredited as to the phenotype relevant with peripheral nervous disease or to the influential gene of the cell of the peripheral nervous system relevant with peripheral nervous disease or by the gene product of described genes encoding, the medicine of production for treating peripheral nervous disease by the method that is defined as above;
Application is accredited as to the phenotype relevant with central nervous system disease or to the influential gene of the cell of the central nervous system relevant with central nervous system disease or by the gene product of described genes encoding, the medicine of production for treating central nervous system disease by the method that is defined as above;
Application is accredited as the coding antigenic gene relevant with cause a disease infection or cancer by the method that is defined as above or by the antigen of described genes encoding, production for treating or prevent the medicine of described infection or cancer;
A kind of non-zoo virus strain of identifying or producing in the methods of the invention by the inventive method;
Application be accredited as by the method that is defined as above enhanced virus anti-tumour effect gene or by the gene product of described genes encoding, the medicine of production for treating or preventing cancer;
A kind of modified non-zoo virus strain that obtains or can obtain by the inventive method by the inventive method;
HSV1 strain JS1 is preserved in European cell culture preservation center (ECACC), and interim preserving number is 01010209; Or by its deutero-HSV1 strain; The medicinal compositions that comprises this virus; This virus that is used for the treatment of the human or animal;
A kind of method for the treatment of the individual in-vivo tumour that needs described treatment, described method comprises the oncolytic virus of the present invention that gives described individual effective dose;
A kind of a kind of gene is passed to the method for the individuality that needs described gene, described method comprises the non-oncolytic virus of the present invention who gives described individual effective dose; With
The method of a kind of treatment or prevention maincenter peripheral nervous disease, described method comprise the peripheral nerve that the neural virus of the present invention parent of significant quantity is needed the individuality of described treatment or prevention.
Description of drawings
Fig. 1. virus
From top to bottom, sketch diagram: laboratory HSV1 strain 17+, clinical strain BL1, clinical strain JS1,17+/ICP34.5-, JS1/ICP34.5-, JS1/ICP34.5-/ICP47-/hGMCSF.
Fig. 2. clinical separation strain is presented at growth enhancing in the tumour cell
(1) growth of 17+, BL1 and JS1.Left figure: U87 cell.Right figure: LNCaP cell.
(2) ICP34.5-17+ and the JS1 growth on tumour cell.Left figure: LNCaP cell.Right figure: MDA-MB-231 cell.
(3) JS1/34.5-does not grow on the non-permissive cells of HSV ICP34.5 mutant strain.
Left figure: 3T6 cell-17+, JS1.Right figure: 3T6 cell-17+, JS1ICP34.5-.
Fig. 3 .ICP34.5 absence type HSV clinical separation strain is presented at cracking enhancing in all tested tumour cells
Tumor cell line carries out simulated infection, infects or with HSV1 strain JS1/34.5-infection with HSV1 strain 17+/34.5-with the MOI of appointment, then after infection in the different time points violet staining, cell is manifested. Each square frame in the photo represents a kind of cell type. From top to bottom, be respectively HT29 colorectal adenocarcinoma, LNCaPFGC prostate cancer, MDA-MB-231 breast cancer, SK-MEL-28 chromoma and U-87MG spongioblastoma astrocytoma. The left frame represents the result of HSV1 strain 17+/34.5-. Right-hand frame represents the result of HSV1 strain JS1/34.5-. The infected cell of middle square frame representative simulation. In each square frame, lastrow represents 24 hours point, and the second row represents 48 hours point, the third line represents 72 hours point, and in each square frame, one hurdle, the left side represents MOI=0.2, a middle hurdle represents MOI=0.1, and one hurdle, the right represents MOI=5.
Detailed Description Of The Invention
A. viral
Strain of the present invention
The present invention generally is applicable to virus. Preferred Strain of the present invention is herpes virus strains, adenovirus strain, picornavirus strain, retroviruse strain or Alphavirus strain. More preferably Strain of the present invention is herpes virus strains. More preferably Strain of the present invention is herpes simplex virus (HSV) strain, is generally HSV1 strain or HSV2 strain again.
When virus of the present invention was herpes simplex virus, described virus can derive from for example HSV1 strain or HSV2 strain or their strain of deriving, preferably HSV1. The strain of deriving comprises and containing from (inter-type) recombinant between the type of the DNA of HSV1 strain and HSV2 strain. Recombinant has description in this area between this type, has described recombinant between this type such as (1988) such as (1998) such as Thompson and Meignier. Derive strain and HSV1 genome or the genomic sequence homology of HSV2 is preferably at least 70%, and more preferably at least 80%, even more preferably at least 90% or 95%. More preferably, the genomic sequence homogeneity of strain and HSV1 genome or HSV2 of deriving is at least 70%, more preferably at least 80% homogeneity, even more preferably at least 90%, 95% or 98% homogeneity.
Provide the BESTFIT program (such as using its default setting) that can be used for calculating homology (Devereux etc. (1984) Nucleic Acids Research 12, the 387-395 pages or leaves) such as the UWGCG program package. PILEUP and BLAST algorithm can be used for calculating homology or with series arrangement (usually using its default setting), for example as at Altschul (1993) J.Mol.Evol.36:290-300; Described in Altschul etc. (1990) J.Mol.Biol.215:403-10.
Be used for carrying out the software public that BLAST analyzes can pass through National Centre for Biotechnology Information (http：//www.ncbi.nlm.nih.gov/) obtain. This algorithm comprise at first be tested and appraised in search sequence with database sequence in the word string of equal length when comparing or coupling or satisfy the short word string of the length W of the minimum score value T of some positive, identify high sub-sequence pairing (HSP). T is called as adjacent word string score value threshold value (Altschul etc., 1990). These original adjacent word strings are hit the seed that contains their HSP as initial search with discovery. Word string is hit with both direction and is extended along each sequence, as long as accumulated sequence comparison score value can increase. Hitting extension in the word string of each direction stops when running into one of following situation: accumulated sequence comparison score value is than the low quantity X of maximum of its acquisition; The accumulation score value be zero or below, this is because due to the accumulation of one or more negative marking residues comparisons; Or arrive the end of each sequence. BLAST algorithm parameter W, T and X determine sensitivity and the speed of described comparison. The default word length (W) that the BLAST program is used is 11, uses BLOSUM62 marking matrix (referring to Henikoff and Henikoff (1992) Proc.Natl.Acad.Sci.USA89: 10915-10919) comparison (B) is 50, and desired value (E) is 10, M=5, N=4, and compare two chains.
The BLAST algorithm carries out statistical analysis to the similitude between two sequences; Referring to for example Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA90: 5873-5787. It is minimum and probability (P (N)) that a kind of similitude that the BLAST algorithm provides is measured, and it provides the accidental index that the probability of coupling occurs between two nucleotide sequences or amino acid sequence. For example, if when a sequence and another sequence are compared minimum and probability less than about 1, preferably less than about 0.1, be more preferably less than about 0.01, most preferably less than about 0.001, then think described First ray and the second sequence similarity.
The strain of deriving can have by nucleotides and replaces for example 1,2 or 3 to 10,25,50 or 100 HSV1 genome sequence or HSV2 genome sequences that replace modification. HSV1 genome or HSV2 genome can or or in addition by one or more insertions and/or disappearance and/or by extending to modify at arbitrary end or two ends.
The characteristic of Strain of the present invention
Strain of the present invention is " non-laboratory " strain. They also can be called as " clinical " strain. Those skilled in the art can be easily make a distinction laboratory strain and non-laboratory strain or clinical strain. In addition, hereinafter provided the characteristic guide that Strain may show.
Key difference between laboratory strain and the non-laboratory strain is that laboratory strain commonly used has at present kept the long duration in culture, be many years in some cases. The cultivation of virus (for example HSV) relates to the technology that is called as continuous passage. In order to make viral growth and maintenance, with the suitable cell of virus infections, virus is in time multiplexed cell system, and then results are viral; Then the new fresh cell of re-infection. This process consists of the one-period of continuous passage. With regard to HSV, each such cycle may need for example several days. As discussed above, this continuous passage may cause the change of Strain characteristic, because the selection to the characteristic (for example quick copy) that is conducive to grow in culture occurs, rather than select and the characteristic that can be used for practical application the ability of for example with regard to HSV with regard to, transporting along aixs cylinder for maintenance.
Strain right and wrong of the present invention laboratory strain is because they derive from the strain of up-to-date separation from infected individual. Compare with original clinical separation strain, strain of the present invention is modified, may need to cultivate a period of time, but it is shorter to cultivate the used time. Prepare strain of the present invention with such method, make it basically keep the ideal characterisitics of the original clinical separation strain that they originate.
Virus of the present invention can effectively infect human target cell. Such virus is just separated from infected individual, then strengthen in external and/or body the required ability in tumour cell and/or other time multiplexed cell system and screen according to comparing with the standard laboratory strain, perhaps (with regard to the neural virus of parent HSV opinion for example) adopts the body inner model to screen according to comparing with the standard laboratory strain to strengthen along neural ability of transporting. Compare with the zoo virus strain, having this viroid of improving characteristic is virus of the present invention. Then can be to having this required improvement characteristic, being undertaken engineered through the virus of identifying by the sudden change of suitable gene, make their optionally killing tumor cells, perhaps with its sudden change, make them in non-oncolytic is used, gene delivery not had poisonous effect to target tissue. These modified viruses also are virus of the present invention. Perhaps, Strain also can be separated from infected individual, and it is expected the sudden change that is applicable to oncolytic method treatment and/or gene delivery. Then these modified viruses are screened according to the required improvement characteristic of comparing with laboratory strain, the virus with this improvement characteristic provides other the present invention's virus.
The following further guide that the possible characteristic of Strain of the present invention is provided.
Preferred Strain of the present invention is separated its not modified clinical precursor strain and is counted and experience below 3 years or 3 years in culture from its host. More preferably strain of the present invention experience below a year or a year in culture, for example below nine months or nine months, six months or below six months, three months or below three months or below two months or two months, one month or below one month, two weeks or two weeks are following or week or below the week. These definition of time refer to the time actual used in culture in culture. Therefore, for example, common way is that Strain is freezing, in order to preserve them. Obviously, preserve by freezing preservation or with equivalent way and do not regard as and in culture, keep strain. Thereby, used time of freezing preservation or preserve the used time with other method and be not included in the scope of above-mentioned definition of used time of culture. Normally actual experience used time of continuous passage, the i.e. time during can selecting undesired feature of used time in culture.
Preferred Strain of the present invention is separated its not modified clinical precursor strain and is counted and experienced 1,000 or continuous passage cycle still less from its host. More preferably it experienced 500 or still less, 100 or still less, 90 or still less, 80 or still less, 70 or still less, 60 or still less, 50 or still less, 40 or still less, 30 or still less, 20 or still less or 10 or such cycle still less.
Preferably, learn check according to canonical statistics and measure, to carry out some ability that can be used for the present function of application strong than having the Reference Lab strain that is equal to modification for the present invention's virus. For example, with regard to being used for the oncolytic virus of oncotherapy, preferred Strain of the present invention than have any tumour cell of Reference Lab virus strain infection that is equal to modification or copy therein, the ability of killing tumor cell or the propagation of the iuntercellular in tissue is strong. More preferably this stronger ability is the stronger ability of tool significance,statistical. For example, according to the present invention, with regard to characteristic to be measured, the ability of Strain of the present invention can be 1.1 times at the most, 1.2 times, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times or 100 times of ability of described reference strain.
Preferably, with regard to one or more were characterized as the characteristic that can be used for using at present, virus of the present invention had the ability of (namely keeping) its not modified clinical precursor strain basically. For example, with regard to the oncolytic virus that plan is used for treating tumour, Strain of the present invention preferably basically have its not modified clinical any tumour cell of precursor virus strain infection or copy therein, ability that killing tumor cell or the iuntercellular in tissue are propagated.
Preferably, according to the present invention, virus keeps the characteristic of its not modified clinical precursor strain basically, if in quantitative test, with regard to characteristic to be measured, it keeps 75%, more preferably 80%, 90%, 95%, 98%, 99% or 100% of not modified clinical precursor strain ability. More preferably with regard to characteristic to be measured, any difference between the modified strain of described not modified clinical precursor strain and the present invention does not have significance,statistical.
Can use standard test, for example t-check, ANOVA or χ2Check is carried out statistical analysis to characteristic described herein. Usually, the significance,statistical level of measurement is p=0.05 (5%), more preferably p=0.01, p=0.001, p=0.0001, p=0.000001.
Modify
Virus of the present invention is compared normally modified with clinical strain before it. Specifically, usually so that some gene nonfunctional, and described virus also comprises heterologous gene. Usually, make viral attenuation of the present invention.
The hiv region that changes according to purpose described herein or can be eliminated (wholly or in part) or become non-functional or replaced, especially replaced by the heterologous gene sequence by other sequence. Can make one or more genes become nonfunctional gene, and can insert one or more heterologous genes.
Embodiment
Oncolytic virus of the present invention
In one embodiment, virus of the present invention is modified non-laboratory oncolytic virus.These viruses can be used for oncolytic ruling by law and treat cancer.This viroid infected tumor cell also duplicates in described tumour cell, kills and wounds described tumour cell subsequently.Therefore, this viroid is the virus with replication.Preferably their selectivity have replication in tumour cell.This means them in tumour cell, to duplicate and in non-tumor cell, do not duplicate that perhaps they more effectively duplicate in non-tumor cell in the tumour cell internal ratio.The mensuration of copy choice ability can be carried out in order to the test of measuring replication and tumor cytotoxicity ability by described herein,, also can analyze it by the statistical technique that this paper enumerates if needed.
Preferred oncolytic virus of the present invention is than the Reference Lab virus strain infection tumour cell with identical modification or duplicate in tumour cell, ability that killing tumor cell or the iuntercellular in tissue are propagated is strong.More preferably this ability is the stronger ability of tool significance,statistical as described herein.Described virus strain can be measured with any method known in the art about the characteristic of tumour cell.
For example, the ability of virus infection tumour cell can be measured the required viral dosage of given cell percentage (for example 50% or 80% cell) and carries out quantitatively by being measured as.The ability of duplicating in tumour cell can be measured by growth, the mensuration (referring to Fig. 2) of for example being carried out in an embodiment is for example by measuring in the time cycle of 6 hours, 12 hours, 24 hours, 36 hours, 48 hours or 72 hours or longer time at intracellular viral growth.
The ability of virus killing tumor cell can be passed through naked eyes roughly quantitative (referring to Fig. 3), and is perhaps quantitative more accurately by the viable count that keeps under point and the MOI in preset time for given cell type is at the appointed time counted.For example, can and use any known cancer cell type to compare in 24 hours, 48 hours or 72 hours.Specifically, can use HT29 colorectum adenocarcinoma cell, LNCaP.FGC prostate cancer cell, MDA-MB-231 breast cancer cell, SK-MEL-28 malignant melanoma cell or U-87MG glioblastoma astrocytoma cell.Any combination of any or these cell type in these cell types can be used, also other tumor cell type can be used.May preferably make up the one group of standard tumor cell type that is used for this purpose.For the viable count that keeps at preset time point is counted, can count the number of the cell (being viable cell) of getting rid of Trypan Blue.Also available fluorescence amplifying cell separator (FACS) or MTT analyze and carry out quantitative assay.Measure the tumor cytotoxicity ability in also can body, for example by measuring because the reducing of the gross tumor volume that specific virus causes.
Virus also can be measured by the cell count of measuring the position that links to each other with original infection site in tissue, the ability especially propagated in solid tissue.
In order to measure the characteristic of virus of the present invention, generally speaking, preferably use standard laboratory reference strain to compare.Can use any suitable standard laboratory reference strain.With regard to HSV, preferably use among HSV1 strain 17+, HSV1 strain F or the HSV1 strain KOS one or more.Described reference strain has the modification that is equal to the present invention's strain to be measured usually.Therefore, described reference strain has the modification of being equal to usually, and for example genetically deficient and/or heterologous gene insert.For example, with regard to the HSV strain, if it is non-functional that ICP34.5 and ICP47 encoding gene are become, it is non-functional then in the reference strain they to be become.Can be identical to the modification that the reference strain carries out with the modification that strain of the present invention is carried out.For this reason, mean that gene disruption in the reference strain is arranged in the position that the gene disruption with strain of the present invention is equal to fully, for example disappearance is for identical size and be positioned at same position.Equally, in these embodiments, heterologous gene can insert in same position, be driven or the like by identical promoters.Yet, not necessarily need to carry out identical modification.Importantly the reference gene has the modification that is equal on the function, for example makes identical gene become non-functional and/or inserts one or more identical heterologous genes.
In oncolytic virus of the present invention, can carry out suitable modification to described virus, make it have oncolytic activity, if it is non-naturally occurring, preferably make it have the selectivity oncolytic activity.
With regard to HSV, provide this class sudden change of selectivity oncolytic activity to comprise ICP34.5, ICP6 and/or thymidine kinase (TK), the preferably sudden change of ICP34.5 encoding gene.ICP34.5 is non-functional any sudden change although can use wherein, and this class sudden change of the ICP34.5 encoding gene of the laboratory strain of relevant HSV has description in Chou etc. 1990, Maclean etc. 1991.
Therefore, in the HSV strain, preferably described virus is modified, make it lack one or more in functional ICP34.5 encoding gene, functional ICP6 encoding gene, functional glycoprotein h encoding gene, the functional thymidine kinase encoding gene; Perhaps in non-HSV strain, described virus lacks the functioning gene that is equal to one of described HSV gene.
More preferably described virus lacks functional ICP34.5 encoding gene.
Also can carry out other modification.Specifically, with regard to HSV, can modify, make it lack functional ICP47 gene described virus.This is because the common function of ICP47 is the intracellular antigen presentation that blocking-up is infected by HSV; produce such virus so it destroys: this virus is not given infected tumour cell particular characteristics, promptly can protect the characteristic of the cell resistance host immune system that is subjected to this HSV infection.
It also is virus of the present invention that the virus of any other genetically deficient/sudden change of oncolytic characteristic (promptly comparing copy choice in tumour with surrounding tissue) is provided, as what one skilled in the art will realize that, above-mentioned tabulation is non-limit, and may point out structure to the evaluation of other gene function of above-mentioned arbitrary virus also is the new virus of virus of the present invention.
Also available technology known in the art and/or technology described herein are inserted into heterologous gene in this viroid of the present invention.In oncolytic virus, described heterologous gene normally strengthens the gene of described virus to antineoplastic ability.Therefore can insert any gene of giving viral antitumor characteristic.Specifically, described heterologous gene can be to improve gene, especially immunostimulation polypeptide for example CD40L, rHuGM-CSF (GMCSF), other cytokine or chemokine (for example RANTES), B7.1 or B7.2 or IL12 at the immunne response of tumour cell in useful mode.Perhaps, can encode prodrug activator, for example nitroreductase or Cytochrome P450 of described heterologous gene.In text of the present invention, imagination is used by described prodrug activator activated prodrug and virus of the present invention and is come the combination therapy tumour.Perhaps, described heterologous gene can codes for tumor arrestin, for example p53.
Other virus strain of the present invention
In other embodiments, non-oncolytic virus meets the requirements.These viruses can be the virus of no replication.Its function normally is passed to individuality with heterologous gene.
Specifically,, can suddenly change, make the expression of viral modulability immediate early gene reduce to minimum in non-oncolytic is used, being used as carrier.Thereby, can separately or unite and make ICP4, ICP27, ICP22 and/or ICP0 encoding gene inactivation or disappearance, perhaps the sudden change in virosome trans-activator vmw65 comprises its trans-activation ability of prevention/reduction.Be used for the particularly preferred embodiment that non-oncolytic is used, make ICP27, ICP0 and ICP4 encoding gene disappearance (having or do not have extra ICP22 and/or ICP47 disappearance/inactivation), perhaps ICP27 and ICP4 are lacked the inactivation that adds among the vmw65, perhaps the ICP4 disappearance adds the inactivation among the vmw65.The example of this viroid comprises the viruses of 1999 reports such as Samaniego etc. 1998, Krisky etc. 1998 or Thomas.
The neural virus of parent
When heterologous gene is passed to neural system, can use parent neural virus, especially hsv for example HSV1 and HSV2 according to the present invention.
According to the virus of this embodiment of the present invention usually than have the neuronic ability of Reference Lab virus strain infection that is equal to modification, the ability or the ability of transporting in aixs cylinder of iuntercellular propagation in nervous tissue is strong.Infecting neuronic ability generally can measure according to above-mentioned described method about cell.The ability that the ability of transporting in aixs cylinder or the iuntercellular in nervous tissue are propagated can be measured by the infection of cell in the neural system of measuring the position that links to each other with original infection site.Can carry out statistical analysis to the result as mentioned above.
In these embodiments, be preferably in the HSV strain, described virus is modified, make it lack in functional ICP27 encoding gene, functional ICP4 encoding gene, functional ICP0 encoding gene or the functional ICP22 encoding gene one, two, three or all; Perhaps in non-HSV strain, described virus lacks the functioning gene that one of a kind of and described HSV gene is equal to; And/or in the HSV strain, described virus lacks functional vmw65 gene, and this is owing to eliminate in described gene due to the sudden change of its transcriptional activation activity; Perhaps in non-HSV strain, described virus lacks the functioning gene that is equal to the vmw65 gene, and this is owing to eliminate in described gene due to the sudden change of its transcriptional activation activity.
It is non-functional that in ICP27, ICP4, ICP0 and the ICP22 encoding gene two or more are become, and more preferably makes three, more preferably makes whole four encoding genes become non-functional again.Preferably, according to these embodiments, virus of the present invention had both lacked functional ICP4 encoding gene, lacked functional ICP27 encoding gene again, and virus of the present invention has an inactivation sudden change of eliminating its transcriptional activation activity in the vmw65 encoding gene.
This viroid can be used for treating peripheral nervous disease or central nervous system disease.Under the situation of central nervous system disease, especially preferably make at least two immediate early genes that are selected from ICP0, ICP4, ICP22 and ICP27 become non-functional.
Immunotherapeutical virus
In the immunotherapy Application Areas, with herpesvirus infection dendritic cell of the present invention or immune other cell, for example scavenger cell.Usually, the herpesvirus infection dendritic cell reduce the ability that described dendritic cell immune stimulatory is replied.Therefore, virus of the present invention is modified, make them can effectively infect dendritic cell, but do not stop described dendritic cell stimulating immune system (WO00/08191).In this application, virus of the present invention comprises the heterologous gene of coding for antigens gene product usually.This may be and infection or the relevant antigen of cancer of causing a disease.Described gene is expressed in dendritic cell, and described gene product on described dendritic cell surface as antigen presentation.This stimulates at described antigenic immunne response by activated T cell, and described T cell is sought and manifested this antigenic cell on its surface, i.e. tumour or pathogen cells or be subjected to the cell of pathogenic infection are then with its destruction.
Preferably, in the HSV strain, described virus lacks functional UL43 gene and/or functional vhs gene, perhaps in non-HSV strain, described virus lack functional UL43 and/or vhs etc. homogenic; And choose wantonly in the HSV strain, described virus is owing to there is a sudden change of eliminating its transcriptional activation activity in the gene that is equal to vmw65, thereby lacks the described functioning gene that is equal to vmw65; Perhaps in non-HSV strain, described virus is owing to have a sudden change of eliminating its transcriptional activation activity in the gene that is equal to vmw65, thereby lacks the described functioning gene that is equal to vmw65; And optional at least one functional immediate early gene that is selected from ICP0, ICP4, ICP22 and ICP27 that lacks.
In text of the present invention, virus preferably of the present invention is stronger than having the ability that the Reference Lab virus strain infection dendritic cell that are equal to modification or immune stimulatory reply, generally speaking, can estimate infection according to above-mentioned described method about cell to dendritic cell.Specifically, non-zoo virus strain of the present invention is compared with the Reference Lab strain, and the percentage that infects dendritic cell when described two kinds of strain consumptions are identical is higher usually.Can carry out statistical analysis with the above method.
B. complementary cell is
When virus of the present invention is the hsv of a kind of shortage particular functionality indispensable gene (for example ICP4 or ICP27 encoding gene), allow virus of the present invention on the clone of expressing this indispensable gene, breed.For example, when virus lacked functional ICP27 gene, described virus can go up breeding at V27 cell (Rice and Knipe, 1990), 2-2 cell (Smith etc., 1992) or B130/2 cell (Howard etc., 1998).When virus lacks functional ICP4 gene, described virus can the clone of expressing ICP4 for example E5 cell (DeLuca etc., 1985) go up breeding.When virus lacked functional ICP4 gene and functional ICP27 gene, described virus was at the clone of expressing ICP4 and ICP27 (E26 cell for example; Samaniego etc., 1995) go up and breed; And when virus also lacked functional vmw65 gene, described virus can go up breeding in the clone (for example deriving from EHV, Thomas etc., 1999) of the non-HSV homologue that also contains vmw65.In the used substratum of viral growth, comprise hexa-methylene bis-dimethylsilyl-acetamide (HMBA), sudden change (MacFarlane etc., 1992) that also can the compensatory vmw65 of part.
Express ICP27 clone can by with comprise can the carrier of the functional HSV ICP27 gene of described cell inner expression preferably plasmid vector encode a kind of selective marker (for example neomycin resistance) carrier preferably plasmid vector cotransfection mammalian cell (for example Vero cell or bhk cell) produce.(for example adopt method known to those skilled in the art then, method described in Rice and the Knipe, 1990), the clone that screening has described selective marker, further determine also expressive function ICP27 of which clone, for example support the ability of ICP27-HSV strain growth to determine based on it.
Produce as mentioned above and can not allow ICP27-mutant HSV strain reply to have the clone of functional ICP27 strain, the carrier of guaranteeing to comprise functional ICP27 gene do not contain with the ICP27 mutant virus in all the other the sequence eclipsed sequences sequence of described sequence homology (promptly with).
Comprise in other indispensable gene (for example ICP4) at HSV strain of the present invention under the situation of inactivation modification, complementary cell system will comprise the functional HSV gene of the modified indispensable gene of complementation with the described same way as of relevant ICP27.For example, in ICP27 and ICP4, all comprise under the situation of HSV strain of sudden change, use the clone of expressing ICP27 and ICP4 (Samaniego etc. for example, 1995 or Thomas etc., 1999 is described).Can with the similar manner of the described mode of relevant ICP27, the HSV strain of other indispensable gene of construction expression.Moreover, if guarantee not have described all the other viral DNAs and be inserted into to supply eclipsed sequence between the clone of viral growth, then the possibility that described virus is replied to defect level reduction form at growing period can be reduced to minimum.
C. mutation method
Can make described various virogene not have the active gene of function by multiple technologies well-known in the art.For example, can or insert by disappearance, replacement, preferably make their no function activity by disappearance.Disappearance can be removed the part of gene or complete gene.For example, can carry out the only disappearance of a Nucleotide, cause frameshit.Yet, preferably carry out bigger disappearance, at least 25%, more preferably at least 50% (perhaps, using absolute term, at least 10 Nucleotide, more preferably at least 100 Nucleotide, at least 1000 Nucleotide most preferably) of for example whole coding and non-coding sequence.Preferred especially described complete gene and some flanking sequence removed.Insertion sequence can comprise one or more heterologous genes hereinafter described.With regard to the vmw65 gene, do not lack whole gene, because the essential structural protein of its coding, but carry out little inactivation sudden change, with the ability of eliminating vmw65 transcriptional activation IE gene (for example, Ace etc., 1989 or Smiley etc., 1997).
According to homologous recombination method well known to those skilled in the art, described simplexvirus is suddenlyd change.For example, with the HSV genomic dna with comprising in abutting connection with the carrier of the mutant nucleotide sequence of HSV homologous sequence, preferably plasmid vector transfection.Described mutant nucleotide sequence can comprise disappearance, inserts or replace, and all these mutant nucleotide sequences can make up by routine techniques.Insertion can comprise selectable marker gene, and for example lacZ or GFP are used for by for example betagalactosidase activity or fluorescent screening recombinant virus.
D. heterologous gene and promotor
Can modify virus of the present invention, to carry one or more heterologous genes.Term " heterologous gene " comprises any gene.Though heterologous gene is non-existent gene in the hsv gene group normally, can use one or more bleb genes, prerequisite is that described encoding sequence is not effectively to connect with its natural relevant described viral control sequence.Described heterologous gene can be any allelic variation body of wild type gene, and perhaps it can be a mutator gene.Term " gene " comprises the nucleotide sequence that can be transcribed at least.Thereby this definition comprises mRNA, tRNA and rRNA encoding sequence.Yet, the present invention relates to polypeptide expression, rather than the expression of tRNA and rRNA.The mRNA encoding sequence is optional to be comprised partly or entirely with the encoding sequence that is translated is natural and links to each other or relative 5 ' and/or 3 ' transcribe but the flanking sequence of untranslated.Can choose wantonly and also comprise the associated retroviral control sequence relevant, for example transcription termination signal, polyadenylation site and downstream enhancer element usually with transcription sequence.
Carry out homologous recombination by HSV strain and the plasmid vector that for example carries in abutting connection with one or more heterologous genes of HSV sequence, described one or more heterologous genes can be inserted in the viral genome.Adopt clone technology well-known in the art, one or more heterologous genes importings can be comprised in the suitable plasmid vector of simplexvirus sequence.One or more heterologous genes can be inserted on virus genomic any position, prerequisite is that described virus still can be bred.Preferably one or more heterologous genes are inserted in the indispensable gene.Can insert heterologous gene on a plurality of sites in described viral genome.
Preferably with the transcription sequence of one or more heterologous genes with allow described one or more heterologous genes at mammalian cell, preferably the control sequence of tumour cell or neural cell inner expression effectively is connected.Term " effectively connect " be meant a kind of side by side, the relation between the wherein said component allows them to work in the mode of its plan." effectively is connected " mode of connection of control sequence with encoding sequence, makes that the expression of described encoding sequence is achieved under the condition of mating with described control sequence.
Described control sequence comprises promotor that allows described one or more allogeneic gene expressions and the signal that is used to stop transcribing.Described promotor is selected from the promotor that function Mammals, is preferably arranged in the cell of human's nervous system or tumour or the immune cell.Described one or more promotors can derive from the promoter sequence of eukaryotic gene.For example, promotor can derive from the cell of described allogeneic gene expression, preferably mammalian cell, the genome of people's cell preferably wherein will take place.With regard to eukaryotic promoter, they can be the promotors (for example promotor of beta-actin, tubulin) that works in ubiquitous mode, or the promotor (for example neuronspecific enolase (NSE) promotor) that works in the tissue specificity mode.They also can be the promotors that particular stimulation is replied, for example in conjunction with the promotor of steroid hormone receptor.Also can use viral promotors, for example Moloney muroid leukosis virus long terminal repeat (MMLV LTR) promotor or other reverse transcription disease virus promoter-people or murine cytomegalovirus (CMV) IE promotor or hsv gene promotor comprise driving the hsv gene promotor of expressing latency-associated transcript.
Adopt conventional clone technology well known by persons skilled in the art (referring to for example Sambrook etc., 1989, Molecular Cloning-A laboratory manual; Cold Spring HarborPress), can make up expression cassette and other the suitable construct that comprises one or more heterologous genes and control sequence.
Described promotor is inducible promoter preferably, makes described expression of heterologous genes level to be regulated in the lifetime of cell.Being meant of induction type can be regulated the employing expression level that described promotor obtained.For example, surpassing a heterologous gene therein is inserted in the genomic preferred embodiment of described HSV, a promotor can comprise promotor (Gossen and the Bujard that the tet repressor/VP16 transcription activating protein fusion rotein to previous report is replied, 1992, Grossen etc., 1995) and drive the promotor of allogeneic gene expression to be regulated.Second promotor can comprise the strong promoter (for example CMV IE promotor) that drives tet repressor/VP16 expressing fusion protein.Therefore, in this embodiment, first expression of heterologous genes will depend on whether there is tsiklomitsin.
Heterologous gene is encoded usually and is had the polypeptide of therepic use.For example, in neural system, when non-oncolytic is used, can regulate the gene stimulating neural regeneration length of pain or prevent neurodegeneration.In oncolytic was used, can encode himself cytotoxic albumen, coding prodrug activating enzyme of heterologous gene maybe can stimulate or strengthen the albumen of anti-tumor immune response.
Heterologous gene can comprise that also marker gene (for example, coding beta-galactosidase or green fluorescent protein or other fluorescin) or its product regulate the gene (transcription regulaton factor that for example comprises above-mentioned tet repressor/vmw65 transactivator fusion protein) of other genetic expression.
Gene therapy and other treatment are used to be starved of and are given several genes.The expression of several genes may be favourable for the various illnesss of treatment.Simplexvirus is particularly suitable for, because they do not have the limited capacity packing of other virus carrier system.Thereby, in its genome, can hold multiple heterologous gene.For example, 2-5 kind gene can be inserted in the described genome.
At least two kinds of modes that wherein can realize this result are for example arranged.For example, can on described virus genomic single site or a plurality of site, more than a kind of heterologous gene and relevant control sequence be imported in the specific HSV strain.Also might use paired promotor (identical or different promotor), described promotor phase is opposite orientation each other, drives the expression of a kind of aforesaid heterologous gene (identical or different heterologous gene) respectively.
E. treatment is used
Virus of the present invention can be used for the treatment of in the method.Specifically, oncolytic virus of the present invention can be used for comprising the application of oncolytic ruling by law treatment cancer, for example by injection in the direct tumour.Comprise in described virus under the situation of heterologous gene of coding prodrug activator, can carry out other prodrug therapy.In addition, can will treat and the stimulation of immunne response associating by any way known in the art.Virus of the present invention can be used for therapeutic treatment Mammals, the intravital any solid tumor of people preferably.The curee that for example, virus of the present invention can be suffered from following disease: prostate cancer, breast cancer, lung cancer, liver cancer, carcinoma of endometrium, bladder cancer, colorectal carcinoma or cervical cancer; Gland cancer; Melanoma; Lymphoma; Neurospongioma; Or sarcoma, for example soft tissue sarcoma and osteosarcoma.
No replication virus of the present invention can be used for gene is passed to the individuality that needs gene therapy.Specifically, the neural virus of parent of the present invention can be used for treating central nervous system disease or peripheral nervous disease.The preferred central nervous system disease that is used for the treatment of or prevents comprises neurodegenerative disease.The particularly preferred central nervous system disease that is used for the treatment of or prevents is apoplexy, Parkinson's disease, presenile dementia, tay-Sachs disease (Tay Sachs disease) and mucopolysaccharidosis.The preferred peripheral nervous disease that is used for the treatment of or prevents comprises motor neuron, chronic pain and peripheral nerve injury.
Immunotherapeutical virus of the present invention can be used for preventing or treat its antigen pathogenic infection or the cancer relevant with the allos encoding gene that is inserted.
F. administration
Therefore, virus of the present invention can be used for the patient of needs treatments, human patients preferably.Virus of the present invention can be used for oncolytic ruling by law and treat cancer, and simplexvirus of the present invention (except that oncolytic is used) for example can be used for treating pain, neural degenerative disease or it is long to be used for stimulating neural regeneration.The purpose of therapeutic treatment is to improve described patient's illness.Usually, adopt viral therapy treatment of the present invention can alleviate the symptom of described disease of patient to be treated or illness.Therefore, comprise according to methods of treatment of the present invention suffer from cancer, the virus of the present invention of the patient treatment significant quantity of pain, neurodegenerative disease or nerve injury.
Give tumour patient with oncolytic virus of the present invention and understand killing tumor cell usually, thereby reduce the tumour size and/or prevent sending out of malignant cell.The patient who virus of the present invention is suffered from other disease (for example pain, degenerative disease or nerve injury) can improve described patient's illness usually.The sex change of the severity that for example eases the pain, the nervous tissue that slows down or promotion neurotization are long.
A kind of method of drug treatment comprises with described virus and pharmaceutically acceptable carrier or thinner combination, with the preparation medicinal compositions.Suitable carriers and thinner comprise etc. and to ooze salts solution, for example phosphate buffered saline(PBS).
Then, can adopt described carrier compositions is injected directly into described target tissue, carry out the oncolytic method and treat and/or gene is passed in the cell for treatment.With regard to HSV, the virus quantity scope that gives is 10
4-10
10Pfu, preferred 10
5-10
8Pfu, more preferably from about 10
6-10
8Pfu.For treatment of oncolytic method or the treatment of non-oncolytic method, when injection, yet the injection consumption of the medicinal compositions of forming by described virus and a kind of pharmaceutically acceptable suitable carrier or thinner 500 μ l, 1-200 μ l, preferred 1-10 μ l usually at the most usually basically, treat application for some oncolytic ruling by law, according to described tumour and inoculation position, also can use the comparatively large vol of 10ml at the most.
Because the technician can easily determine best route of administration and dosage, so described route of administration and dosage are only as guide.Described dosage can be according to various parameters, especially determine according to the severity and the route of administration of patient's age to be treated, body weight and illness, disease or illness.
For the preferred route of administration of cancer patients is to be injected directly in the tumour.Also can system give described virus, or described virus be given in the blood vessel to described tumor feeding by injection.The suitableeest route of administration will depend on the position and the size of described tumour.Described dosage can be according to various parameters, especially determine according to the position of tumour, the size of tumour, patient's age to be treated, body weight and illness and route of administration.
G. non-treatment aspect
Also providing can be for the authentication method of the suitable clinical strain of modifying according to the present invention.In addition, the method that also provides target to confirm.These relate to being applicable to that the gene that aforesaid the present invention treats application identifies.
The preparation method of virus of the present invention also is provided.
Following examples explanation the present invention.
Before shown that wherein neurovirulence factor ICP34.5 was all instructed tumour-specific lysis in two kinds of tumor models in vitro and in vivo by the I herpes simplex virus type (HSV1) of inactivation.In the I clinical trial phase that this viroid carries out in by the brain that is injected directly into neuroglia cancer patient in late period, it is safe also demonstrating.
The laboratory strain isolated (deriving from the virus of HSV1 strain 17+ or HSV1 strain F) that connects the HSV1 that goes down to posterity is used in early stage research always, compares with up-to-date clinical separation strain, can expect that the cracking ability of described virus aspect human tumor cells weakened.
In target is to produce in the research of the ICP34.5 absence type HSV with enhanced oncolytic potential and antitumor potential, we make the HSV1 clinical separation strain lack ICP34.5, and relatively compare with HSV1 strain 17+ (standard laboratory strain) in the various human tumor cell type, duplicate potential and cracking potential.
Virus formulation (referring to Fig. 1)
Used virus or based on HSV1 strain 17+ (standard laboratory strain) or based on deriving from herpes labialis, deriving from two strain clinical separation strains of the common resurrection thing (frequent re-activator) of HSV1.These strain called after BL1 and JS1.Make strain 17+ and JS1 lack ICP34.5 and insertion CMV-GFP box fully.By inserting human GM-CSF or mouse GM-CSF, so that displacement ICP34.5 gene also carries out further engineered to JS1.Therefore, BL1 and JS1 are clinical separation strain or " non-laboratory " strain.The JS1 discussed in this article also right and wrong laboratory strain of strain of deriving, i.e. the non-laboratory strain that the present invention is modified.
The growth (referring to Fig. 2) of virus in tumour cell
17+ compares with HSV1ICP34.5 absence type strain, and when at 72 hours build-in tests, JS1 and BL1 are presented at growth enhancing (Fig. 2) in some tested person's tumour cell.Select JS1 for further studying usefulness, and it is carried out aforesaid modification (referring to Fig. 1 and above).
The cracking ability (referring to Fig. 3) of virus
In all tested tumor cell lines, strengthen cracking (cell killing) ability with the non-laboratory strain of JS1 deutero-derived virus.More particularly, with reference to Fig. 3, JS1/ICP34.5-virus, promptly remove the JS1 of ICP34.5, be presented at cracking ability enhancing in the following cell: HT29 colorectum gland cancer, LNCaPFGC prostate cancer, MDA-MB-231 mammary cancer, SK-MEL-28 malignant melanoma and U-87MG glioblastoma astrocytoma by disappearance.
17+ compares with strain, after infecting with BL1, JS1, gets rid of and measures by infected cell being carried out Trypan Blue, also estimate various dosage and time at SK-MEL-28, MDA-MB-231 and the intracellular cracking ability of HT29.Viable cell is got rid of Trypan Blue, therefore, can estimate remaining viable count in culture by this method.With MOI is 0.1 or 1 usefulness or 17+, BL1 or JS1, and the tumor cell line of cultivating in the duplication hole of six hole plates was infected 24 hours, 48 hours or 72 hours, then viable count is counted.Be shown in Table 1 at the viable count percentage that does not infect in the control wells that is equal to.
Therefore, when in human patients, being used for the treatment of cancer, in all cases, kill more tumour cell with clinical separation strain virus BL1 and JS1 than laboratory strain isolated 17+, reaching oncolytic activity increases, and therefore uses up-to-date clinical disease strain and may strengthen the modified anti-tumor capacity that duplicates the virus of (for example passing through disappearance ICP34.5) to obtain tumor-selective of this class.
Table 1
| Clone | Infect the back time | JS1 MOI＝0.1 MOI＝1 | 17 MOI＝0.1 MOI＝1 | BL1 MOI＝0.1 MOI＝1 | |||
| The percentage of viable count in the control wells that does not infect | |||||||
| SK-MEL-28 | 24h duplication sample | 41 33.7 | 8 7 | 57.3 62.6 | 19 19.3 | 43.7 39 | 6.67 6.33 |
| 48h | 5.51 5.05 | 1.9 0.8 | 7.4 7.1 | 3.7 2.6 | 4.5 4.8 | 0.8 1.1 | |
| | 0 0 | 0 0 | 0 0 | 0 0 | 0 0 | 0 0 | |
| MDA-MB- 231 | 24h | 44.91 44.02 | 16.7 16.96 | 69.37 65.8 | 36.34 34.55 | 55.63. 60.45 | 26.79 25.27 |
| 48h | 14.1 13.5 | 4.7 3.8 | 27.9 27 | 8.3 8.5 | 18 20 | 6.7 8.3 | |
| | 0 0 | 0 0 | 2.91 2.91 | 0.73 1.27 | 1.46 1.64 | 0 0 | |
| HT-29 | 24h | 37.53 39.24 | 15 15 | 47.28 45.76 | 23.61 24.24 | 42.22 43.04 | 22.15 21.33 |
| 48h | 13.2 14 | 2.3 3 | 29.4 27.7 | 4.2 4.7 | 18.4 21.2 | 4.4 3.7 | |
| | 0 0 | 0 0 | 1.57 1.89 | 0 0 | 1.64 1.57 | 0 0 | |
Further enhanced anti-tumor activity
If these viruses are used for transmitting the gene with anti-tumor activity, then also can expect further enhanced activity.Such gene comprises coding prodrug activator or the proteic gene of immunostimulation.
For this reason, we have been made up the ICP34.5 absence type clinical separation strain of the HSV1 of a kind of expressing human GM-CSF or mouse GM-CSF by JS1, and GM-CSF is a kind of effective immunostimulation albumen.This virus design strengthens anti-tumor immune response in order to injection back in tumour.
Reference
Chou etc., 1990, Science 250:1262-1266
Maclean etc., 1991, J.Gen.Virol.72:631-639
Samaniego etc., 1998, J.Virol.72:3307-3320
Krisky etc., 1998, Gene Therapy 5:1593-1603
Thomas etc., 1999, J.Virol.73:7399-7409
MacFarlane etc., 1992, J.Gen.Virol.73:285-292
Howard etc., 1998, Gene Therapy 5:1137-1147
Samaniego LA etc., 1995, J.Virol.69:5705-5715
Ace CI etc., 1989, J.Virol.63:2260-2269
Smith IL etc., 1992, Virol.186:74-86
Rice, SA and Knipe DM, 1990, J.Virol.64:1704-1715
DeLuca NA etc., 1985, J.Virol.56:558-570
Gossen M and Bujard H, 1992, PNAS 89:5547-5551
Gossen M etc., 1995, Science 268:1766-1769
Smiley, J.R. and Duncan J., 1997, J.Virol.71:6191-6193
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Preservation information
HSV1 strain JS1 is preserved in European cell culture preservation center (ECACC) January 2 calendar year 2001, CAMR, and Salisbury, Wiltshire SP4 0JG, Britain, interim preserving number is 01010209.
Claims (3)
1. measure the sick HSV strain in non-laboratory for the adaptive method that is used to prepare modified oncolytic HSV for one kind, described method comprises:
(i) randomly from the host, separate the HSV strain;
Described HSV strain (ii) is provided;
Estimate (iii) that described HSV strain duplicates or the ability of killing tumor cell in tumour cell; And,
(iv) be chosen in duplicate in the tumour cell or the ability of killing tumor cell be higher than among HSV1 strain 17+, HSV1 strain F and the HSV1 strain KOS one or more for the HSV strain of modifying usefulness.
2. method for preparing modified oncolytic HSV strain, described method comprises:
(i) provide a kind of HSV strain of selecting by the method for claim 1;
(ii), described strain is modified by making one or more genes that are selected from ICP34.5, ICP6 and thymidine kinase encoding gene become no function; With
(iii) estimate the ability of described modified HSV oncolytic strain killing tumor cell.
3. the method for a claim 2, wherein by comparing with the tumor cytotoxicity ability that has identical gene or become the Reference Lab strain of non-functional gene, measure the tumor cytotoxicity ability of described modified HSV oncolytic strain, wherein said Reference Lab strain is selected from HSV1 strain 17+, HSV strain F and HSV strain KOS.
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| WO2020109389A1 (en) | 2018-11-28 | 2020-06-04 | Innovative Molecules Gmbh | Helicase primase inhibitors for treating cancer in a combination therapy with oncolytic viruses |
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|---|---|---|---|---|
| CN107354136A (en) * | 2017-06-15 | 2017-11-17 | 杭州睿可特生物科技有限公司 | Recombinant herpes simplex virus and its preparation method and application |
| BR112021017551A2 (en) * | 2019-03-05 | 2021-11-09 | Amgen Inc | Use of oncolytic viruses for the treatment of cancer |
| CN110982795B (en) * | 2020-03-05 | 2020-06-16 | 北京唯源立康生物科技有限公司 | Herpes simplex virus and application thereof |
| CN110982794B (en) * | 2020-03-05 | 2020-06-16 | 北京唯源立康生物科技有限公司 | Modified herpes simplex virus |
| CN113943752A (en) * | 2020-07-15 | 2022-01-18 | 东莞市东阳光生物药研发有限公司 | Constructs, oncolytic viruses with improved sensitivity and uses thereof |
-
2000
- 2000-01-21 GB GB0001475A patent/GB0001475D0/en not_active Ceased
-
2001
- 2001-01-22 CN CNA2009101295146A patent/CN101560502A/en active Pending
- 2001-01-22 CN CN 200610003779 patent/CN1865449A/en active Pending
-
2002
- 2002-07-08 ZA ZA200205439A patent/ZA200205439B/en unknown
- 2002-07-09 ZA ZA200205470A patent/ZA200205470B/en unknown
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106075422A (en) * | 2005-11-29 | 2016-11-09 | 衣阿华州立大学研究基金公司 | Qualification of protective antigenic determinants of porcine reproductive and respiratory syndrome virus (PRRSV) and application thereof |
| WO2020109389A1 (en) | 2018-11-28 | 2020-06-04 | Innovative Molecules Gmbh | Helicase primase inhibitors for treating cancer in a combination therapy with oncolytic viruses |
Also Published As
| Publication number | Publication date |
|---|---|
| ZA200205470B (en) | 2003-10-29 |
| GB0001475D0 (en) | 2000-03-15 |
| CN101560502A (en) | 2009-10-21 |
| ZA200205439B (en) | 2003-09-04 |
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Application publication date: 20061122 |