Multipurpose cancer-specific promoters and using them in anti-cancer therapy

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, in particular to tumour-specific promoters, and can be used in the anti-cancer therapy. There are constructed the broad-spectrum tumour-specific promoters providing the therapeutic gene expression inside a cancer cell. The invention also involves expression cassettes, expression vectors, pharmaceutical compositions, methods of treating cancer and using the expression cassettes and vectors.

EFFECT: promoters of the present invention provide a high expression level of the operatively linked therapeutic gene in the cancer cells of different origin, wherein the normal cell expression is absent or low.

29 cl, 19 dwg, 4 tbl, 20 ex

 

The technical field to which the invention relates.

The present invention relates to biotechnology, in particular genetic engineering, artificial DNA structures, demonstrating the high transcriptional activity of the promoter, whose expression is below an arbitrary nucleotide sequence that is transcribed by RNA-polymerase II. The activity of the promoters is limited only by cancer cells.

Promoters can be used for gene therapy of tumors of different nature.

The level of technology

Cancer is currently the most serious problem in medicine. It is increasingly clear that prevailed until recently, the concept of molecular targeting (targeting) of anticancer drugs on the estimated key molecules of cancer cells, which are involved in tumor development, is in a crisis state (Salk JJ and Fox EJ et al. (2010) Annu Rev Pathol Mech Dis 5:51-75). The vast majority of agents in this category who have passed pre-clinical and clinical trials proved ineffective (Hambley TW and Hait WN (2009) Cancer Res 69:1259-62). It is quite expected the crisis: cancer as a disease combines the complexity of cellular organization, inherent and other diseases, with the growing complexity of the system, capable adaptivetec effects of drugs by the existence of microheterogeneity inside the tumor (Merlo LM and Pepper JW et al. (2006) Nat Rev Cancer 6:924-35). This leads to the fact that most cancer genes only take a small part in the development of cancer. Consequently, they are difficult to identify for use as targets of action of anticancer agents (Salk JJ and Fox EJ et al. (2010) Annu Rev Pathol Mech Dis 5:51-75). The result in cancer therapy critical situation: on the one hand, medicine has a fairly effective, but highly toxic means of chemotherapy, with the other low-toxic, but ineffective means of molecular targeted therapy (MTT).

In the last decade actively began to develop gene therapy of cancer (GT). The General principle GT consists of the delivery of regulated genetic material in cancer cells, where the produced products, capable of destroying cancer cells. The GT approaches can be divided into two broad categories (Sverdlov ED (2009) Mol Gen Mikrobiol Virusol 24:93-113) / (http://link.springer.com/article/10.3103%2FS089141680903001X). First use a strategy of targeted therapy. When the target agent is a gene product, enter in any way into the cell tumors, which is an inhibitor of a product, the concentration of which in cancer cells is increased, and it is one of the causes of cancer. This category can be attributed reverse technology is shipping in about whole gene, the product which compensates for the lack of a certain protein in cancer cells. In both cases, the target exposure is a certain link in the signal systems of the cell, which changes when cancerous degeneration and promotes him. Options GT, based on the targeted principle, suffer from the same shortcomings that imtt.

The first gene therapeutic virus for therapy of head and neck cancer was approved for clinical use in China in 2003 under the name Genticin (Gendicine). The virus contains as a therapeutic gene p53 (Peng Z (2005) Hum Gene Ther 16:1016-27, Wilson JM (2005) Hum Gene Ther 16:1014-5). In combination with radiotherapy of Genticin caused complete regression of tumors in 64% of patients and partial in 29%, while one radiotherapy was given full regression 19% and partial in 60%, which, apparently, shows a marked improvement in outcome with combination therapy (Peng Z (2005) Hum Gene Ther 16:1016-27). But these results also show that only 64% respond to treatment, and the subsequent behavior of the disease is unknown. It is reasonable to expect the appearance of secondary tumor growth, since it has been shown that with the introduction of p53 in tumors, in which he corrupted source, causes the emergence of resistant p53 variants (Martins CP and Brown-Swigart L et al. (2006) Cell 127:1323-34). The data obtained with the use of GT as a molecular target therapy presupposes ageut, that as well as classical MTT and due to the same reasons, it is unlikely to be efficient or versatile.

The second strategy of gene therapy, aimed at the destruction of tumor cells, such as through the use of their properties, which are characteristic of all cancer cells, for example, an increased rate of mitotic divisions, in this respect like chemotherapy. However, unlike the latter, the toxin that kills cancer cells through inhibition of the replication systems, is formed inside them, so characteristic of chemotherapy toxicity in this case is sharply reduced. This approach is known as gene-directed enzyme proletarienne therapy, GDEPT (Gene-directed enzyme prodrug therapy (GDEPT) or gene therapy using genes suicide tumors (suicide gene therapy) (Altaner C (2008) Cancer Lett 270:191-201, Fillat With and Carrio M et al. (2003) Curr Gene Ther 3:13-26, Portsmouth D and Hlavaty J et al. (2007) Mol Aspects Med 28:4-41, Seth P (2005) Cancer Biol Ther 4:512-7). The approach is not molecular targetirovannye and therefore devoid of all flaws MTT.

One of the important elements of the success of cancer gene therapy is an adequate system of expression of therapeutic genes because these genes must run in cancer cells and not work in normal cells of the body. In the ideal case, the control system of therapeutic genes in the body must ensure that (i) is strictly conspecifics and (ii) sufficiently strong transgene expression, so on the one hand to provide security, and system efficiency. When designing vectors used promoters and enhancers, working in specific tumors in this tissue (Robson T and Hirst DG (2003) J Biomed Biotechnol 2003:110-137, Saukkonen K and Hemminki A (2004) Expert Opin Biol Ther 4:683-96). However, all natural ofwholesale promoters have two major drawbacks: on the one hand, are weak, and on the other, have a high level of activity only in certain cancer cell lines, i.e. are not universal. So, the AFP promoter is most active in carcinoma cells of the liver, the PSA promoter in the tumor cells of the prostate, the promoter SOH-2 in cancer cells of the stomach and duodenum, MK promoter in neuroblastoma cells (Adachi Y and Reynolds PN et al. (2001) Cancer Res 61:7882-8).

Most ofwholesale promoters has low activity in comparison with the strong constitutive promoters such as the promoters of viruses SV40 and CMV (Van Houdt WJ and Haviv YS et al. (2006) J Neurosurg 104:583-92, Lu and Makhija SK et al. (2005) Gene Ther 12:330-8, Rein DT and Breidenbach M et al. (2004) J Gene Med 6:1281-9).

There is another problem. Even relatively strong tumor-specific promoters such as the promoter of the gene BIRC5 (hSurv), encoding an inhibitor of apoptosis, survivin, and the promoter of the gene for reverse transcriptase of the human telomerase (hTERT) with a fairly wide spectrum of the act is Vesti, show her not all cancer cells. For example, the promoter hSurv active in tumors only about 60 - 80% of patients with non-small cell lung cancer (NSLC), and the TERT promoter in 60% of patients with NSLC (Hsu CF and Miaw J et al. (2003) Eur J Surg Oncol 29:594-9). While there is considerable variability in the relative activity of these promoters in various tumor cell lines. Thus, the activity of the promoter of survivin ranges from 0.3 to 16% of the activity of the CMV promoter (Chen JS and Liu JC, et al. (2004) Cancer Gene Ther 11:740-7, Konopka and To Spain With et al. (2009) Cell Mol Biol Lett 14:70-89, Zhu ZB and Makhija SK et al. (2004) Cancer Gene Ther 11:256-62), and the efficiency of the hTERT promoter can vary up to 20 times depending on the cancer cell lines (Gu J and Fang (2003) Cancer Biol Ther 2:S64-70).

The natural variability ofwholesale promoters in different tumors complicates the selection of the doses of the prodrug to obtain a therapeutic effect and changes therapeutic index of the drug from tumor to tumor.

To increase the efficiency of tumor-specific expression of therapeutic genes using combined and double (chimeric) promoters. Chimeric promoters may include combinations of known promoters with each other or with a separate heterologous regulatory elements to increase the strength and specificity of expression in cancer cells (With Wu and Lin J et al. (2009) Mol Ther 17:2058-66) an example of a chimeric promoter may serve as a combination PhTERT with minimal cytomegalovirus promoter (PhTERT-CMV) (Davis JJ and Wang L et al. (2006) Cancer Gene Ther 13:720-3); or the TATA box, which is absent in native PhTERT. As a rule, the authors of the various studies aimed at efficient use of hybrid promoters are ways to maximize the efficiency and specificity of expression in certain types of cancer. An example of this approach is the work now et al. (Now TT and Pedersen N et al. (2008) Cancer Gene Ther 15:563-75). In this study we identified two genes, vysokoagressivnyh in small cell lung cancer (SCLC). One of them encodes a transcription factor hASH1, and the other, EZH2 is a member of the Polycomb family. When the promoters of these genes were combined in one design, the resulting chimeric promoter was able to cause a strong transgene expression in specific cells of SCLC. Such a promoter is capable of initiating gene expression-killers in SCLC, but not in other cancer types.

This ideology maximum specificity of promoter activity in a particular type of cancer is common (see, for example, Farokhimanesh S and Rahbarizadeh F, et al. (2010) Biotechnol Prog 26:505-11). Use is strictly specific for this tumor promoters and other regulatory elements has as advantages the maximum reduction of side effects due to the reduction of transgene expression in normal tissues. However, the drawback of such approaches is their non-generic is aracter and the associated inevitable increase in the cost of drugs, based on these promoters. A compromise is to use a more universal tumor-specific promoters that can operate in a wide range of tumors but not in normal cells. A few increasing the risk of destruction of normal tissue, this approach is more cost effective: the same design can be used in treating a wide spectrum of tumors. There is another important consideration in the use of promoters a wider range of actions. It is poorly understood specificity of gene expression in metastases of this tumor. There is no strict guarantee that uzkospetsialnyh the promoter that works well in the primary tumor, will retain this ability in all its metastases. The use of universal promoters reduces the likelihood of inactivation of the promoter in metastases.

Disclosure of inventions

The present invention aims at eliminating the drawbacks inherent in the promoters of the prior art, strictly specific type of cancer and create a universal promoters with a broad spectrum of activity against different types of cancers and their metastases, while still retaining the ability to function essentially only in the tumor but not in normal cells.

The present image is the buy provides artificial universal ofwholesale dual promoters, highly active in a broad spectrum of cancer cells regardless of their type.

Ofwholesale dual promoters created on the basis of modified promoters of genes of the reverse transcriptase of the human telomerase (hTERT) and survivin human and mouse (Surv). The gene of surviving person (BIRC5) encodes a protein of survivin, which belongs to the family of protein inhibitors of apoptosis and is one of the key participants of tumor formation. The promoter of survivin has a high tumor-specificity and active in the vast majority (85-90%) tumors (Takakura M and Kyo S et al. (1999) Cancer Res 59:551-7, Ambrosini G, Adida and With et al. (1997) Nat Med 3:917-21, Fukuda S and Pelus M (2006) Mol Cancer Ther 5:1087-98). Gene hTERT encodes the catalytic subunit of human telomerase. The transcriptional activity of this gene occurs during embryonic development and is often approximately 85% of cases present in the tumor cells, whereas in the vast majority of normal cells, the expression of hTERT is suppressed (Cong YS and Wright WE, et al. (2002) Environ Mol Biol Rev 66:407-25).

It is shown that the established promoters on quantitative and qualitative criteria are different from their constituent hTERT promoters and Surv.

In experiments on transient transfection of cancer cell lines plasmid vectors containing reporter gene Firefly luciferase under the control created promotional the Directors, demonstrated high activity created promoters, exceeding the activity as a strong constitutive promoter of the SV40 virus, and single promoters of genes hTERT and Surv. In experiments using normal cells (lung fibroblasts) proven tumor-specificity created dual promoters.

Also on the basis of the created dual promoters derived therapeutic constructs containing the gene-killer or gene-killer in combination with a cytokine gene.

Panel of cancer and normal cell lines demonstrated tumor-specific therapeutic effect obtained structures.

On the basis of the created dual promoters can be obtained plasmid, viral or other vector designs for ofwholesale expression of any gene or different genes in cancer cells. The amount of promoter may be increased, and the nucleotide sequence of the promoter can be added other regulatory and genetic elements that enhance the activity of the promoter.

Thus, the present invention in its first aspect relates to opukholespetsificheskaya promoter consisting of:

A) the nucleotide sequence of SEQ ID NO:2 or its functional derivative, and

B) a nucleotide sequence selected from the group, what with:

(i) the nucleotide sequence of SEQ ID NO:1 or its functional derivative;

(ii) the nucleotide sequence of SEQ ID NO:3 or its functional derivative.

In the promoter of the present invention the sequence of SEQ ID NO:2 may be located above (in 5'-direction) relative to the sequence SEQ ID NO:1 or SEQ ID NO:3, or the sequence SEQ ID NO:2 may be located below (in 3'-direction) relative to the sequence of SEQ IDNO:1 or SEQ ID NO:3. The promoter of the present invention may contain other nucleotide sequences that do not affect its activity, for example the linker sequence.

The most preferred sequence of the promoters of the present invention is selected from the group comprising SEQ ID NO:4 - 7.

In its second aspect the present invention relates to an expression cassette, containing in the direction from 5' to 3' promoter of the present invention, the coding sequence functionally linked to a promoter, and a polyadenylation signal, and the expression of the coding sequence is under control of the specified promoter.

In the expression cassette of the present invention, the coding sequence may encode therapeutic proteins or peptides, antigens, antisense RNA or ribozymes. In particular, Express the ion cartridge can contain the coding sequence, which includes genes of therapeutic intervention selected from the group including: dominant negative mutants (BAT) genes involved in oncogenesis; genes killer genes inhibitors of angiogenesis, genes oncosuppressor, genes Immunostimulants, siRNA genes.

Negative dominant mutants (BAT) genes can be selected from the group including NDM gene of survivin, NDM gene c-Jun, BAT the oncogene RAS. Genes killer can be selected from the group comprising the genes of toxin genes and enzymes that can turn a non-toxic agent (prodrug) in the toxin. The latter can be selected from the group comprising a gene timedancing of herpes simplex virus and yeast cytosine deaminase gene or E. coli. Genes angiogenesis inhibitors can be selected from the group comprising the genes of proteins angiostatin and endostatin. Genome-oncosuppressor may be, for example, a gene of p53 protein. Genes Immunostimulants can be selected from the group comprising the genes IL-1, IL-2, IL-4, IL-6, TNF, GM-CSF, or IFN-γ. Genes siRNA can be selected from the group comprising siRNA for gene of survivin.

In the third aspect of the present invention relates to an expression vector containing an expression cassette of the present invention. This expression vector of the present invention may be viral or non-viral. In the case when the vector of the present izopet the Deposit is a viral vector, it can be selected from the group including retrovirus, adenovirus, or adeno-associated herpes virus vector. Non-viral vector may be represented, for example, a plasmid.

In the fourth aspect of the present invention relates to pharmaceutical compositions containing a therapeutically effective amount of the expression cassette or vector of the present invention and a pharmaceutically acceptable excipient, which without limitation may be represented by a carrier, solvent, excipients, bulking agent, buffer agent, stabilizer, preservative, etc.

The pharmaceutical composition of the present invention may be used to treat a wide range of cancers and disease without the constraint may be selected from the group comprising lung cancer, pancreatic cancer, melanoma, fibrosarcoma, sarcoma, cancer of the head and neck.

In the fifth aspect of the present invention relates to a method of treating cancer comprising the administration to a patient the pharmaceutical composition of the present invention.

The treatment method of the present invention may include without limitation, intravenous, intratumoral, intramuscular, intraperitoneal, subcutaneous, oral administration of the pharmaceutical composition of us who Otsego invention. The treatment can be used to treat cancer, without limitation, selected from the group comprising lung cancer, pancreatic cancer, melanoma, fibrosarcoma, sarcoma, head and neck cancer.

Finally, in the sixth aspect of the present invention relates to the use of the expression cassette or vector of the present invention for the manufacture of a medicinal product for the treatment of cancer. Cancer without restrictions may be selected from the group comprising lung cancer, pancreatic cancer, melanoma, fibrosarcoma, sarcoma, head and neck cancer.

Brief description of drawings

Fig.1. Schematic representation of expression constructs PhSurv269-pGL3. PhSurv 269 - a fragment of the promoter region of the gene of surviving person; LUC - luciferase gene of the Firefly, polyA site of SV40 virus polyadenylation.

Fig.2. Schematic representation of expression constructs. On the left is the name of the structures. PhSurv269 - fragment of the promoter of the gene of surviving person; PhTERT-fragment of the promoter of the gene reverse transcriptase telomerase person; LUC - luciferase gene of the Firefly.

Fig.3. Schematic representation of expression constructs PhTSurv269-HSVtk-pGL3. PhTERT - fragment of the promoter region of the gene reverse transcriptase of the human telomerase (hTERT); PhSurv269 - promotor fragment R is the region of the gene of surviving person; HSVtk gene timedancing of herpes simplex virus type 1; SV40 late poly(a) -polyadenylation signal of the SV40 virus; Amp(r) - gene p-lactamase.

Fig.4. Schematic representation of expression constructs PhTSurv269-HSVtk-mGM-CSF-pGL3. PhTERT - fragment of the promoter region of the gene reverse transcriptase of the human telomerase (hTERT); PhSurv269 - fragment of the promoter region of the gene of surviving person; HSVtk gene timedancing of herpes simplex virus type 1; IRES - plot planting ribosomes virus encephalomyocarditis person; mGMCSF gene granulocyte-macrophage colony-stimulating factor mouse; SV40 late poly(A) - polyadenylation signal of the SV40 virus; Amp(r) gene of β-lactamase.

Fig.5. Schematic representation of expression constructs PhTSurv269-FCU1-mGM-CSF-pGL3. PhTERT - fragment of the promoter region of the gene reverse transcriptase of the human telomerase (hTERT); PhSurv269 - fragment of the promoter region of the gene of surviving person; FCU1 - hybrid gene sitoindosides/uralspetstransmash; IRES - plot planting ribosomes virus encephalomyocarditis person; mGMCSF gene granulocyte-macrophage colony-stimulating factor mouse; SV40 late poly(A) - polyadenylation signal of the SV40 virus; Amp(r) - gene p-lactamase.

Fig.6. Diagram of the modified system PhTSurv269 Cre-LoxP//pCMV-Stop-FCU1. While getting into a tumor cell killing and activation vectors will be the INIC is to aromatise the following cascade of events:1) activation of the promoter of the gene PhTSurv269, which will lead to expression of Cre recombinase; 2) established the Cre recombinase will learn loxP sites in the composition of the killing vector, and then cut fragment, flanked by these sites; 3) by deleting "Stop"signal, CMV will "run" time killer protein CD-UPRT (the expression product FCU1). Stop sequence length 705 p. O., consisting of the triple repeat (235 p. O. ×3) the polyadenylation signal of the SV40 virus. NLS (Nuclear Localization Signal) - the nuclear localization signal. Black arrows indicate LoxP sites, empty arrows - promoters, empty rectangles - structural part of the gene, an empty circle marked Stop sequence.

Fig.7. Chart of the relative activity of the studied promoters in different cell lines. The names of the promoters are given at the bottom of the figure. The ordinate axis shows the relative luciferase activity, normalized to luciferase activity in cells transfected with the construct PV-pGL3. The names of the cell lines shown on the x-axis. Calu-1 - epidermoid carcinoma of the lung human A375 melanoma human A - carcinoma of the lung man, PANC1 - carcinoma of the exocrine part of the pancreas, NT - fibrosarcoma.

Fig.8. Activity generic ofwholesale promoters in different cell lines. Axis about which dinat shows the luciferase activity relative luciferase activity in cells transfected vector PV-pGL3. The abscissa axis indicates the types of cell lines, which were carried out measurement of promoter activity. In the top right corner of the names of the promoters used in the work. The height of the columns represents the average luciferase activity of three independent experiments are shown with standard errors of the mean (SEM). PV - promoter of SV40 virus, mSurv - fragment of the promoter region of the gene of survivin mouse, hTERT - fragment of the promoter region of the gene reverse transcriptase telomerase person.

Fig.9. Activity created ofwholesale promoters in different cell lines. The ordinate axis shows the luciferase activity relative luciferase activity in cells transfected with the vector PV-pGL3. The abscissa axis indicates the types of cell lines, which were carried out measurement of promoter activity. In the top right corner of the names of the promoters used in the work. The height of the columns represents the average luciferase activity of three independent experiments are shown with standard errors of the mean (SEM).

Fig.10. Western blot analysis of protein FCU1 in cell line S37, transfected with constructs expressing FCU1 gene. 1 - pCMV-FCU1; 2-3 - different ratios of vectors PhTSurv269-Cre and pCMV-STOP-FCU1 (cotransfection): 2:2, 1:3. The quality is TBE primary antibodies used sheep IgG (Santa-Cruz) (1:5000). The secondary antibody to sheep IgG were conjugated with horseradish peroxidase (Santa-Cruz). Cell lysates pre-normalized by total protein.

Fig.11. Survival of the cancer cell line S37 (mouse sarcoma) in the presence of 5-fertilizin, transfected with the following vectors: vector pCMV-FCU1 (positive control), a system of vectors PhTSurv269-Cre (activator)/pCMV-STOP-FCU1 (effector) at a ratio activator/effector 2:2 and 1:3 by the number of micrograms.

Fig.12. Survival of the cancer cell line S37 (mouse sarcoma) in the presence of 5-fertilizin, transfected with the following vectors: vector pCMV-FCU1 (positive control), a system of vectors PhTSurv269-Cre/pCMV-STOP-FCU1 and PhSurv-Cre/pCMV-STOP-FCU1.

Fig.13. Schematic of dual promoters and appropriate transcripts. The rectangles marked promoters PhTERT, PhSurv, PhSurv269, PmSurv and luciferase gene of the Firefly - Luc. On the left lists the names of the dual promoters. Under the scheme each of the double promoter lines indicate the transcripts initiated from these promoters. The thick part of the line indicates the sequence of the transcript complementary to the sequences of the promoters or gene luciferase, thin - linker sites. Broken arrow denotes the point of transcription initiation. Arrows indicate the primers. Above the arrows indicate the names of the primers.

When used and primer pairs TSL-F/hSurv_150R, TSL-F/hS269_122R and TSL-F/mS_122R it provided amplification of only the transcripts initiated from the distal promoters. Primer pair UPF/Luc_202R allows you to amplify the total transcripts.

Fig.14. Electrophoregram products obtained by RT-PCR and initiated with the distal and proximal promoters. The names of the dual promoters listed above. The position in relation to controlled individual gene promoter elements that make up the double promoter, and the number of PCR cycles is indicated to the left and bottom, respectively. Distal products of amplification of transcripts initiated from the promoter PhTERT using primers TSL-F/hSurv_150R, TSL-F/hS269_122R for promoter PhTS and primers TSL-F/mS_122R for promoters, PhTSurv269 and PhTmS. Proximal - amplification products using primers UPF and Luc_202R total transcripts and/or transcripts initiated from the distal promoter.

Fig.15. Survival in the presence of ganciclovir cancer cell line NT (human fibrosarcoma), transfected with vectors: PhSurv-HSVtk, PhTSurv269-HSVtk, control.

Fig.16. Survival in the presence of 5-fertilizin cancer cell line Calu-1, transfected with vectors: PhSurv-FCU1-mGM-CSF, PhTSurv269-FCU1-mGM-CSF, CMV-FCU1-mGM-CSF, control.

Fig.17. Western blot analysis of HSVtk protein in the cell line NT, transfected constructs, expressyou the mi HSVtk gene. 1 - pCMV-HSVtk; PhSurv-HSVtk, PhTSurv269-HSVtk. As the primary antibodies used sheep IgG (Santa-Cruz) (1:5000). The secondary antibody to sheep IgG were conjugated with horseradish peroxidase (Santa-Cruz), Cell lysates pre-normalized by total protein.

Fig.18. The influence of transformation of cells of Lewis lung carcinoma mouse (LLC) gene-inzhenernye designs on the growth rate of tumors caused by transplantation of these cells in mice C57B1/6. Nontransgenic (Control), transformed design CMV-HSVtk-mGM-CSF-pGL3 (CMV), transformed design PhSurv-HSVtk-mGM-CSF-pGL3 (PhSurv) and transformed design PhTSurv269-HSVtk-mGM-CSF-pGL3 (PhTSurv269) cells transplanted mice. Intraperitoneal injections of ganciclovir (GCV) in an amount of 75 mg/kg twice a day for 10 days starting from the third day after transplantation of the cells. Tumor size was measured 6 days after transplantation of the cells. Data represent average values for a group of 10 animals. The abscissa axis indicates the time elapsed since transplantation of cells.

Fig.19. The influence of transformation of cells of Lewis lung carcinoma mouse (LLC) genetically engineered designs on the growth rate of tumors caused by transplantation of these cells in mice C57B1/6. Transformed design CMV-HSVtk-mGM-CSF-pGL3 (CMV), transformed design PhSurv-HSVtk-mGM-CSF-pGL3 (PhSurv) and the transformer is new design PhTSurv269-HSVtk-mGM-CSF-pGL3 (PhTSurv269) cells transplanted mice. Intraperitoneal injections of ganciclovir (GCV) in an amount of 75 mg/kg twice a day for 10 days starting from the third day after transplantation of the cells. Tumor size was measured 6 days after transplantation of the cells. Data represent average values for a group of 10 animals. The abscissa axis indicates the time elapsed since transplantation of cells.

The implementation of the invention

Created dual promoters are chimeric dual promoters, in which the promoters of genes hSurv and hTERT are in two different positions relative to each other. In the first case, the promoter of the gene hSurv is above the hTERT promoter relative to the start codon controlled gene, in the second case, the promoter of hTERT gene is located upstream of the promoter hSurv relative to the start codon of the controlled gene. Created promoters are highly active in cancer cells, the activity of the promoters is higher than the activity of the promoter of the SV40 virus. The promoters may have a functional derivative, i.e. modification of the promoters as a result of deletions, substitutions, insertions, or other mutations in the original promoter, not changing or retaining a fairly high level of activity compared with the original promoter (examples 2 and 3, table 2).

Received promoters can be used for preferential transcription of various the ENES in cancer cells comprising the expression cassette. The expression cassette (EC), designed for the expression of therapeutic genes in mammalian cells, is a fragment of DNA containing all of the necessary genetic elements for expression embedded in his genetic information. The expression cassette in the direction from 5' to 3'-end, consists of 1) promoter (promoter); 2) one or more genes, the expression of which is expected to provide; 3) the polyadenylation signal, necessary for the completion of transcription, posttranscriptional processing of RNA. In the structure of EC may also contain additional elements that provide adjustment of the conditions of expression of the gene of interest. So, to increase expression of therapeutic gene is sometimes used chimeric intron located between the promoter sequences and therapeutic gene (Gross MK and Kainz MS et al. (1987) Mol Cell Biol 7:4576-81, Buchman AR and Berg P (1988) Mol Cell Biol 8:4395-405). Used the polyadenylation signal may also have an effect on the level of gene expression by affecting the stability of the synthesized transcript (Azzoni AR and Ribeiro SC (2007) J Gene Med 9:392-402).

As genes controlled by ofwholesale the promoters comprising expression cassettes typically use genes of therapeutic intervention following groups:

1. Negative dominant mutants (BAT) genes, membership is involved in the oncogenesis

2. Genes killer

3. Genes angiogenesis inhibitors

4. Genes oncosuppressor

5. Genes Immunostimulants

6. Genes siRNA

1. Negative dominant mutants (BAT) genes are used to suppress the actions of proteins, genes involved in oncogenesis, such as oncogenes or inhibitors of apoptosis. For example, the introduction of the NDM gene of surviving mouse in cancerous prostate cells inhibits their growth (Pan L and Peng XC, et al. (2011) J Cancer Res Clin Oncol 137:19-28). Introduction NDM gene C-Jun in colon tumor leads to its partial regression (Suto R and To Tominaga et al. (2004) Gene Ther 11:187-93). BAT of RAS oncogene suppresses the development of several lines of cancer cells, such as pancreatic cancer, colon cancer, language.

2. Genes killers are toxin genes (type I), as well as genes for enzymes capable of transforming a non-toxic agent (prodrug) in toxin that kills cancer cells (type II). The most efficient genes of the type II genes are timedancing of herpes simplex virus (HSVtk) and yeast cytosine deaminase or E. coli. Sitoindosides - enzyme that catalyzes the hydrolytic deamination of 5-fertilizin with the formation of 5-fluorouracil (5-FU), which kills cancer cells. Antitumor activity of combination sitoindosides/5-fluorouracil has been shown in animals with such tumors, as fibrosarcoma (Mullen CA and Coale MM et al. (1994) Cancer Res 54:1503-6), carcinoma (HuberBE and Austin EA et al. (1994) Proc Natl Acad Sci US A 91:8302-6, Bentires-Alj M and Hellin AC, et al. (2000) Cancer Gene Ther 7:20-6, Huber BE and Austin EA et al. (1993) Cancer Res 53:4619-26, Ohwada A and Hirschowitz EA et al. (1996) Hum Gene Ther 7:1567-76, Kanai F and Lan KH et al. (1997) Cancer Res 57:461-5), glioma (Ichikawa T and Tamiya T et al. (2000) Cancer Gene Ther 7:74-82, To Ge and Xu L et al. (1997) Int J Cancer 71:675-9) and others.

Thymidine kinase of herpes simplex virus capable of transforming non-toxic antifungal agent Ganciclovir (GCV) into a toxic metabolites, which are incorporating into the growing DNA chain of cancer cells during division, inhibit its synthesis, thus ensuring the death of the cancer cell. HSVtk/GCV is the only gene therapeutic combination, under the third phase of clinical trials (Immonen A and Vapalahti M et al. (2004) Mol Ther 10:967-72, Evrard A and Cuq P, et al. (1999) Int J Cancer 80:465-70). In phase I clinical trials of eight patients with local recurrence of prostate cancer previously undergone hormonal therapy was administered adenovirus carrying the expression cassette with the gene of HSVtk. The subsequent introduction of GCV resulted in significant partial regression of the tumor (Nasu Y and Saika T, et al. (2007) Mol Ther 15:834-40).

Research phase II clinical trials, in which as a vector for HSVtk was used replication defective adenovirus showed that the combined use of standard and gene therapy leads to a statistically significant increase in average survival: from 38 to 62 weeks (Immonen A and Vapalahti M et al. (2004) Mol Ther 10:97-72).

3. Gene expression inhibitors of angiogenesis in tumor cells leads to suppression of the formation of new blood vessels in tumors leads to slower growth rates and degradation due to lack of nutrients and oxygen. Inhibitors of angiogenesis genes are protein angiostatin (suppresses the development of lymphoma EL-4) and endostatin (an increase of carcinoma of the kidney, melanoma, colorectal carcinoma) (Sun X and Kanwar JR et al. (2001) Gene Ther 8:638-45, Shi W and Teschendorf C et al. (2002) Cancer Gene Ther 9:513-21, Cichon T and Jamrozy L et al. (2002) Cancer Gene Ther 9:771-7).

4. Genes oncosuppressor play a key role in maintaining the stability of the cellular genome and the regulation of cell division and apoptosis (programmed cell death). Tumor cells suppress genes-oncosuppressor and so on. avoid apoptosis. The introduction of genes oncosuppressor in cancer cells leads to the suppression of their growth and death. Protein p53 is the most well-known tumor suppressor. The world's first gene therapy drug "Gendicine ™" (Chinese Shenzhen SiBiono Genetechnologies), created on the basis of rereplacenocase adenovirus that carries the gene p53, was introduced into clinical practice in 2003 in China and is used for treatment of squamous cell cancer of the head and neck.

5. Genes immunostimulators able to activate cells of the immune system. It is assumed that the introduction of the data the genes in cancer cells provides stimulation of the immune system to recognize tumor cells. Examples of genes Immunostimulants are IL-1, IL-2, IL-4, IL-6, TNF, GM-CSF, gamma interferon.

For example, the use of GM-CSF in combination with suicide genes leads to an increase in the effectiveness of cancer treatment. In a number of studies have shown that regression of the tumor increases significantly when combined gene-killers and GM-CSF (Hamilton JA (2002) Trends Immunol 23:403-8, Hamilton JA and Anderson GP (2004) Growth Factors 22:225-31). Moreover, several studies have demonstrated the development of specific antitumor immunity after co-expression of HSVtk and GM-CSF in tumor cells (Guo SY and Gu QL et al. (2003) World J Gastroenterol 9:233-7).

6. Introduction to cancer cells gene siRNA can suppress the expression of oncogenes and as a consequence induces cell death. For example, the introduction of cancer cells siRNA for gene of survivin member recombinant plasmid suppresses gene expression of survivin, stimulates apoptosis of cancer cells and inhibits their proliferation.

For the transport of therapeutic gene comprising the expression cassette inside cancer cells using expression vectors.

The expression vector is a nucleic acid molecule capable of transporting expression cassette into the cell. Typically, the expression vector is a plasmid or other DNA or RNA, have the th capacity for Autonomous replication in certain cell. Vector molecule must possess certain properties: 1) the vector must a long time to exist in the population of host cells, i.e., to replicate autonomously or together with the chromosomes of the cells; 2) any vector must be biochemical or genetic markers that would allow to detect its presence in the cells; 3) the structure of vector molecules must allow embedding in her alien of a nucleotide sequence without disrupting its functional integrity.

For the purposes of gene therapy uses several types of vectors, featured by way of delivery of genetic material. Thus, the vectors are viral and non-viral. Currently, the method of delivery of DNA in cancer cells using viruses is the most common. So, 76% of gene therapy trials conducted with different kinds of viral vectors and 24% non-viral. The viral vector is a type of vector, in which for the delivery and implementation of its genetic information genetic apparatus of any virus. Type used viral vectors are most commonly used retroviral, adenoviral, adeno-associated and herpes virus vectors (Balicki D and Beutler E (2002) Medicine (Baltimore) 81:69-86, Kovesdi I and Brough DE, et al. (1997) Curr Opin Biotechnol 8:583-9).

For replication and packaging of adenovirus D Is necessary To only a small sequence at both ends, containing the field started replication, and sequence-specific DNA packaging. Therefore, almost the entire viral genome can replace alien DNA, growing hybrid virus in the presence of virus-assistant. One of the drawbacks of such a system is a certain difficulty in getting rid of the virus helper. When using retroviruses are commonly used viruses have been removed genes for structural proteins and revertase. The most progressive as delivery systems are vectors based on adeno-associated viruses, these viruses do not cause disease in humans and are therefore very promising from the point of view of safety of use. The disadvantages of this system need to include a small container for the introduction of genetic information which is limited to 4800 p. O. Dignity viral systems is their high efficiency. The disadvantage of immunogenicity, and the complexity and high cost of production (Prestwich RJ and Errington F (2008) Clin Med Oncol 2:83-96).

As a vector for non-viral delivery are commonly used plasmids, which can be produced in large quantities in bacterial cultures.

Plasmid should have a special sequence required for developments outside the body, is the replication origin, the gene of resistance to the antibiotic, site m the divine clone (MCS). In contrast to recombinant viruses, plasmids quite simple to design and to produce in large quantities. In addition, plasmids provide a fairly high level of safety when used in comparison with viral vectors (Williams PD and Kingston PA (2011) Cardiovasc Res 91:565-76).

In addition to plasmid and viral systems in recent times, there are other delivery systems. For example, the proposed system bacterial delivery, which use natural property of bacteria to accumulate in cancer cells.

For delivery of plasmid vectors in tumor cells used two approaches.

The first is the conclusion of plasmids in artificial lipid vesicles - liposomes are able to penetrate through the plasma membrane. The second way is to create a complex of DNA with positively charged carrier, for example by polyethyleneimine or poly-liposomes, however, the formation of larger complexes, which are difficult to deliver into the cell (Wagstaff KM and Jans DA (2007) Biochem J 406:185-202).

Also for delivery using peptides/proteins, penetrating into cells (RAF). These peptides/proteins in the formation of complex with DNA provide direct delivery into the nuclei of cells (Wagstaff KM and Jans DA (2006) Curr Med Chem 13:1371-87, Gupta In and Levchenko TS, et al. (2005) Adv Drug Deliv Rev 57:637-51, Morris MC and Chaloin L et al. (2000) Curr Opin Biotechnol 11:461-6, Wagstaff KM andJans DA (2007) Biochem J 406:185-202, WagstaffKM and Fan JY et al. (2008) FASEB J 22:2232-42). CPP does not have strong antigenic properties and are able to migrate into the cell larger DNA molecules (Balicki D and Putnam CD, et al. (2002) Proc Nati Acad Sci USA 99:7467-71). For the delivery of DNA into the nuclei of cells can be used proteins-histones. The best activity against the expression of delivered genes reported to possess histones H2A and H2B as well as (Kaouass M and Beaulieu R et al. (2006) J Control Release 113:245-54). The big advantage of histones over other gene delivery systems is their low toxicity.

In General, for efficient non-viral delivery vector should possess a combination of properties such as the ability to form a complex with DNA condensing DNA into a more compact state, to protect it from the action of nucleases, to ensure effective passage through cellular and nuclear membranes and not to inhibit the transcription of DNA. Gene anticancer drugs can be administered to a patient intravenously, intraperitoneally, nutripure, subcutaneously or intramuscularly depending on tumor localization and prevalence of the disease. An example of the effective use of intratumoral injection of genetic constructs can serve as a test adenoviral preparation containing bacterial genes sitoindosides and timedancing of herpes simplex virus - Ad5-CD/TKrep. report was injected into the prostate and then therapy 5-fertilizing and ganciclovir (Freytag and SO Khil M et al. (2002) Cancer Res 62:4968-76). For the treatment of prostate tumors also used subcutaneous and intravenous introduction of genetic constructs (Small EJ and Carducci MA, et al. (2006) Mol Ther 14:107-17) (http://www.asco.org/ASCOv2/Meetings/Abstracts?&vmview=abst_detail_view&confID=26&ab stractID=890).

In the treatment of malignant pleural mesothelioma and lung cancer using intrapleural injection of structures (Sterman DH and Recio A, et al. (2005) Clin Cancer Res 11:7444-53, Tan Y and Xu Metal. (1996) Anticancer Res 16:1993-8).

Often, gene therapeutic drug is liofilizovannye DNA, because the DNA in the form of liofilizirovannogo powder is the most convenient for storage and use as injection. To prevent the loss of transfection activity of DNA during lyophilization use mono and disaccharides, such as lactose, glucose and sucrose as an additive to the solution of plasmid DNA before lyophilization process. Sugar is also used in lyophilization of complexes of DNA and polycation.

Thus, gene therapeutic pharmaceutical composition should contain: 1) the expression cassette comprising the expression vector carrying therapeutic gene under the control of the promoter; 2) the media to ensure penetration expression vector containing an expression cassette, through the cell membrane. The carrier may be a virus. In the case of non-viral n is sites he must be positively charged (liposomes, polyethylenimine and other polycation). Auxiliary components in gene therapeutic pharmaceutical compositions can be sugars (mono and disaccharides), buffer solutions, antibacterial agents, and others.

Examples

The following examples are intended solely to illustrate specific preferred embodiments, but should not be construed as limiting scope of the present invention.

Example 1. Creating design PhSurv269-pGL3

Fragment 269 p. O. proximal promoter region of the gene BIRC5 (survivin - Surv) amplified by PCR using as template genomic DNA of the human brain and synthesized primers (pSurv269For-BglII CGGAGATCTCGCGTTCTTTGAAAGCAGTCGA, pSurv269Rev-HmdIII CCCAAGCTTGCCGCCGCCGCCACCTCTG), flanking fragment 269 p. O. and containing the recognition sites of restricted BglII and HindIII (Fermentas, Canada). The obtained PCR product intermediate cloned in the vector pAL-TA (Evrogen, Russia) and the resulting intermediate structure PhSurv269-pAL-TA sequenced to exclude errors Taq polymerase (Evrogen, Russia). Next, the intermediate design PhSurv269-pAL-TA were treated with restrictase BglII/HindIII required box 269 p. O. was isolated from agarose gel and cloned into the vector pGL3 carrying the reporter gene Firefly luciferase, also treated restrictase BglII/HindIII. It was further determined nucleotide after outermost final design PhSurv269-pGL3 to exclude nucleotide substitutions (Fig.1).

Example 2. Creating design PhSurv269+130-pGL3, functional derivative promoter PhSurv269 modified by adding insertions

Was established promoter PhSurv269+130, which is the functional derivative of the promoter PhSurv269. The modified promoter differs from the original promoter PhSurv269 the presence of insertions size 130 p. O. the Insertion is located in a proximal position relative to the 5'-end of the promoter PhSurv269 and is a fragment of a+269/+399 sequence of the native promoter of survivin person. The insertion is located in the "direct" position, i.e. in the same orientation as in the native promoter.

A fragment of the proximal promoter region of the gene BIRC5 (survivin, Surv) amplified by PCR using as template genomic DNA of the human brain and synthesized primers (pSurv269+130For-BglII CATAGATCTATTTTTAGTAGAGACAAGGTTTCACCGTG and pSurv269+130Rev-HindIII CCCAAGCTTGCCGCCGCCGCCACCTCTG) containing the recognition sites of restricted BglII and HindIII (Fermentas, Canada). The obtained PCR product intermediate cloned in the vector pAL-TA (Evrogen, Russia) and the resulting intermediate structure PhSurv269+130-pAL-TA sequenced to exclude errors Taq polymerase (Evrogen, Russia). Next, the intermediate design PhSurv269+130-pAL-TA were treated with restrictase BglII/HindIII, the desired insert was isolated from agarose gel and cloned in the vector pGL3 that carries a reporter gene is seperate Firefly also treated restrictase BglII/HindIII. It was further determined the nucleotide sequence of the final design PhSurv269+130-pGL3 to exclude nucleotide substitutions.

Example 3. Comparison of activity PhSurv269+130 modified by adding insertions, with the unmodified promoter PhSurv269

To compare the functional activity of the derived promoter PhT269 (PhT269+130) and unmodified variants PhT269 we used the following cancer cell lines of various origins: Calu-1 (lung carcinoma human), A (melanoma human skin), A (human lung adenocarcinoma), PANC-1 (pancreatic carcinoma cancer man), HepG2 (human hepatocellular carcinoma), LLC (epidermoid carcinoma, Lewis lung).

The experiment was carried out as described in Example 9.

The relative activity of the investigated promoters in different cell lines are shown in table 1. As can be seen from table 1, the activity of promoters PhT269 and PhT269+130 are almost the same in all cell lines tested, while the activity of promoters PhT269 and PhT269+130 differ in different cell lines, each cell line activity of these promoters are practically identical. Thus, the functional derivative of the promoter PhT269 has the same activity as the original promoter PhT269.

The obtained data allow to make the e l e C what possible use of functional derivatives promoters, characterized by almost the same or a sufficiently high activity compared with the original promoter.

Table 1
The relative activity of the investigated promoters in different cell lines. Values represent the activity of the promoters as the ratio of luciferase activity was studied promoters to the activity obtained with plasmid PV-pGL3 containing only the SV40 promoter. Standard error of the mean (±SEM) calculated from three independent experiments
AACalu1HepG2PANC-1LLC
PhT269of 2.26±0,36,11±2,69,11±1,01,76±0,32,96±0,15,38±0,3
PhT269+1302,58±0,4to 5.93±2,710,16±1,41,71±0,2 3,25±0,15,38±0,6

Example 4. Construction of expression vectors based on the dual promoters PhTSurv269 and PhSurv269T

A fragment of the promoter of the gene of survivin person, PhSurv269 (SEQ ID NO: 1) length 269 p. O. cloned into the design PhTERT-pGL3 derived from the plasmid pGL3-basic (Promega, USA), bearing a fragment of the promoter of the reverse transcriptase telomerase person (PhTERT) and reporter gene Firefly luciferase. The promoter PhSurv269 inserted into the design PhTERT-pGL3 in direct orientation in two positions relative to the promoter PhTERT (SEQ ID NO:2). For this he received cDNA promoter region of the gene of survivin person length 269 p. O. as a result of hydrolysis of plasmid PhSurv269-pGL3 the enzymes BglII and HindIII, followed by processing the fragment maple. Then the obtained fragment PhSurv269 ligated with the vector PhTERT-pGL3, previously linearized site recognition by the restriction enzyme HindIII, or on the website of recognition by the restriction enzyme XhoI and processed large subunit of DNA polymerase I of E. coli (Fermentas, Canada). Thus, in the first case was received vector PhTSurv269-pGL3 carrying the luciferase gene under control of the double promoter PhTSurv269, where the promoter PhTERT is above promoter PhSurv269 relative to the start codon of the gene of Firefly luciferase. In the second case, the received vector PhSurv269T-pGL3, in which the promoter PhSurv269 is above promoter PhTERT otnositelno start-codon of the gene luciferase. The structure of the resulting expression cassette is illustrated in Fig.2.

Example 5. Construction of expression vectors based on the dual promoters PhTmSurv and PmSurvhT

A fragment of the promoter of the gene of survivin mouse (PmSurv) length 197 p. O. (SEQ ID NO:3) was cloned into the design PhTERT-pGL3, created on the basis of plasmid pGL3-basic (Promega, USA) previously, carrying the promoter of the reverse transcriptase telomerase person (PhTERT) and reporter gene Firefly luciferase. The promoter PmSurv cloned into the design PhTERT-pGL3 in direct orientation in two positions relative to the promoter PhTERT. For this he received the cDNA of survivin mouse (pmSurv) as a result of hydrolysis of plasmid PmSurv-pGL3 the enzymes HindIII, followed by processing the fragment maple and New members (Fermentas, Canada). Then the resulting fragment containing the cDNA of the gene of survivin mouse (420 p. O.), ligated with the vector PhTERT-pGL3, previously linearized site recognition by the restriction enzyme XhoI and processed large subunit of DNA polymerase I of E. coli, and then treated with restriction enzyme NotI. Thus was obtained the vector PmSurvhT-pGL3 carrying the luciferase gene under control of the double promoter PmSurvhT, where the promoter PhTERT is below the promoter PmSurv relative to the start codon of the gene of Firefly luciferase.

To obtain the plasmid carrying the gene luciferase under the control of the double promoter PhTmSurv, where the promoter PhTERT is what ISE promoter pmSurv relative to the start codon of the gene luciferase Firefly received cDNA reverse transcriptase telomerase person (PhTERT) length 243 p. O. as a result of hydrolysis of plasmid PhTERT-pGL3 the enzymes HindIII, followed by processing the fragment maple and NotI. The resulting fragment, containing PhTERT (437 p. O.), ligated with the vector pmSurv-pGL3, pretreated with enzymes BglII, followed by processing the fragment maple and NotI. Thus was obtained the vector PhTmSurv-pGL3 carrying the luciferase gene under control of the double promoter PhTmSurv, where the promoter PhTERT is above promoter PmSurv relative to the start codon of the gene of Firefly luciferase.

Example 6. Construction of expression of suicide vectors based on the double-reconstituting promoter PhTSurv269: PhTSurv269-HSVtk and PhTSurv269-HSVtk-mGM-CSF

cDNA gene timedancing of herpes simplex virus type 1 (HSVtk) cloned into the design PhTSurv269-pGL3-based plasmid pGL3-basic (Promega, USA), bearing a double promoter consisting of the promoter region of the gene reverse transcriptase of the human telomerase (hTERT) and a shorter promoter region of the gene of surviving person (PhSurv269, coordinates -268+1 [where +1 adenine triplet encoding iniciarse methionine]). cDNA HSVtk gene was inserted into the design PhTSurv269-pGL3 in direct orientation. For this vector PhTSurv269-pGL3 were treated with restrictase NcoI/BamHI (Fermentas, Canada), the resulting vector has been completely deleted the selected cDNA gene Firefly luciferase. Plasmid PhSurv-HSVtk-pGL3 obtained above was treated with restrictase NcoI/BamHI. Then the resulting fragment containing the sequence of HSVtk, ligated with the linearized vector PhTSurv269-pGL3. The result was obtained design PhTSurv269-HSVtk-pGL3. The sequence of the resulting construction was confirmed by the method of sequencing by Sanger. The structure of the resulting expression cassette is illustrated in Fig.3.

Insert HSVtk-IRES-mGM-CSF containing gene timedancing of herpes simplex virus type 1 (HSVtk), IRES (plot planting ribosomes virus encephalomyocarditis person) and gene mGM-CSF (granulocyte-macrophage colony-stimulating factor mouse), cloned into the design PhTSurv269-pGL3. The specified fragment was cloned into the design PhTSurv269-pGL3 in direct orientation. For this vector PhTSurv269-pGL3 were linearizable by processing the restriction enzyme BamHI in combination with a fragment of maple and then by restriction enzyme NcoI. Fragment of HSVtk-IRES-mGM-CSF was obtained by the hydrolysis of plasmid CMV-HSVtk-IRES-mGM-CSF-pGL3 restriction endonucleases NcoI/XhoI with "zatuplenie" remainder of the XhoI site of the large subunit maple. The obtained DNA fragment was Legerova in PhTSurv269-pGL3. The result was obtained design PhTSurv269-HSVtk-IRES-mGM-CSF-pGL3. The sequence of the obtained structures was confirmed by the method of sequencing by Sanger. The structure of the resulting expression cassette proell is reported in Fig.4.

Example 7. Creation of expression vectors carrying the gene cassette FCU1-mGM-CSF under the control of the double promoter PhTSurv269

Design PhTSurv269-FCU1-mGM-CSF consists of: 1) promoter PhTSurv269; 2) chimeric intron length 133 p. O., located between the promoter and the FCU1 gene; 3) cDNA FCU1 gene (fragment of the gene sitoindosides (FCY1) and a fragment of the gene uracil-phosphoribosyltransferase (UPRT, FUR1) from an organism Saccharomyces cerevisiae, the coordinates 1002-2120 p. O.) ; 4) IRES (plot planting ribosomes virus encephalomyocarditis person) and 5) cDNA gene mGM-CSF

To obtain design PhTSwv269-FCU1-mGM-CSF-pGL3 plasmid pCMV-FCU1-pGL3 obtained previously, was linearizable by processing restrictase HindIII (with subsequent processing of the fragment maple) and NotI (Fermentas, Canada). Insert PhTSurv269 was obtained when processing vector PhTSurv269-pGL3-restrictable NcoI (with subsequent processing of the fragment maple) and NotI (Fermentas, Canada). Then, the resulting fragment, containing the promoter PhTSurv269, provided with the linearized vector CMV-FCU1-pGL3. The result was obtained design PhTSurv269-FCU1-pGL3. The sequence of the resulting construction was confirmed by the method of sequencing by Sanger.

Target design PhTSurv269-FCU1-mGM-CSF (Fig.5) was prepared as follows: plasmid PhTSurv269-FCU1-pGL3, was treated with restriction enzyme BamHI (Fermentas, Canada), the resulting vector was deleted part of the FCU1 gene and signal polyadenylate the Oia. Insert FCU1*(ahfuvtyn)-mGM-CSF was obtained by treatment of the vector pCMV-FCU1-IRES-mGM-CSF-pGL3 the restriction enzyme BamHI. Then, the resulting fragment, containing the fragment of the FCU1 gene, IRES, mGM-CSF, and the polyadenylation signal provided with the linearized vector PhTSurv269-FCU1-pGL3 (from which the cut part of the FCU1 gene and the polyadenylation signal). When legirovanii were recovering holistic FCU1 gene (gene contains the restriction site BamHI). The result was obtained design PhTSurv269-FCU1-mGM-CSF-pGL3. The sequence of the resulting construction was confirmed by the method of sequencing by Sanger.

Example 8. Creating design PhTSurv269-Cre

To enhance tumor-specific expression of the hybrid gene sitoindosides/uralspetstransmash (FCU1) in tumor cells, we created a modified system Cre-LoxP. Were constructed two vectors: PhTSurv269-Cre-pQC (activator) and CMV-LoxP-Stop-LoxP-FCU1-pGL3 (effector).

First, the activation vector of the binary system carries the gene Se-recombinase with a nuclear localization signal, under control we created opukholespetsificheskaya promoter PhTSurv269. Second, effector, or killer, the vector contains the promoter of cytomegalovirus (CMV), separated from the FCU1 gene "Stopw-CHraanoM, which consists of three tandem polyadenylation signals of the SV40 virus. In addition, a "Stop"signal on both sides flanked in one'or the orientation sequences, recognizable protein Cre - LoxP-sites. Schematic diagram of the gain expression of suicidal FCU1 gene using the Cre/LoxP system shown in Fig.6.

Design PhTSurv269-Cre was obtained on the basis of plasmids PhSurv-Cre-pQXIX and PhTSurv269-pGL3 obtained previously.

DNA plasmids PhSurv-Cre-pQXIX hydrolyzed by site recognition by restrictase BglII/NotI, and then processed large subunit of maple. With the remaining part of the vector, provided the insert PhTSurv269 obtained by the hydrolysis of DNA plasmids PhTSurv269-pGL3-restrictable XhoI/NcoI, followed by processing the fragment maple. Selection of clones in the correct orientation was performed by PCR using specific primers complementary to the gene Cre-recombinase (CreSeq-Rev!) and promoter PhSurv269 (Surv269For). The correctness of the resulting construction was confirmed by determining the nucleotide sequence using specific primers UP-L1, Surv269For and CreSeq-F2.

Example 9. The determination of the activity of the universal ofwholesale promoters PhTSurv269 and PhSurv269T in cancer cells

To test the activity of the obtained promoters were used the following cancer cell lines of various origins: Calu-1 (lung carcinoma human), A (melanoma human skin), A (human lung adenocarcinoma) NT (human fibrosarcoma), PANC-1 (carcinoma of the pancreas of a person).

Eucario the practical cells were cultured at 37°C in CO 2the incubator to achieve 90% of confluently 25 cm2the culture vial. Next was preparing solutions containing 9 μg plasmids of Examples 1, 4, or 5, or a control plasmid BV-pGL3, PV-pGL3 and 1 µg normalization plasmid pRL-TK, carrying luciferase gene Remlla reniformis, 200 μl of serum-free medium Opti-MEM (Invitrogen, USA). Further to the solution of plasmid was added 200 μl of a solution LFA2000 in Opti-MEM (25 μl LFA+175 μl of Opti-MEM), incubated 5 min at room temperature. Eukaryotic cells incubated with the received lipoplexes within 3-4 hours depending on the cell line.

48 hours after introduction into cells of one of the structures of Examples 1,4 or 5, the control plasmids BV-pGL3, PV-pGL3 and normalization plasmid pRL-TK was measured activity of Firefly luciferase and R. reniformis in the cellular extract using an assay kit Dual-Luciferase Reporter Assay System (Promega, USA). The values of the activity of Firefly luciferase was normalized relative luciferase activity of R. reniformis plasmid pRL-TK to reduce the error associated with different efficiency of delivery of expression constructs into cells in a series of independent experiments. To increase the visibility and further reducing the error of the experiment, the data was normalized by the activity of the control structures BV-pGL3 and PV-pGL3.

The average luciferase activity and the standard error of renego was calculated by the formula S x-bar=(s2/n)1/2where s is the total variance, n is the number of observations.

In the result of the analysis it was found that the activity of the dual tandem promoters PhTSurv269 and PhSurv269T surpasses the activity of single PhTERT and PhSurv269 promoter in all cell lines except line Calu-1. The relative activity of the investigated promoters in different cell lines is shown in Fig.7, 8 and table 2. As can be seen from table 2, the activity of the double promoter PhTSurv269 and PhSurv269T correspond approximately to the sum of the activities of individual promoters. This is especially observed for PhTSurv269 tandem. Activity PhSurv269T tandem is usually slightly lower than the activity PhTSurv269 tandem.

It was also shown that the activity of the double promoter PmSurvhT in cancer cells 2-3 times higher than the activity of a single promoter PmSurv depending on the type of cell line (Fig.7).

The data obtained allow to conclude that the use of tandem promoters for gene expression rather than using each of the single promoters, and PhTSurv269 tandem preferable PhSurv269T tandem activity expression in different types of cancer cells (Fig.8).

3,96±0,1
Table 2
The relative activity of the investigated promoter is in different cell lines. Values represent the activity of the promoters as the ratio of luciferase activity was studied promoters to the activity obtained with plasmid PV-pGL3 containing only the SV40 promoter. Standard error of the mean (±SEM) calculated from three independent experiments. The "+" sign in the first column means that the data were obtained by arithmetic addition. The average activity of the promoters was calculated for the five cancer cell lines (Calu-1, A, A, PANC-1 and NT) as the sum of activities in each line, divided by 5. The rightmost column is the normal lung fibroblasts, the first line shows the p53 status of the cell lines, null p53 protein is not expressed in cell lines, mut - mutant p53 protein, wt - wild type p53 protein
Calu-1A375A549PANC-1HT1080The average activity in cancer cellsIVL-11NS
P53 statusnullwtwtmutwtwt
PhTERT 0,80±0,12,38±0,21,68±0,20,78±0,11,23±0,21,370,02±0,002
PhSurvof 6.31±0,71,46±0,21,15±0,20,38±0,10,84±0,22,030,12±0,014
PhSurv2699,1±1,02,20±0,26,11±2,72,98±0,11,08±0,3of 4.380,19±0,014
PhTERT+PhSurv26910,85±1,14,58±0,47,79±2,93,76±0,22,31±0,35,860,21±0,016
PhTSurv2698,80±0,13,89±0,810,91±0,93,96±0,25,47±1,56,610,40±0,034
PhSurv269T2,87±0,28,97±0,453,85±0,13,68±0,74,670,23±0,04

Example 10. Comparison of the effectiveness of dual promoters PhSurv269T and PhTSurv269 efficiency dual promoters PhTS and PhST, designed by us previously

For comparison of promoter activity, we used the following cancer cell lines of various origins: Calu-1 (lung carcinoma human), A549 (human lung adenocarcinoma) HT1080 (human fibrosarcoma), PANC-1 (carcinoma of the pancreas of a person).

The experiment was carried out as described in Example 9. In Fig.9 shows that in all investigated cell lines, the activity of the promoter PhTSurv269 higher than the activity of single promoters PhSurv, PhTERT and activity of artificial double promoters PhTS and PhST.

Example 11. Detection of the hybrid protein sitoindosides/ uralspetstransmash (FCU1) using Western blot analysis of transfected cancer cells

In the first stage, carried out the transfection of the cell line S37 genetically engineered designs PhTSurv269-Cre//pCMV-STOP-FCU1 (cotransfected vectors pCMV-STOP-FCU1 and PhTSurv269-Cre). Transfection of cells was performed in the culture flasks T-25 Lipofectamine 2000 ("Invitrogen, USA) according to the recommendations of the producer is. For transfection is typically used 10 μg of plasmid DNA. After lipofectin cells were cultured in DMEM/F12 (1:1) without antibiotics containing 10% fetal calf serum for 48 hours, then got cellular precipitation for Western blot analysis.

As a vector for comparison, we used the vector pCMV-FCU1, in which the FCU1 gene expression was sent a strong constitutive promoter pCMV. 48 hours after injection of the constructs cells were detached from the surface of the culture flasks by trypsin solution, and then suspended in the DMEM:F12 with antibiotics and determined the number of cells in the resulting suspension using a counting chamber Neubauer, adding to the aliquot of cell suspension tripney blue in a 1:1 ratio. Further centrifuged 5 minutes at 7°C and 160g, cellular precipitation twice washed with 1X phosphate-saline buffer (1.7 mm KN2RHO4, 5.2 mm Na2HPO4,150 mm NaCl) (see below).

The cells suspended in the buffer for the application (the concentration of cells in the buffer should be 107cells/ml) and incubated for 5 minutes at 95°C.

The resulting cell lysates were subjected to denaturing electrophoresis of proteins in polyacrylamide gels by the method of laemmli's method (Laemmli U. K. (1970) Nature, Aug 15;227(5259):680-5) with subsequent transfer of the fractionated proteins from the gel to the membrane is wound. The amount of protein FCU1 in the lysates (normalized for total protein) transtitional cell lines were analyzed using Western blot analysis. As the primary antibodies used sheep IgG ("Abeam"), bind to the N-terminal region of the protein FCU1. The secondary antibody to sheep IgG were conjugated with horseradish peroxidase (Santa-Cruz). To control the amount of protein in cell extracts used color with antibodies to actin. The result of the Western blot analysis shown in Fig.10.

In order to determine the most effective ratio of vector-effector and vector-activator, we performed transient transfection vectors PhTSurv269-Cre and pCMV-STOP-FCU1 in ratios by the number of micrograms 2:2 and 1:3. The maximum amount of DNA that could be taken for transfection is 4 g (according to the manufacturer's recommendations). The selected ratio of effector (pCMV-STOP-FCU1) and activator (PhTSurv269-Cre) vectors describe two possible situations: 2:2 for the effective operation of the system must equal the number of effector and activator of vectors; 1:3 - for the best possible FCU1 gene expression requires excessive effector gene.

In lysates of cells S37, transfected with the vector pCMV-FCU1, the amount of protein FCU1 more than in lysates of cells of the same lines, cotransfection binary system of vectors PhTSurv269-Cre/pCMV-STOP-FCU1 used in both ratios, however, the ratio activator/effector 1:3 is somewhat more preferable from the viewpoint of production of the protein FCU1.

Example 12. Determination of cytotoxicity of the established system of vectors PhTSurv269-Cre//DCMV-STOP-FCU1

To determine the cytotoxicity of the system PhTSurv269-Cre//pCMV-STOP-FCU1 measured the sensitivity of transfected cells S37 (mouse sarcoma) to 5-forcasino (5-FT). To quantify the sensitivity of the cells was determined by the percentage of surviving cells at different concentrations of 5-FC. Comparing the survival rate of cells transfected with the vector pCMV-FCU1 and transfected with the binary system of vectors PhTSurv269-Cre//pCMV-STOP - FCU1, it is possible to draw conclusions about the effectiveness of structures from a therapeutic point of view.

Cells of the transient was transfusional the following expression constructs: system vectors PhTSurv269-Cre//pCMV-STOP-FCU1 (cotransfected, the ratio of vectors 2:2 and 1:3 by the number of micrograms) and the vector pCMV-FCU1 (positive control). Next transfetsirovannyh cells within 120 h were incubated in the medium with different concentrations of 5-FC (0, 50, 200, 500 and 1000 μm). As control was used nitrostilbene cells S37.

Cell survival was assessed using the MTS test according to the manufacturer's recommendations (Promega, USA). The efficiency of transfection was determined after FACS analysis (Fluorescence-Activated Cell Sorting, FACS) cells, t is inficirovannyh reporter vector pEGFP.

As a result of experiments it was shown that the effect of the system of vectors PhTSurv269-Cre//pCMV-STOP-FCU1 comparable with the effect caused by the construction of pCMV-FCU1. When the concentration in the medium for culturing cancer cells to 5-fertilizin 200 microns 90% of cancer cells are killed after treatment system PhTSurv269-Cre//pCMV-STOP-FCU1 (Fig, 11).

Example 13. Comparison of cytotoxicity of systems of vectors Promoter-Cre//pCMV-STOP-FCU1, where Promoter - PhSurv or PhTSurv269

To compare the cytotoxicity systems PhTSurv269-Cre//pCMV-STOP-FCU1, PhSurv-Cre//pCMV-STOP-FCU1 cell line S37 was transfusional obtained by the systems and control design CMV-FCU1-pGL3, then transfetsirovannyh cells were treated with a solution of 5-fertilizin different concentrations and after 196 hours after treatment was measured and determined the percentage of surviving cells in each group.

Cell survival was assessed using the MTS test according to the manufacturer's recommendations (Promega, USA). The efficiency of transfection was determined after FACS analysis (Fluorescence-Activated Cell Sorting, FACS) cells, transfected with the reporter vector pEGFP.

It is shown that suicidal effect of the system of vectors PhTSurv269-Cre//pCMV-STOP-FCU1 exceeds the effect of the system PhSurv-Cre//pCMV-STOP-FCU1 and is comparable with the effect caused by the construction of pCMV-FCU1 (Fig.12).

Example 14. Determination of the transcriptional activity of the double promoter PhTSurv269, PhTmSurv

Cell lines A (melanoma of the skin man) was transfusional designs PhTSurv269-pGL3, PhTmSurv-pGL3 and control design PhTSurv-pGL3, 48 hours after injection of cell designs collected and isolated total RNA using a kit RNeasy Mini Kit (Qiagen, USA) and then were treated with Dnazol I (Quiagen, USA) according to the manufacturer's protocols. The obtained total RNA was used for cDNA synthesis with a seed consisting of statistical hexamers (Perkin Elmer, USA). This cDNA was used to detect the presence of transcripts with promoters PhTERT, PmSurv and PhSurv269 in the composition of the tandems. To determine whether transcripts from promoters has been used technique is semi-quantitative RT-PCR as described previously (Pleshkan VV and Vinogradova TV et al. (2008) Biochim Biophys Acta 1779:599-605).

To determine whether transcripts from the distal promoter PhTERT tandem picked up a General primer TSL-F, which is located in the linker immediately during the analyzed promoter PhTERT and paired him reverse primers for each gene promoters of survivin: primer hS269_128R for constructs with promoter PhTSurv269, primer mS_122R for constructs with promoter PhTmSurv and primer hSurv_150R for constructs with promoter PhTS. Reverse primers were located in the proximal promoter at a distance of not more than 150 p. O. from its 5'-end, obviously to sites of transcription initiation of the proximal promoter (Fig.13). The resulting polymerase chain reaction products were analyzed by the use of electrophoretic separation in agarose gel. For each construct was analyzed at least three independent results FROM RT-PCR.

In the analysis, it was shown that the structures PhTSurv269-pGL3 and PhTmSurv-pGLS works distal PhTERT promoter, while the transcripts of the distal PhTERT promoter in the design PhTS-pGL3 not detected (Fig.14, panel "distal").

To detect the presence of transcripts initiated from the proximal and/or both promoters in tandem, used a different primer pair. Used direct UPF primer, located just beyond the proximal promoter and the reverse primer to him Luc_202R. Transcripts were detected as in the case of structures PhTSurv269-pGL3 and PhTmSurv-pGL3, and in the case control design PhTS-pGL3 (Fig.14, panel "proximal").

Thus, we have shown that in the investigated structures PhTSurv269-pGL3 and PhTmSurv-pGL3 work both promoter.

Example 15. Functional test for cytotoxicity HSVtk, controlled, double tandem promoter PhTSurv269, with subsequent treatment with ganciclovir

Eukaryotic cells have transfusional in sectionone tablets design PhTSurv269-HSVtk-mGM-CSF-pGL3 and control design PhSurv-HSVtk-mGM-CSF-pGL3 using Lipofectamine-2000 (Invitrogen, USA). After 48 hours after transfection, cells were added to the solution of ganciclovir ("Cymeven, Roche, Switzerland) in DMEM/F12 (1:1) and what tibeticum at a concentration of 0, of 12.5, 50 μm. After 192 hours after the addition of ganciclovir conducted MTS-test to determine the number of living cells (Promega, USA). The results of the experiment are shown in Fig.15. In cell lines NT it was shown that the survival rate of the control group cells (nitrostilbene cells) at a concentration of ganciclovir from 0 to 50 μm was approximately 100%, the survival rate of cells transfected with the design PhTSurv269-HSVtk-mGM-CSF-pGL3, was in each group is approximately 2 times lower than the survival rate of cells transfected with the design PhSurv-HSVtk-mGM-CSF-pGL3.

Thus, in this experiment it is shown that the use of dual-tandem promoter PhTSurv269 for control of therapeutic genes more promising than the use of full-length promoter of survivin.

Example 16. Functional test for cytotoxicity FCU1, controlled, double tandem promoter PhTSurv269, during subsequent processing 5-fortitudinem

Eukaryotic cell line Calu-1 was transfusional 25 cm2the bottle design PhTSwv269-FCU1-mGM-CSF-pGL3 and control constructs pCMV-FCU1-mGM-CSF-pGL3 and PhSurv-FCU1-mGM-CSF-pGL3 using Lipofectamine-2000 (Invitrogen, USA). After 48 hours after transfection, cells were added to a solution of 5-fortitudine (Sigma-Aldrich, USA) in DMEM/F12 (1:1) with antibiotics at a concentration of 0, 10, 50, 200, 500 μm. After 144 hours after added the I 5-fertilizin conducted MTS-test to determine the number of living cells (Promega, USA). The results of the experiment are shown in Fig.16. It was shown that the survival rate of the control group cells (nitrostilbene cells) at a concentration of 5-fertilizin from 0 to 500 ám was approximately 100%, the survival rate of cells transfected with the design PhTSurv269-FCU1-mGM-CSF-pGL3, was lower than the survival rate of cells transfected with the design PhSurv - FCU1-mGM-CSF-pGL3.

Thus, the use of dual tandem promoter PhTSurv269 for control of therapeutic genes is more preferable than the use of full-length promoter of survivin (PhSurv). In conjunction with this property are double tandem promoter PhTSurv269, as greater tumor specificity, it is especially important.

Example 17. Determining the amount of secreted cytokine GM-CSF under the control of the dual tandem promoter PhTSurv269

Eukaryotic cells have transfusional in sectionone tablets design PhTSurv269-HSVtk-mGM-CSF-pGL3 and control design PhSurv-HSVtk-mGM-CSF-pGL3 using Lipofectamine-2000 (Invitrogen, USA). After 48 hours after transfection were collected, air-conditioned environment to determine the amount of protein GM-CSF.

The definition of production of GM-CSF in transfected cells was performed by the method of enzyme immunoassay culture fluid using a commercial ELISA kit production R&D Systems (USA). The results of exp is riment are shown in table 3. It was shown that under the control of the promoter PhTSurv269 when using design PhTSurv269-HSVtk-mGM-CSF-pGL3 depending on the cell line is synthesized in 2.5-4 times more protein GM-CSF, than under the control of the promoter PhSurv when using design PhSurv-HSVtk-mGM-CSF-pGL3.

Table 3
The number of mGM-CSF in ng produced 105cell lines Calu-1, HT1080, LLC, transfected with constructs containing the HSVtk cassette-mGM-CSF under the control of the CMV promoters, PhSurv, PhTSurv269, per day
DesignCell line
Calu-1HT1080LLC
CMV6.512.53.77
PhSurv0.570.270.38
PhTSurv2691.431.0970.41

Example 18. Determining the amount of secreted cytokine GM-CSF design PhTSurv269-FCU1-mGM-CSF-pGL3

Eukaryotic cell line Calu-1 transfiere the Ali designs PhTSurv269-FCU1-mGM-CSF-pGL3, PhSurv-FCU1-mGM-CSF-pGL3 and control design CMV-FCU1-mGM-CSF-pGL3 using Lipofectamine-2000 (Invitrogen, USA). After 48 hours after transfection were collected, air-conditioned environment to determine the amount of protein mGM-CSF.

Product definition mGM-CSF in transfected cells was performed by the method of enzyme immunoassay culture fluid using a commercial ELISA kit production R&D Systems (USA). The results of the experiment are shown in Table 4. It was shown that under the control of the promoter PhTSurv269 when using design PhTSurv269-FCU1-mGM-CSF-pGL3 depending on the cell line synthesized 2.6 times more protein GM-CSF, than under the control of the promoter PhSurv when using design PhSurv-FCU1-mGM-CSF-pGL3.

Table 4
The number of mGM-CSF in ng produced 105cell line Calu-1, transfected with constructs containing the cartridge FCU1-mGM-CSF under the control of the CMV promoters, PhSurv, PhTSurv269, per day
DesignCell line Calu-1
CMV1.05
PhSurv0.007
PhTSurv2690.02

Example 19. The determination of the number of HSVtk synthesized designs PhTSurv269-HSVtk-mGM-CSF-pGL3 and PhSurv-HSVtk-mGM-CSF-pGL3

At the first stage, the cells were transfusional in the culture flasks T-25 Lipofectamine 2000 ("Invitrogen, USA) according to the manufacturer's recommendations. For transfection is typically used 10 μg of plasmid DNA. After lipofectin cells were cultured in DMEM/F12 (1:1) without antibiotics containing 10% fetal calf serum for 48 hours, the cells were detached from the surface of the culture flasks by trypsin solution, and then suspended in the DMEM:F12 with antibiotics and determined the number of cells in the resulting suspension using a counting chamber Neubauer, adding to the aliquot of cell suspension tripney blue in a 1:1 ratio. Then centrifuged 5 minutes at 7°C and 160g, cellular precipitation twice washed with IX phosphate-saline buffer (1.7 mm KH2PO4, 5.2 mm Na2HPO4, 150 mm NaCl). The cells suspended in buffer for drawing, and incubated 5 minutes at 95°C. Normalized to the concentration of protein samples was fractionally by electrophoresis in 12-13% SDS page under denaturing conditions. As a token used a set of proteins with molecular masses of 6.5-200 kDa. After fractionation in PAG proteins from the gel were transferred to PVDF membrane Immobilon-P (the Millipore", England), using the instrument Bio-Rad Trans-Blot SD cell. The membrane was pre-treated with methanol for 1 min, washed with bidistilled water for 3 minutes and placed in a buffer for transfer. Next, the membrane was incubated with antibodies to timedancing of herpes simplex virus HSV-tk (SC-28038) and GAPDH (SC-for 47724) (Santa Cruz Biotechnology, USA) at a dilution of 1:1000 for 16 hours. Then washed the membrane IX phosphate-saline buffer (PBS) containing 0.5% Tween-20, 4 times in 10 minutes. Next, the membrane was incubated with secondary antibodies donkey, conjugated with horseradish peroxidase, against immunoglobulins goats in the case of a protein of HSV-tk and antibodies goat against mouse IgG in the case of protein GAPDH (SC-2302) (Santa Cruz Biotechnology, USA) at a dilution of 1:1000 and washed as described above. Proteins on the membrane were detected by chemiluminescent method using the reagents of the company Bio-Rad (USA). Calculation of the amount of protein was performed using the program Quantity One 4.6.1 (Bio-Rad, USA). The results of the experiment shown in Fig.17. It is seen that the number of HSVtk, accumulating under the control of the double promoter PhTSurv269 (track 3) is higher than the number of HSVtk, accumulating under the control of the promoter PhSurv (lane 2).

Example 20. A comparative analysis of the effectiveness of the following promoters: CMV (cytomegalovirus promoter), the promoter of the gene of surviving person (PhSurv), artificial double promoter (PhTSurv269) ex vvo in mice

To compare the effectiveness of CMV promoters, PhSurv and PhTSurv269-based vector pGL3 (Promega, USA) were created designs containing a cassette of therapeutic genes HSVtk-mGM-CSF under the control of these promoters. You have created the following design CMV-HSVtk-mGM-CSF-pGL3, PhSurv-HSVtk-mGM-CSF-pGL3, PhTSurv269-HSVtk-mGM-CSF-pGL3 (design vector described in Example 4). Cassette therapeutic genes HSVtk-mGM-CSF contained gene timedancing of herpes simplex virus (HSVtk). The product of this gene is able to fosforilirovanii ganciclovir, non-lethal analogue of guanine. Cells transformed HSVtk, perish in the presence of ganciclovir, because cellular kinase turn phosphorylated ganciclovir in triphosphates that during cellular division are incorporated into newly synthesized DNA and cut it further synthesis. The gene product granulocyte-macrophage colony-stimulating factor (GM-CSF) are able to activate the growth, development and differentiation of granulocytes and macrophages, thereby stimulating an immune response.

In the experiment were used:

1) Mouse (female) line C57B1/6, average weight at the beginning of the experiment and 17.9±1,4 (hereinafter mean ± standard error of the mean) were kept on a standard granulated dry food.

2) Cell line LLC (Lewis lung carcinoma mouse).

3) Design CMV-HSVtk-mGM-CSF-pGL3, PhSurv-HSVtk-mGM-CSF-pGL3, PhTSurv269-HSVtk-mGM-CSF-pGL3

Cell line LLC was transfusional in the culture flasks (75 cm2) plasmids CMV-HSVtk-mGM-CSF-pGL3, PhSurv-HSVtk-mGM-CSF-pGL3, PhTSurv269-HSVtk-mGM-CSF-pGL3, nitrostilbene cells were used as control. For transfection were using 30 μg of plasmid DNA and 75 μl of Lipofectamine-2000 per culture plate. The transfection was carried out for 3 hours, 24 hours after transfection the cells were detached from the surface of the culture flask using trypsin solution and suspended in DMEM/F12 with the antibiotic, the cells are transferred into vials and centrifuged (1000 rpm, 160g,) 5 minutes the cells were twice washed in PBS and prepared cell suspension LLC in PBS with a concentration of 2·105cells/ ml for the introduction of animals.

Cell transplantation to female mice of C57B1/6 was carried out on day 1 subcutaneously in the hind part of the back at 2·105cells in 100 µl PBS. 48 hours after inoculation of the tumor began the introduction of a solution of ganciclovir (GCV) intraperitoneally for 10 days at the rate of 75 micrograms per gram weight (2 introduce 100 μl of 1 mouse per day, the concentration of ganciclovir 5 mg/ml) 2 times a day.

Starting from the 5th day after transplantation of the cells was the measurement of tumor size in 2 dimensions (less () and more (In) diameter of the tumor) electronic caliper and weight of the animals. The tumor size was calculated according to the formula A·B2/2. Measurement of the diameters of the tumor was performed every other day. Criterion euthanasia was the achievement of a tumor larger than 10% by weight of the animal.

Just been formed 4 groups of animals: 1) mice transplanted with cells LLC, transient transfitsirovannykh design CMV-HSVtk-mGM-CSF-pGL3, 2) mice transplanted with cells LLC, transient transfitsirovannykh design PhSurv-HSVtk-mGM-CSF-pGLS, 3) mice transplanted with cells LLC, transient transfitsirovannykh design PhTSurv269-HSVtk-mGM-CSF-pGL3, 4) control group (nitrostilbene cells LLC). Each group consisted of 10 animals. Animals were evenly distributed in groups according to their weights.

The results of the experiment shown in Fig.18 and 19. As can be seen in Fig.18, 19, tumor growth in the control group starts on day 10 of the experiment, whereas in the experimental groups, the tumor appears in animals only 20 days from the beginning of the experiment. The average tumor size on the 20th day of the experiment in the control group was 1343 mm3that was the basis of breeding animals of group of experiment (tumor weight in animals exceeded 10% of body weight). On the 36th day of the experiment in the CMV group, the tumor even royalty in 1 of 10 animals in group PhSurv - 2 animals in group PhTSurv269 was not observed in animals with palpable tumors. Thus, artificial the tandem promoter PhTSurv269 in the system with therapeutic genes HSVtk-mGM-CSF has high efficiency. He is ofwholesale unlike CMV and active in a wider range of cancer cell lines than the promoter PhSurv.

Listing free text sequences:

SEQ ID nos:1 -

Human survivin gene promoter fragment sequence (PhS269, positions -268 to +1, wherein +1 corresponds to adenin of an initiating methionine encoding triplet)

SEQ ID NO:2 -

Human telomerase reverse transcriptase gene promoter fragment sequence (hTERT)

SEQ ID NO:3 -

Murine survivin gene promoter fragment sequence

SEQ ID nos:4 -

PhTSurv269 promoter sequence

SEQ ID NO:5 -

PhSurv269T promoter sequence

SEQ ID NO:6 -

PhTmSurv promoter sequence

SEQ ID NO:7 -

PmSurvhT promoter sequence

1. Opukholespetsificheskaya promoter containing:
A) the nucleotide sequence of SEQ ID NO:2 or its functional derivative; and
B) the nucleotide sequence selected from the group consisting of:
(i) the nucleotide sequence of SEQ ID NO:1 or its functional derivative; and
(ii) the nucleotide sequence of SEQ ID NO:3 or its functional derivative,
connected directly or through a linker sequence.

2. The promoter under item 1, in which the sequence of SEQ ID NO:2 is located above (in 5'-direction) relative to the sequence SEQ ID NO:1 or SEQ ID NO:3.

3. The promoter under item 1, in which the sequence of SEQ ID NO:2 is located below (in 3'-direction) relative to the sequence SEQ ID NO:1 or SEQ ID NO:3.

4. The promoter according to any one of the preceding paragraphs, to the which further comprises a linker sequence between the sequence according to subparagraph (a) and a sequence according to subparagraph (B).

5. The promoter under item 2, the sequence of which is presented in SEQ ID NO:4 or SEQ ID NO:5.

6. The promoter under item 3, the sequence of which is presented in SEQ ID NO:6 or SEQ ID NO:7.

7. Expression cassette for directed expression in tumor cells antitumor agents containing in the direction from 5' to 3' promoter according to any one of paragraphs.1-6, coupled with the encoding DNA sequence which when transcribed produces the messenger RNA encoding antitumor agent.

8. Expression cassette under item 7, in which the coding sequence encodes a therapeutic protein or peptides, antigens, antisense RNA or ribozymes with antitumor activity.

9. Expression cassette under item 8, in which the coding sequence includes genes of therapeutic intervention selected from the group including: dominant negative mutants (BAT) genes involved in oncogenesis; genes killer; genes angiogenesis inhibitors; genes oncosuppressor; genes Immunostimulants; siRNA genes.

10. Expression cassette under item 8, in which dominant negative mutants (BAT) genes selected from the group including NDM gene of survivin, NDM gene c-Jun, BAT of RAS oncogene.

11. Expression cassette under item 8, in which the genes murderers selected from the group comprising the genes of toxin genes and enzymes capable of reversat non-toxic agent (prodrug) in the toxin.

12. Expression cassette under item 11, in which the genes of the enzymes capable of converting a non-toxic agent (prodrug) in toxin selected from the group containing the gene timedancing of herpes simplex virus and yeast cytosine deaminase or E. coli.

13. Expression cassette under item 8, in which genes angiogenesis inhibitors selected from the group comprising the genes of proteins angiostatin and endostatin.

14. Expression cassette under item 8, in which genes oncosuppressor selected from the group containing the gene p53.

15. Expression cassette under item 8, in which genes Immunostimulants selected from the group comprising the genes IL-1, IL-2, IL-4, IL-6, TNF, GM-CSF, interferon-gamma.

16. Expression cassette under item 8, in which the genes siRNA selected from the group comprising siRNA for gene of survivin.

17. Expression vector containing an expression cassette according to any one of paragraphs.7-16.

18. Expression vector under item 17, which is a viral vector.

19. Expression vector for p. 18, where the vector is selected from the group including retroviral, adenoviral, adeno-associated, herpes virus vector.

20. Expression vector under item 17, which represents a non-viral vector.

21. Expression vector for p. 20, where the vector is selected from the group comprising plasmid.

22. The pharmaceutical composition intended for the treatment of cancer is of deseases, containing a therapeutically effective amount of the expression cassette according to any one of paragraphs.7-16 or vector according to any one of paragraphs.17-21 and pharmaceutically acceptable auxiliary substance.

23. The pharmaceutical composition according to p. 22, intended for the treatment of cancer.

24. Pharmaceutical composition for p. 23, where the disease is selected from the group including: lung cancer, pancreatic cancer, melanoma, fibrosarcoma, sarcoma.

25. A method of treating cancer comprising the administration to a patient the pharmaceutical composition according to any one of paragraphs.22-24.

26. Method of treatment for p. 25, where the pharmaceutical composition is administered intravenously, nutripure, intramuscularly, intraperitoneally, subcutaneously, orally.

27. The method of treatment according to any one of paragraphs.25-26, where the cancer is selected from the group including: lung cancer, pancreatic cancer, melanoma, fibrosarcoma, sarcoma.

28. The use of the expression cassette according to any one of paragraphs.7-16 or vector according to any one of paragraphs.17-21 for the manufacture of a medicinal product for the treatment of cancer.

29. Application on p. 28, where the cancer is selected from the group including: lung cancer, pancreatic cancer, melanoma, fibrosarcoma, sarcoma.



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. There are presented: an antibody binding to interleukin-17 (IL-17) characterised by 6 CDR of a light and heavy chain, as well as a coding nucleic acid and a vector for expression of the above antibody. What is described is a pharmaceutical composition for treating a patient with multiple sclerosis, rheumatoid arthritis, psoriasis, Crohn's disease, chronic obstructive pulmonary disease, asthma, graft rejection on the basis of the above antibody. What is disclosed is a method for preparing the antibody by means of expressing the respective nucleic acid and recovering the antibody from a cell culture or a cell culture supernatant.

EFFECT: using this invention provides the antibody with IC50 twice as much as shown by in vitro IL-6 and IL-8 neutralisation as compared to the known NVP-AIN-497 antibody, which binds human IL-17A and IL-17F that can find application in medicine in therapy of various inflammatory diseases.

9 cl, 6 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to biotechnology. Claimed is cell of mycelial fungus Penicillium canescens with removed catabolite repression and arabinose induction, producing xylanase and laccase. Cell is transformed with plasmid, containing promoter of gene bgaS, bgaS leader peptide, xylanase-coding DNA fragment, bgaS terminator, β-lactamase gene and pMB1 replicon, and plasmid, which contains promoter of gene bgaS, bgaS leader peptide, laccase-coding DNA fragment, bgaS terminator, β-lactamase gene and pMB1 replicon. Also claimed is method of enzyme preparation of xylanase and laccase with application of said cell of Penicillium canescens.

EFFECT: group of inventions provides increased output of target enzymes.

10 cl, 9 dwg, 3 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to nucleic acids, coding functional AID mutants, to vectors and cells, including said nucleic acids. Claimed invention also relates to methods of applying mutants of AID protein.

EFFECT: obtaining functional mutants of protein of activation induced cytidine deaminase (AID), which possess higher activity in comparison with wild type AID protein.

39 cl, 11 dwg, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to virology and implies using the similar mini-antibodies for the prevention and therapy of influenza. What is declared is a similar mini-antibody specifically binding to a certain epitope of influenza A (H5N2) hemagglutinin and suppressing the influenza A (H5N2) infection. What is created is an adenovirus viral vector expressing the similar mini-antibody, which can effectively bind to the certain epitope of influenza hemagglutinin and thereby block the influenza progression. What is presented is a composition containing an effective amount of the similar mini-antibody and the viral vector expressing this similar mini-antibody.

EFFECT: what is presented is a method for the prevention and therapy of the influenza A (H5N2) infection providing administering the preventive or therapeutic effective amount of the pharmaceutical composition intranasally in the form of drops or spray into the patient in need thereof.

9 cl, 10 dwg, 2 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a polypeptide containing two binding fragments presented by antibodies; the first of them binds to CD3e(epsilon) chain epitope of a human or a primate, other than a chimpanzee, particularly Callithrix jacchus, Saguinus oedipus and Saimiri sciureus; the second one - to EGFR, Her2/neu or IgE of a human or a primate, other than a chimpanzee, with the above CD3e epitope containing the amino acid sequence Gln-Asp-Gly-Asn-Glu. There are also disclosed a coding sequence of the nucleic acid, a vector, a host cell and a method for preparing the above peptide, as well as a pharmaceutical composition and using the polypeptide in preventing, treating or relieving a proliferative disease, a malignant disease or an immunological disorder.

EFFECT: invention provides the clinical improvement of T-cell redistribution and the enhanced safety profile.

17 cl, 8 tbl, 26 dwg, 26 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, in particular, to method of producing amino acids derived from aspartate with application of recombinant microorganism, possessing at least the following properties in comparison with initial microorganism: (a) increased activity of glucose-6-phosphatedehydrogenase, (b) increased activity of fructose-1,6-biphosphatase, (c) weaker activity of isocitrate dehydrogenase, (d) increased activity of diaminopimelate dehydrogenase and (e) increased activity of aspartate kinase, where microorganism represents Corynebacterium.

EFFECT: invention makes it possible to produce amino acids of aspartate family, in particular L-lysine with high degree of efficiency.

15 cl, 35 dwg, 20 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a combined recombinant protein of the formula: S-L-R, including SR10, SR13, SR15, SdR10, SdR13 or SdR15, which specifically recognises melanoma cells, where S - streptavidin monomer, L - linker having amino-acid sequence Ser-Arg-Asp-Asp-Asp-Asp-Lys containing a restriction site with enteropeptidase and marked as "d", or amino-acid sequence Ser-Arg-Ala-Gly-Ala,R - melanoma-addressing oligopeptide representing R10 having amino-acid sequence Asp-Gly-Ala-Arg-Tyr-Cys-Arg-Gly-Asp-Cys-Phe-Asp-Gly, or R13 having amino-acid sequence Leu-Ser-Gly-Cys-Arg-Gly-Asp-Cys-Phe-Glu-Glu, or R15 having amino-acid sequence Asp-Gly-Phe-Pro-Gly-Cys-Arg-Gly-Asp-Cys-Ser-Gln-Glu. This invention also describes recombinant plasmid DNAs pSR and pSdR for expression of the specified combined proteins, bacterial strains Escherichia coli MG1655/pSR and MG1655/pSdR, producers of the specified combined proteins and a producing method of melanoma-addressing oligopeptide R from combined recombinant proteins SdR10, SdR13 or SdR15.

EFFECT: invention allows producing combined proteins that provide selective and effective binding to receptors on the surface of melanoma cells and can be used in diagnostics and therapy of cancer of a human being.

9 cl, 7 dwg, 5 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a constructed plasmid for expression in a cell of a Chinese hamster, in the following sequence, which mainly contains the following elements: pUC plasmid replication beginning region; an open reading frame (ORF) of beta-lactamase providing immunity to ampicillin; procaryotic gene promoter bla; a section of terminal repetition of Epstein-Barr virus of a human being; a functional gene promoter of elongation factor 1 alpha of the Chinese hamster, 5' non-translated region of this gene and a non-transcribed region flanking this gene, coding Kozak sequence for cap-dependent initiation of translation; ORF of the gene of subunit 1 of complex of 2,3-epoxyreductase of vitamin K (VKORC1) of the Chinese hamster with stop codon; a functional terminator and a signal of polyadenilation of the gene of elongation factor 1 alpha of the Chinese hamster, 3' non-translated region of this gene and a non-transcribed region flanking this gene; a promoter of early genes of virus SV40; gene of immunity to a selective agent; and a polyadenilation signal and terminator of virus SV40. Cells of the Chinese hamster - producer of protein with Gla-domain are transformed by the obtained plasmid, which are used in a method of recombinant obtainment of proteins with Gla-domain.

EFFECT: invention allows increasing productivity of the above cells owing to increasing activity of native VKORC1.

12 cl, 12 dwg, 2 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to succinic acid-producing mutant microorganism, which is able to apply simultaneously saccharose and glycerine as carbon sources. Mutant microorganism is obtained by weakening of mechanism of saccharose-mediated catabolite repression of glycerol by removal of gene, which codes fructosephosphotransferase, or by introduction of gene, which codes glycerolkinase. Mutant microorganism is selected from the group, which consists of Mannheimia sp., Actinobacillus sp. and Anaerobiospirillum sp. Also claimed is method of obtaining mutant microorganism and versions of method of obtaining succinic acid with application of claimed microorganism.

EFFECT: group of inventions provides obtaining succinic acid with high output 1,54 mol/mol.

11 cl, 4 dwg, 2 tbl, 5 ex

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