EA016223B1 - Method and gene structure for high specific inhibiting of unwanted cell growth - Google Patents

Abstract

The present invention relates to molecular biology and gene therapy of diseases at which an unwanted cell proliferation happens. The present invention relates to a gene structure comprising 3'-untranslated region of the gene transcript selected from the gene DNA-topoisomerase II alpha (TOP2A) of a human and the gene DNA-(cytosin-5)-methyltransferase 1 (DNMT1) of a human; kDNA at least one protein inhibiting cell growth; and a promoter, transcriptional activity of which is specific for target cells. Excessively proliferating neoplastic or nonneoplastic cells serve as the target cells for administering said structure. The present invention also relates to a method for increasing specificity of inhibiting and to a method of properly inhibiting of unwanted growth of target cells by administering therein said gene structure.

Description

The present invention relates to the field of molecular biology and gene therapy of diseases in which undesired cell proliferation occurs.

BACKGROUND OF THE INVENTION

A number of diseases are characterized by undesired cell proliferation. In the case of neoplasms, abnormal cell proliferation is caused by the ability of the cells to grow autonomously. Neoplasms can be benign, precancerous and malignant. Pathological cell proliferation may not be a tumor process, but refer to hyperplasia, in which pathological proliferation of normal, unchanged cells occurs under the influence of external stimuli (for example, Cushing's syndrome, neoangiogenesis, which is one of the causes of age-related retinal degeneration, etc.). Obstruction of abnormal cell proliferation is the main task in the treatment of such pathologies. So, the effect of most antitumor drugs is aimed at inhibiting uncontrolled proliferation and destroying tumor cells. Another example is the treatment of age-related retinal dystrophy, which is based on the inhibition of growth signals for endothelial cells.

Various methods can be used to inhibit cell growth, in particular, to ensure the production of exogenous protein in them, the presence of which leads to inhibition of their growth. In particular, inhibition of cell growth can be achieved by inducing their death due to the production of a specific exogenous protein in them. Examples of such proteins, the production of which in tumor cells leads to suppression of their proliferative activity and / or to their death, are tumor suppressors, apoptosis inducers, as well as cytotoxic proteins.

Tumor suppressors are proteins whose production is often reduced or absent in tumor cells, or they are mutated, which contributes to the processes of the onset and progression of the tumor. The restoration of the production of functional suppressors of tumor growth in the defective tumor cells allows them to inhibit the process of tumor progression. As one of the most illustrative examples of the use of restoration of the functions of tumor suppressor suppressors as antitumor therapy, one can cite a strategy based on the restoration of the expression of a normal (unmuted) tumor suppressor p53 in tumor cells. p53 is a transcription factor that regulates the expression of genes that induce cell cycle arrest, apoptosis, DNA repair processes, etc. The presence of a functionally active p53 protein may be necessary for apoptotic cell death. p53 is often mutated or its expression is suppressed in tumor cells, and restoration of the expression of wild-type p53 protein in p53-defective tumor cells suppresses their growth, sensitizes to the action of other antitumor agents and leads to suppression of the tumor phenotype and even tumor regression in mice (Beute, A. 1. p53, 111th se11u1ag yes! Seerer Gog dgoMy apb άίνίδίοη. Se11.1997; 88: 32331; Nath S.S., No11kh! Et M. S11shsa1 prprbsaboph oG 1Ne p53 1tog-hirrgehhog depe.Yet Ep. Möb. 1993; 329: 1318-27; Syep, R.-L., Syep Υ., Vookhljet V., Lee ^ .- N. Oepeis t skashkhth o! 1tog hirrgehjup b le le yitap p53 dep. 8c1epse. 1990; 250: 1576-80; UepShga A., Kihsy Ό.Ο., MsayidYt M.E., Titekhop Ό.Α., Sptt 1., baai b. , Le ^ tap 1., Res / ek E.E., \ Ve1hh1ebeg V., 1askh T. VekhLgayop o! P53 Gipsyop 1eabh U 1itoig gedgehhup sh tao. Lalge. 2007; 445: 661-5; bw V., Ζΐκ -πΐβ N., 2oyo O., X1e Υ., Nao 1., Οίιι V., Eiap X., 21ui O. Abepot1hih-tebaa! eb p53 depe yaphGeg hepkhTs / / ex yer! os11i1ag sagstota se11kh Yu Neatuup gab1ayop . 1. Sah1 2007; 42: 140-5, etc.). Therefore, the restoration of p53 functions is considered as a promising method of antitumor therapy (Voiske! V.R., Be Rgotep! E1 S.S., Rshkh1eih A., Oa1tapsh S.M. p53 ah a 1agde (Gog apy-sapseg bgid beta1ortep1. Sgy. Bev. Opso1. Neta1o1. 2006; 58: 190-207) Currently, a preparation based on recombinant replicatively defective adenovirus for p53 production (ΙΝΟ201, 1p! Yeardep) is undergoing various phases (including phase III) of clinical trials for the treatment of various types tumors: In addition to p53, numerous studies have shown the beneficial effect of restoring ment of expression of a number of other tumor suppressors, some of which are now considered as the basis for the creation of gene therapy of anticancer drugs.

The initiation of apoptotic cell death is one of the mechanisms for controlling cell proliferation. Therefore, the induction of apoptotic cell death through the production of proteins capable of activating apoptotic cell death, apoptosis inducers, is one of the experimentally confirmed methods for eliminating undesirable cell populations. An example of the use of apoptosis inducers for the destruction of tumor cells is the approach based on the exogenous production of the apoptosis inducer TB1AB, the production of which can effectively induce apoptosis of tumor cells (Cag1o-8! E11a C., L-ata / ha C., Eosa! E. A., U1dapo L., 61app1 A.M., O1apsh L. Tagdeypd TVA1b adoshkhes geserLgh Gog sapseg Legara. C1L Sapseg. Vekh. 2007; 13: 2313-7).

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Cytotoxic proteins are another type of protein whose exogenous expression in target cells can be used to destroy them. Examples of such proteins that can be used for gene therapy are thymidine kinase of the herpes simplex virus type 1 and cytidine deaminase E.co11. These enzymes, which are absent in mammalian cells, are capable of converting specific non-toxic substances (prodrugs) into toxic substances, the presence of which in the cell leads to their death (AdN1 M., Nosniegd E., VgakeG1e1b Kh.O. Rtobtid asbuaboi ephutez ίη sapezeg depe (Negaru 1. Sepe Meb. 2000; 2: 148-64). Herpes simplex virus type 1 thymidine kinase (H8U4k) is 1000 times more active than mammalian thymidine kinase in the monophosphorylation of ganciclovir. Subsequent phosphorylation of gancicloviron monophosphate leads to In a cell of ganciclovir triphosphate, which is a toxic analogue of thymidine triphosphate, the inclusion of which in DNA during replication leads to a halt in the replication and death of cells producing H8U4k, as well as nearby cells (L3l run esGes!). The use of cytosine deaminase is based on the same principle, but 5-fluorocytosine convertible to cytotoxic 5-fluorouracil.

For the production of exogenous protein in target cells in order to inhibit their proliferation, gene constructs are used that contain the cDNA of the exogenous protein in combination with other elements necessary for expression, namely, a transcription promoter and a 3'-untranslated region that provides transcription termination and polyadenylation of the transcript.

The gene construct that provides the production of exogenous protein must be delivered to the target cells and get into them. Various methods are used to deliver gene constructs, including electroporation of the gene construct in the form of plasmid DNA, delivery of the gene construct in the form of plasmid DNA as part of polyplexes, delivery of the gene construct in the form of plasmid DNA using polycationic lipids, delivery of the gene construct in the recombinant virus genome (e.g. , adenovirus), etc. However, the delivery of the gene construct by any means in the body is associated with the problem of the specificity of delivery specifically to target cells, i.e. gene constructs only in target cells, but not in other cells. Currently, several approaches are used to ensure the specificity of the delivery of the gene construct to the target cells, for example, by modifying the capsid of the recombinant adenovirus containing the gene construct, which ensures its infectivity only with respect to target tumor cells due to interaction with specifically highly represented only target cells with proteins. However, using selective delivery of the gene construct to target cells, it is currently not possible to ensure high specificity of expression of the gene construct in target cells, which may be due, for example, to the presence, albeit in smaller amounts, of such proteins on normal cells. Therefore, to ensure the specificity of exogenous protein production in target cells, promoter elements are used as part of the gene construct, the transcriptional activity of which is abnormally high and / or is present only in target cells. In the case where the target cells are tumor cells, promoter regions of genes are used whose transcriptional activity in the vast majority of normal cells in the body is absent or very low and is often activated in tumor cells. Examples of such promoter elements that ensure transcription specificity in tumor cells are fragments of the promoter regions of 6ΊΈΚ.ΊΈΚ human telomerase reverse transcriptase genes and VZh.C5 survivin gene.

Transcription of the 6ΤΕΚ.Τ gene is absent in the vast majority of normal human cells. Moreover, in tumor cells, 11ΤΕ.Τ transcription is often observed (about 85%) (Ci 1., Epd. V. Te1oshegae rgoto (er-bpuep sapseg depe (Negaru. Sapseg. Vu1. Tbet. 2003; 2: 864-70). The analysis of the transcriptional activity of fragments of the 6ΤΕΚ.Τ gene promoter showed that the promoter fragment about 200 bp above the transcription initiation site is active and sufficient to ensure specific transcription in 6ΤΕΚ.Τ-positive, but not in ΙιΤΕ.Τ-negative cells (Matzppag A.8., NidNez Ό.Ε., P1sP181e1peg 8.R., апддддΖ,,,,,, еЬЬкоткотезез111.8., Wa88go (A.R. P1e (e1otega8e eeege bässpärté rtoto1e bbüe eGhysasyuiz (the result of the big depe in 11ΤΕ.Τpositive tumor cells (C. 1., Eapd V., rhyme of rgoto (er-bpuep sapseg depe (Negaru. Sapseg Vu1. P1eg. 2003; 2: 864-70). In a large number of experimental studies, it was demonstrated that the gene promoter 6ΤΕΚ.Τ really allows for the expression of various proteins ov in tumor ΙιΤΕΒΤ-positive. but not in ΗΤΕ.Τ-negative normal cells (8opd 1.8., K1t N.R., Uoop ν.8., Lee Κ.ν., K1t M.N., K1t Κ.Τ., K1t H. 8. , K1t Υ.Τ. Abepow | gi8-teb1a (eb zhabe depe (Negaru izd (Ne Nitap (e1tega8e sa! VyusNo. 2003; 67: 2344-50; KateazYta та., Kadatea 8., KohauazN Н., 8Н1гак1ua уа., Iteoka Τ., ^ Πιίδΐιί E., ΤηΕί M., Kuo 8., Gapaka Ν., Eirteata ата. Ge1ote ^ azezres ^ G ^ with gerNsabop-ze1esbuye ubo (Negaru Gog Nitap sapseg. Sbp. Sapseg. Kez. 2004; 10: 285-9; Lapzop Ν.Α. 1g., Epeblapet R.,

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8cN \ gag / epBegdeg R., Ko11k 1.K., \ ¥ apd 6. KernSabop οί ap abepogt wes (og sop (go11eb bj (Not Nitap (e1otegake gsusks) (hapkspr (ake rgoto! Eh saikek! Summary-ke1esN 1uk1k. Sapseg Kek. 2003; 63: 7936-41) In particular, a number of studies demonstrated the effectiveness of using the herpes simplex virus type 1 thymidine kinase gene (H8U- (k) under the control of the NTECT gene promoter as a method of ganciclovir-specific destruction of tumor cells w U1 (th and w y1uo. The NTEKT gene promoter is 6 / C-rich and does not contain canonical TATA and CAAT motifs (Sop d8.8., \ ¥ en 1., VassNesh 8. TNe Nitap 1eotetake sa! a1ubs kiipy NTEKT: dvogs / a (undep apb sNagas (en / a (un oe (Not. Mo1. Sepe !. 1999; 8: 137-42. Adding a synthetic TATA box to a fragment of the NTEKT promoter allows one to increase its activity without loss of specificity for NTEKT-positive cells (\ ¥ H (H T., 2epbet. , 8cNiNe V., Mipb! V., P1ep (x K., Kibo1rN K.B., Mappk M., Kiyska 8., ΚϋNie1 E. A (e1tegene-bebpebep Sapseg. Sapseg. Kek. 2003; 63: 3181-8). A similar effect is achieved by the introduction of a minimal early promoter of the cytomegalovirus virus (SMU) (Eau1k I, ^ apd b., Eopd E., 2Napd b., Sio ^., Tega1kN1 E., Hee K., L. b., Eapd V. Opso1uk1k apb kirrtekkup oE (the result of dGouN bou a SER-extektkt opso1ubs abepougik sop (go11eb bup ap NTEKT apb SMU Nubb rgoto (eg. Sapseg Sepe TNG. 2006; 13: 720-3). Embedding in the promoter area. ) the fragment of the NTECT gene immediately after the site of transcription initiation did not affect the specificity of the promoter, but significantly increased its activity in cells, with type E6 protein-containing human papillomavirus (NRU) type 16 (Ve1btai T., Nopka \ ha I., Watte !! EC '.. 8cH1dee1 K. Tgapkspr (jupa1 acNgabop oE (Not (e1tegake NTECT depe Nu nitap rarhota 16 e6 opsorgo (et. 1. UNO1. 2001; 75: 4467-72), which, given its oncogenicity, allows us to expect increased efficiency of gene-therapeutic constructs based on the NTEKT promoter in NRU-positive tumors.

Another example of a tumor-specific promoter, the use of which is promising for ensuring the expression of exogenous therapeutic proteins in tumor cells, is the promoter of the B1KS5 gene encoding the survivin protein. Survivin is often expressed at a high level in tumor, but not in normal tissues (Antibody S., Abba S., AShep E. S. Ape1 ap (1-aror (ok1k depe, kshuMp, exheckkeb sapseg apb 1utrNota. No. (. Mob. 1997; 3: 917-21), and the survivin promoter is able to successfully control transgene expression specifically in tumor cells (Vao K., Soppo11u E.S., MigrNu M., Szheep 1., \ ¥ etc (eH1 1.K ., P1kags1k E.A., NatShop T.S. Asbgabop oE sapseg-kresEys depe e hrgekkup bj (Not kshuMp rgoto (ex. 1. No. 1 (1. Sapseg. 1pk (. 2002; 94: 522-8), which makes it possible to use it, like the promoter of the NTEKT gene, when the development of antitumor gene therapeutic approaches.

However, despite the high specificity of the transcriptional activity of target cell-specific promoters, these promoters can exhibit transcriptional activity, albeit substantially lower, in cells other than target cells (Kapihopo 1., N ada 8., MitoEiky Υ. , Kotlua 8., Eiretata N., MakhshkN1 T., Koka1 K. the NTEKT gene in NTEKT-negative non-tumor normal cells in the genes x constructs nevertheless possesses, although to a small extent, the transcriptional activity of (Ma_) itbag A.8., NidNek E.E., YsyKktet 8.R., ^ apd Ζ., Herbko \ rk1b 18., Wakkogo! A. R. TNe (e1otegake gssusks (hapkspr (ake rgoto (er bggek eEsasyuyik (total of khs1be depe determined by the presence of a set of transcription factors in certain types of cells, sufficient to ensure transcription from this promoter, and nonspecific transcriptional activity ugih DNA sequences consisting of a gene construct that may serve as ectopic promoter. Thus, in spite of the pronounced activity in NTECT-positive target cells, the promoter of the NTECT gene does not provide absolute transcription specificity in target cells, and, in general, the use of promoters that are specifically active only in target cells does not allow absolute specificity in the strength of the above reasons. As a result of the background activity of the promoter in normal cells, a protein can be produced, the presence of which in them can have undesirable consequences. In this regard, there is a need to further increase the specificity of exogenous protein production in target cells, which will minimize the production of exogenous protein in cells other than target cells and reduce the likelihood and potential severity of side effects resulting from the production of exogenous protein in cells, different from target cells.

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Disclosure of the present invention

The increase in the specificity of exogenous protein production in target cells can be achieved in various ways by increasing the specificity at any stage of the life cycle of the gene construct used to produce the exogenous protein, including delivery of the gene construct, its transcription, transcript lifetime, protein translation efficiency and its stabilization . It is known that the transcript lifetime in a cell can be regulated. Specific sequences in the composition of the transcript can determine its lifetime in the cell, for example, causing rapid degradation of the transcript. Obviously, with a selective increase in the transcript life time in target cells compared to other cells (or its reduction in cells other than target cells), the amount of transcript in target cells will specifically increase (or decrease in cells other than target cells) , which will have a corresponding effect on the level of exogenous protein in cells, the translation of which depends on the amount of transcript in the cell.

One of the common differences between abnormally proliferating cells, regardless of the cause of this proliferation, from most other body cells is their frequent division. Therefore, one of the ways to increase the specificity of production can be selective stabilization of the transcript transcribed from the gene construct in dividing cells, and accordingly its relative destabilization in non-dividing cells.

It is known that in the process of cell division, a number of proteins are necessary only in certain phases of the cell cycle. A cell uses various mechanisms to control the presence of various proteins in certain phases of the cell cycle. In particular, such proteins are DNA methyltransferase 1, encoded by the gene ΌΝΜΤ1 (8kuG M., Βοζονίο V., Tashdata S. Sgo \\ 111 ged1a1yup οί shoike с ts111u11gap5Gsga5C depexphexop. 1. ΒίοΙ 26. 30), and topoisomerase II α, encoded by the TOP2A ^ ο5 \ ναιηί R.S. gene, Βοίί Βοίί РЬ., ΗιιπΙ S.V. YOU DO NOT SE11 SUS1E-SOIR1EB EXHERGKKUP ί ί ΐ ο δ ^ δ ο ο 11 11 11 11 11 ип ип ип ип ип п а а а а а а а а 18 18 18 18 18 18 18 18 18!!!!! δοsk ογ ίοπίζίπβ gab1a1yup. Μο1. Ce11. Βίο1. 1996; 16: 1500-8; Scrapi R.S., Ηί11 Μ., I Nd ^ Shkibu V., \\ pd1n No..Ό., Βοίί Βοίί 1.B. THERE ARE NOT FIRST LITTLE 8UP1LE818 ORDER AND WRITING HERE! We have all the best for your life! Wab1a! Sun 8. 1992; 132: 162-7; Nesk Μ.Μ., H1e1tap ^ .Ν., EapnNshu ^ .C. 01P'egep11a1 expr88yup οί ΌΝΑ ίοрο ^ δοе ^ аδеδ I apb II bippd 1ее еика ^ Is ce11 сус1е. Rgos. No. 111. Asab. 8sk I8A. 1988; 85: 1086-90). Their presence in dividing cells is at least partially controlled by selective stabilization of the corresponding transcripts in the 8-phase and 8- and C2 / M phases of the cell cycle, respectively (phases of the cell cycle associated with cell division) and destabilization of transcripts in other phases of the cell cycle. Stabilization of the ΌΝΜΤ1 and TOP2A transcripts during cell division is mediated by their 3'-untranslated regions. Moreover, the addition of the Z'-untranslated region of the ΌΝΜΤ1 or TOR2A transcript to the heterologous cDNA (β-globin or β-galactosidase cDNA, respectively) leads, as in the case of endogenous transcripts, to stabilization of their transcripts at certain stages of the cell cycle in dividing cells in comparison with non-dividing cells (Ee11cN Ν., WattsNapbap 8., 8ζуί А. And skinny-eb 3'-1181ап818181аеб е е е111еди г г г111 г г гедиедиедиедиедиедиедиедиедиеди а асНАНАНА ап ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^. ^.... ^ ^. ^ ^...................................... I. Βίο1. SN. 2001; 276: 24881-90; ^ δλνηιηί R.S., 8 Negep I., A1bee L.E., Ragaap A., 81t S.Ι., Ibadit L.A., Η ^ yes δН ^ ^ ки В. В. В. В. В. V., Sg8 С., Nip! S.V., 8p ^ ίζ Ό.Β. Ce11 ss1e-sóir1eb uapa! Uup sh ίοрο ^ δοе ^ аδе Pa1рНа тВNΑ ίδ гед1а! Fuck LU 1Not 3'-un1gap81a1eb gedup. Ροδδ ^ b1е go1e οί ^ οοοx-δеηδ ^ ι ^ νе рго! Ешшшшш ш тΒNΑ assit1a1yup. I. Βίο1. SN. 2000; 275: 38. This fact is the basis for the use of fragments of 3'-untranslated regions of transcripts ΌΝΜΤ1 and TOP2A as part of gene constructs for the selective stabilization of exogenous transcripts in actively dividing cells and their destabilization in non-dividing cells, which will increase the specificity of exogenous protein production in proliferating target cells.

Fragments of the 3'-untranslated region of the transcript ΌΝΜΤ1 (8ЕО Ю ΝΟ: 1) or ТОР2А (8ΕΟ Ш ΝΟ: 2) were used in gene constructs containing cDNA of the protein of the destabilized green fluorescent protein (BESER) under the control of the early cytomegalovirus promoter (Fig. 1) . The resulting expression constructs rbESER-ΟΝΜΤ! and rbESER-TOR2A transfected lung cancer cells of the ΝΟ-Η1299 line. As a comparison control, the plasmid rBECEP-ΝΊ was used, containing the signal for terminating the transcription and polyadenylation of the 8U40 virus, which was not able to selectively stabilize the transcript of which it is included in non-dividing cells (Fig. 1). As a result of the analysis of the level of expression of BESEP protein in transfected cells, it was found that the use of the TOR2A transcript insignificantly (3 times) reduces the level of protein production, and the use of the 3'-untranslated region of the ΌΝΜΤ1 transcript not only does not reduce the level of protein production, but also increases its ~ 1.6 times (Fig. 2). Thus, the introduction of the 3'-untranslated regions of transcripts ΌΝΜΤ1 or ТОР2А into the expression construct does not significantly affect the expression efficiency of the protein encoded by it in actively proliferating cells, despite their prior art ability to destabilize the transcript in non-dividing cells.

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Fragments of the 3'-untranslated region of the ΌΝΜΤ1 or TOP2A transcript were used in gene constructs containing the H8U-! C cDNA under the control of the cTEKT gene promoter fragment (8ES GO N0: 3), a combination of which is used to selectively kill the kTEKT-positive tumor cells in the presence of ganciclovir ( plasmids rTETEKT-TK-ΌΝΜΤΙ and rkTEK.T-TK-T0P2A, respectively; Fig. 3). The presence in the construction of the 3'-untranslated region of the ΌΝΜΤ1 transcript or the TOP2A transcript, as follows from the prior art (ЭееЦΝ,., Kashskapbash 8., 8ζνΓ Μ. apb ίπ1ιίΕίΐ5 55 1gap8Gogtd asbuyu. 1. Vu1. Sket. 2001; 276: 24881-90; Co5 \\ wsh R.S., 8kegei 1., A1kee LGO., Ragaap A., 81t EE., KGBpoig L.A. , Shdazykio K .., S1U5 Ό., Nip! S.K., 8ρίΐζ EK Ce11 ss1e-soir1eb sapabop 1n 1oro15ega5e 11a1rka m ^ A 15 gediaeb ebu! Ke 3'-un1gap51a1e1b1eaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa from ... -5ep5 у р р!! Ет! . Vu1. Sket. 2000; 275: 38384-92), leads to the selective stabilization of the transcript containing it in dividing cells, which is often not provided by the expression termination transcription and polyadenylation of 8U40 virus, which was used as a control (rkTEK plasmid). T-TK-8U40) (Fig. 3). Therefore, on the basis of the known data (OeSk Ν., Kashskapbash 8., 8zyr Μ. And with a 3'-un1gap51a1eb e1teep! Teb1a! 2001; 276: 2488190; Co5 \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ndgrowth, 1.Albee LGO., Ragaap A., 81t EE., KGBpoig L.A., Shdakykyo K., C1I5 Ό., Nip! ., 8] ΐίΙζ Ό.Ρ .. Ce11 ss1e-soir1eb uapabop w 1poorotega5e 11a1kka m ^ A 15 hed1a! Eb bk! Vu1. Sket. 2000; 275: 38384-92) it can be argued that the replacement of the transcription termination signal and polyadenylation Nia virus 8U40 fragmented 3'-untranslated regions of transcripts ΌΝΜΤ1 TOR2A or lead to destabilization of the relative transcript N8U-1k in non-dividing (normal) cells compared with actively dividing (tumor) cells.

The introduction of fragments of 3'-untranslated regions of transcripts ΌΝΜΤ1 or TOP2A into the structure of the gene construct allows us to increase the relative specificity of the production of exogenous protein encoded by the gene construct in dividing cells compared to non-dividing ones. However, it remained unclear whether the replacement of the 3'-untranslated region would lead to a decrease in the overall efficiency of the gene construct, since in this case, a decrease in the production efficiency of exogenous protein can have a significantly greater negative value than the positive effect of increased specificity. The results of analysis of the production of the BESEP protein, the transcript of which contained 3'-untranslated regions of transcripts ΌΝΜΤ1 or ТОР2А, suggest that the efficiency of the gene constructs will not change significantly when 3'-untranslated regions of transcripts ΌΝΜΤ1 or ТОР2А are introduced into their composition. To confirm this hypothesis, a comparative analysis of the efficiency of the gene constructs rkTEKT-TK-8U40, ρкΤЕК.Τ-ΤК- ^ NΜΤ1 and pkTEKT-TK-T0P2A in the ability to cause ganciclovir-dependent cell death of lung cancer cells of the Νί.Ί-Η1299 line was performed. The cells of the Νί.Ί-Η1299 line are positive-positive (bt 8.S., b1 \ U.S., 8k1k Εν., Nopd K.E., Rap Υ.Ρ .., bn 1.1. Tke! Ea ro1urkepoo 15 ECC apb ECC gerge55 t ^ A exrge55yup oG kitap! e1otega5e geuegke 1gap5spr1a5e (kTECT) t saggshot se115. Sapseg ley. 2006; 236: 80-8), and, as expected, the region of the kTEKT + -6 gene promoter (+20 ) (8ЕО GO Ν0: 3) is transcriptionally active in them (Fig. 4). Accordingly, transfection of ΝΟ-Π1299 cells with the plasmid rkTEKT-TK-8U40 led to their efficient death in the presence of ganciclovir, while this effect was not observed in the absence of ganciclovir (Fig. 5). When cells were transfected with plasmids ρкЕК.Τ-ΤК- ^ NΜΤ1 or ркТЕКТ-ТК-Т0Р2А containing fragments of 3'-untranslated regions leading to destabilization of the transcript in non-dividing cells in the presence of ganciclovir, cell death efficiency did not decrease compared to cells transfected with plasmid rkTEKT-TK-8U40, and in the case of the expression construct containing the 3'-untranslated region of transcript No. 1, it even increased slightly (Fig. 5), which correlates with increased expression of the BESEP protein when replaced in ec the compression construction of the transcription termination and 8U40 virus polyadenylation signal to the 3'-untranslated region of transcript No. 1. This result allows us to conclude that the use of fragments of 3'-untranslated regions of transcripts ΌΝΜΤ1 and TOP2A does not lead to a general decrease in the efficiency of the action of gene constructs in target cells.

Summarizing, the introduction into the expression construct of a fragment of the 3'-untranslated region of the ΌΝΜΤ1 or ТОР2А transcript does not significantly affect the expression efficiency of the protein encoded by it (shown on the example of the BESEP protein) and its total activity (shown on the example of the herpes simplex virus thymidine kinase that causes ganciclovir-dependent cell death). Moreover, as follows from the prior art, the introduction of a fragment of the 3'-untranslated region of the transcript ΌΝΜΤ1 or TOP2A allows you to selectively destabilize the transcript in non-dividing cells, thereby weakening the expression of exogenous protein (and, accordingly, its total activity) in them and increasing the specificity of production exogenous protein in dividing target cells.

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Thus, the present invention relates to a gene construct for inhibiting undesired growth of target cells, comprising:

a) the 3'-untranslated region of the transcript of the gene selected from the human DNA topoisomerase II α (TOR2A) gene and the human DNA- (cytosine-5) methyltransferase 1 (ΌΝΜΤ1) gene;

b) cDNA of at least one protein that inhibits cell growth;

c) a promoter whose transcriptional activity is specific for target cells.

Since the effect of including fragments of the 3'-untranslated regions of transcripts ΌΝΜΤ1 or TOR2A consists in the destabilization of the transcripts containing them and is due to specific features of the nucleotide sequences of the 3'-untranslated regions of transcripts ΌΝΜΤ1 or TOR2A (OsISP Ν., RatsNapyash B., BhuG Μ 3 sopket. -np1gap51a1sy e1shept1 te1a1e8 dt ^ 1st tedi1a1yup οί ΌΝΑ teShuNtapkGegake 1 apy shyyyk (κ (hapkGogtd asyuyu. I. Vyu. A., B1t Ι.Ε., Shpoig L.A., Nfakıkкиo V., Szk Ό., Nip! S.V., 8ρίΐζ Ό .Ce11 ss1e-sour1e uapayop t (orokotegake 11a1pPa tvla to the hedgehog bj (Pe 3'-np1gap81a geschop. Po881b1e gogo-kepkshue tgloit btb2 382). then the type (type) and method of delivery of the expression construct, the specific structure of the promoter element in its composition and the exogenous protein encoded by it are not essential for the manifestation of these properties.

So, one of the possible embodiments of a promoter whose transcriptional activity is specific for target cells is the promoter of the human telomerase reverse transcriptase gene (11ΤΕΡΤ). Another embodiment of a promoter whose transcriptional activity is specific for target cells is a hybrid promoter consisting of an 11отор gene promoter and a minimal early SMC promoter or synthetic TATA box. Another embodiment of a promoter whose transcriptional activity is specific for target cells is the promoter of the human survivin gene (B1BC5).

The present invention equally relates to gene constructs encoding various proteins that inhibit cell growth. Thus, a cell growth inhibitory protein may be, without limitation, a tumor suppressor and / or an apoptosis inducer and / or a cytotoxic protein. According to a preferred embodiment, the cytotoxic protein is an enzyme (e.g., herpes simplex virus type 1 thymidine kinase) that converts a substrate (e.g. ganciclovir) into a toxic compound causing cell death.

The positive effect on specificity from the use of fragments of the 3'-untranslated regions of transcripts ΌΝΜΤ1 or TOP2A as part of expression constructs is due to the degradation of the transcripts containing it in normal non-dividing cells and, accordingly, does not depend on the type of proliferating target cells, since it is inextricably linked to the process of cell division.

One preferred embodiment of the target cells are tumor cells that are characterized by abnormal proliferation. Another preferred embodiment of the target cells are non-tumor target cells susceptible to abnormal proliferation, in which transcripts containing fragments of the 3'-untranslated regions of the ΌΝΜΤ1 or TOR2A transcripts will also be stable due to their active division.

In addition, the present invention relates to a method for increasing the specificity of inhibiting undesired growth of target cells, comprising delivering to a target cell a gene construct comprising:

a) the 3'-untranslated region of the transcript of the gene selected from the human DNA topoisomerase II α (TOR2A) gene and the human DNA- (cytosine-5) methyltransferase 1 (ΌΝΜΤ1) gene;

b) cDNA of at least one protein that inhibits cell growth;

c) a promoter whose transcriptional activity is specific for target cells.

In the context of the present invention, the term specific inhibition of unwanted growth of target cells means the acquisition by a population of such cells of the ability to reduce their proliferation, including death, as a result of their expression of proteins encoded by the gene construct introduced into them, which cause a decrease in proliferation, achieved either by a direct decrease in proliferative activity (i.e., the process of cell division), either directly by starting the process of cell death (for example, by the mechanism of apoptosis), or by rotating an additionally introduced prodrug into a cytotoxic compound, or by increasing the sensitivity of cells to the effects of other antiproliferative agents, or by inhibiting other undesirable properties of cells characterized by undesirable growth, for example, their mobility and invasion in the case of tumor cells, and only excessively proliferating cells selectively acquire this property.

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Therefore, the present invention also relates to a method that implements the property described above for controlling overly proliferating cell populations, namely, a method for highly specific inhibition of undesired growth of target cells, comprising delivering a gene construct containing a 3 ′ untranslated region of the transcript to target cells a gene selected from a human DNA topoisomerase II α (TOR2A) gene and a human DNA- (cytosine-5) methyltransferase 1 (ΌΝΜΤ1) gene, cDNA of at least one growth inhibitory protein cells, and a promoter whose transcriptional activity is specific for target cells, and (if necessary for cytotoxicity) the delivery to the target cells of a substrate converted by a protein encoded by the cDNA contained in the specified gene construct into a toxic compound that causes cellular death.

In the context of the present invention, the term “necessary for cytotoxic action” refers to the situation described above, when the protein encoded by the gene construct introduced into the target cells does not directly initiate the cell death process, but is an enzyme that converts the additionally introduced prodrug into a cytotoxic compound. One of the preferred embodiments of such an enzyme-prodrug pair is respectively herpes simplex virus type 1 thymidine kinase and ganciclovir.

It will be obvious to a person skilled in the art that, depending on the cDNA used in the inventive gene construct, the claimed method of increasing the specificity of inhibition and the method of actually inhibiting the undesired growth of target cells will either be carried out simultaneously (for example, when the cDNA encodes a protein that directly starts the process of cell death ( for example, by the mechanism of apoptosis)), or they will be spaced in time (for example, when cDNA encodes a protein, which is an enzyme that additionally converts Qdim prodrug into a cytotoxic compound).

It will also be obvious to a person skilled in the art that the claimed method for increasing the specificity of inhibiting and actually inhibiting the unwanted growth of target cells as a result of the production of exogenous protein in dividing target cells using fragments of 3'-untranslated regions of transcripts ΌΝΜΤ1 or ТОР2А is not limited to the above example consisting in use:

(1) tumor cells of the N0-41299 line;

(2) the promoter of the 11ΤΕΡΤ gene as an element that provides specific transcription in target cells;

(3) herpes simplex virus thymidine kinase type 1 as a cytotoxic protein;

(4) plasmids as carriers of the expression construct;

(5) transfection as a method of delivering an expression construct to target cells.

Brief Description of the Drawings

FIG. 1. Schemes of plasmids ρΦΕ6ΡΡ-ΌΝΜΤ1 (A), ρΦΕ6ΡΡ-ΤΘΡ2Α (B) and ρΦΕ6ΡΡ-Ν1 (C).

Thick lines show: light gray — early cytomegalovirus promoter (Ρο _Μ ν); dark gray color - ΦΕΟΡΡ (ΦΕΟΡΡ) cDNA and neomycin phosphotransferase II cDNA under the control of the virus promoter δν40 (ΝΡΤ); in black are the sequences of fragments of the 3'-untranslated regions of the transcripts ΌΝΜΤ1 (ΌΝΜΤ1; A), TOR2A (TOR2A; B) and the sequence for terminating the transcription and polyadenylation of the virus δν40 (3δν40; C). Thin arrows indicate the direction of DNA fragments in the expression vectors from the 5'- to the 3'-end. The positions of restriction endonuclease recognition sites flanking the 3'-untranslated regions in expression constructs are also shown.

FIG. 2. Analysis of the production of ΦΕΟΡΡ protein in the cells of the Νί.Ί-Η1299 line. transiently transfected with plasmids ρΦΕ6ΡΡ-Ν1 (δν40), ράΕ6ΡΡ-ΌΝΜΤ1 (ΌΝΜΤ1) or ρΦΕ6ΡΡ-ΤΘΡ2Α (Τ0Ρ2Α).

The results of Western blot analysis of cell lysates using antibodies against ΕΟΡΡ (ΦΕΟΡΡ) and antibodies against neomycin phosphotransferase II (ΝΡΤ) to control the amount of applied lysates and normalize the efficiency of transfection are shown. Below are data on the quantitative determination of the level of ΦΕΟΡΡ normalized to the amount of neomycin phosphotransferase in the samples.

FIG. 3. Schemes of plasmids ρΗΤΕΚΤ-ΤΚ-ΌΝΜΤ1 (A), ρΗΤΕΚΤ-ΤΚ-Τ0Ρ2Α (B) and ρΗΤΕΚΤ-ΤΚ-δν40 (C).

The vector ρΒΙιιοδοπρΙ II 8Κ (+) is shown by a thin line. Thick lines show: light gray — fragment of the 11ΤΕΡΤ gene promoter (-206 - +37) (Ρ | _ιΤ ι: .ι < _Τ ); dark gray color - herpes simplex virus type 1 thymidine kinase cDNA (H8U-1k); in black, the sequences of fragments of the 3 'untranslated regions of the transcripts ΌΝΜΤ1 (ΌΝΜΤ1; A), TOR2A (TOR2A; B) and the sequence for terminating the transcription and polyadenylation of the virus δν40 (3δν40; C). Thin arrows indicate the direction of DNA fragments in the expression vectors from the 5'-to 3'-end. The positions of restriction endonuclease recognition sites flanking the ΗΤΕΚ.ΗΤΕΚ gene promoter, thymidine kinase cDNA, and 3'-untranslated regions in expression constructs are also shown.

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FIG. 4. Analysis of the activity of the promoter of the iTERT gene (-206 - +37) in the cells of the линии.Ί-Η 1299 line.

The results of measuring firefly luciferase activity normalized to the Kesh11a luciferase activity (in relative luminescence units) for cells transfected with the plasmid pKb-CMU together with the indicated reporter plasmids (pCb3-Ba51e, pCb3-Coi1go1 and pCb3-thTEKT) are shown. Data are given as the average of measurements in 3 wells ± standard deviation.

FIG. 5. Analysis of ganciclovir-dependent cell death of ΝΟΙ-Η1299 cells transfected with the plasmid pB1is5epr1 II 8K (-) (Control), rjTERT-TK-8U40 (8U40), rjTECT-TK-ΌΝΜΤΙ (ΌΝΜΤ1) or rjTERT-TK-TOR2A ( TOR2A).

Transfected cells were treated with ganciclovir at the indicated concentrations for 6 days. Cell viability was determined using the Ce11Tyeg-C1o® Eatshexei kit! Ce11 U1aY1yu Akkau (Rgoteda, USA) and is indicated in relative luminescence units, the value of which is proportional to the number of viable cells in the well. Data are given as the average of measurements in 3 wells ± standard deviation.

Examples of preferred embodiments of the invention

Example 1. The creation of plasmids rBESPP-EMMT1 and rbESPP-TOR2A.

The plasmids rBESPP-OMMT1 and rbESPP-TOR2A were created on the basis of the plasmid rBESPP-U (C1O! Esy, USA) and contain the 6ESEP protein cDNA under the control of the early promoter of cytomegalovirus with a fragment of the 3'-untranslated region of the ΩΝΜΜ transcript or TEP2NA transcript on 3 instead of the sequence for terminating the transcription and polyadenylation of 8U40 virus in the plasmid rDEPP-L1 (Fig. 1). A fragment of the 3′-untranslated region of the ΩΝΜΜ transcript (SeieVaik Ass. No. ΝΜ_001379, NT 5108-5409) with recognition sites for restriction endonucleases No. I at the 5'-end and A1Sh and Xa1 at the 3'-end was obtained by PCR on a genomic DNA matrix human peripheral blood lymphocytes with primers 5'-a1dsdddssds1dShs1dddsassaddaa 1-3 'and 5'-ic1adasyaad1dddSha1addadada Sha-3'. A fragment of the 3'-untranslated region of the TOR2A transcript of 1033 nt in size, starting with nt 4696 (SeeieVaik Ass. No. ΝΜ_001067), with recognition sites for restriction endonucleases No. P at the 5'-end and A1Sh and Xba1 at the 3'-end were obtained by PCR on the genomic DNA matrix of human peripheral blood lymphocytes with primers 5'-a (dsddssdsdad (cada (daada (yes (c (d (-3 'and 5'-ic1adasyaadsays1a1dddddsa1d1-3'). The resulting PCR fragments were cloned into plasmid vectors sequences of cloned fragments of 3'-untranslated regions of transcripts Ό ΜΡ1 TOR2A and confirmed by sequencing. Cloned fragments are 3 'untranslated regions of transcripts ΩΝΜΉ TOR2A and cut with restriction endonucleases and №11 A1SH and cloned into plasmid rbESRR-Ν 1 between the recognition sites of these restriction endonucleases, replacing a transcriptional termination sequence and polyadenylation virus 8U40.

Example 2. Analysis of the production of 6ESPP protein in cells of the ΝΟ-Η1299 line transiently transfected with plasmids rBESPP-L1, rbESPP-EMMT1 or rbESPP-TOR2A.

YC1-H1299 cells were scattered into the wells of a 24-well plate and transfected with plasmid rbESPP, rbESPP-EMMT1 or rbESPP-TOR2A using ishGes1sh-56 transfection reagent (IIES! Sgoir, Russia). After 48 hours, the cells were lysed and the amount of 6ESPP protein in the lysates was analyzed by Western blotting using antibodies against ECPP. Western blot analysis was also performed using antibodies against neomycin phosphotransferase II encoded by the same plasmid to normalize the amount of 6ESPP protein in the sample and to exclude the effect of transfection efficiency with different plasmids on the results of analysis of the amounts of 6ESPP protein in lysates. The antibodies bound to the proteins were visualized using the ECB + chemiluminescent reagent (CE Neisage, USA) and the SyetShos XP8 photo-documentation system (WiKab, USA). The amounts of 6ESPP and neomycin phosphotransferase II proteins in the samples were determined using the OiaiSu Opera 1ΌAi1u515 8oP program (VuKab, USA). As a result of the analysis, it was found that the expression of the 6ESPP protein does not significantly change when the 8U40 virus transcription and polyadenylation termination signal is replaced by a 3'-untranslated region of the ΩΝΜΜ or TOR2A transcript and is respectively x 1.6 and x0.3 with respect to the 6ESPP level when using signal transcription termination and polyadenylation of the virus 8U40 as part of the expression construct (Fig. 2). Thus, substitution of the 8U40 virus transcription and polyadenylation termination signal with the 3'-untranslated region of the ΩΝΜΉ or TOR2A transcript in the expression construct does not significantly affect the expression level of the protein encoded by it in dividing cells.

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Example 3. The creation of the plasmid ρ! ΤΕΚΤ-ΤΚ-ΌΝΜΤ1.

Plasmid ρ1ΤΕΚΤ-ΤΚ-ΌΝΜΤ1 was created on the basis of the vector pB1is5SG1p1 δΚΙΙ (+) (§1га1адее, USA) and contains the herpes simplex virus type 1 thymidine kinase cDNA under the control of the 11ΤΕΡΤ gene promoter fragment with the 3 ′ untranslated region of the ΌΝΜΤ1 transcript 3 the 'end of thymidine kinase cDNA (Fig. 3). Herpes simplex virus thymidine kinase cDNA with recognition sites for Bd111 restriction endonucleases at the 5'-end and ΝοίΙ at the 3'-end was obtained using the polymerase chain reaction (PCR) with primers 5'-a1dd1assdasssdssyasdss1ss1ss1ss1ss1d1s1d1s1d1s1d1s1d1s1d1s1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1d1dd1 the matrix of the plasmid άίδΓυρΙίοη оТ 11е с-аЫ рГОУ-опсодепе. Сe11. 1991 1ip. 28; 65 (7): 1153-63). herpes simplex virus thymidine kinase cDNA type 1. A fragment of the MERG gene promoter (-206 - +37) (Cepewapc Ass. No. AE128893. Nt 110006-11248) was amplified on a human blood peripheral lymphocyte genomic DNA matrix with primers 5'daysddsddsadasadasd-3 'and 5'-dadsadass1-dasadas1-dasadas1-dasdass1-dasadas1-dassadass1-dasadass1 subsequent re-amplification with primers 5'-ca1dd} ass1sdadsassadasssssaddassdsds1-3 'and 5'-a1dda1ssada1c1ssssad1dsdsadsadda-3' for the introduction of recognition sites for the restriction endonucleases Κρη Х and X1 and ото ото ото ото ото конце конце конце ото ото ото ото ото конце конце конце конце конце конце конце конце конце конце конце конце конце конце конце The preparation of a fragment of the 3'-untranslated region of the ΌΝΜΤ1 transcript with recognition sites for restriction endonucleases No. 5 at the 5'-end and Αί1ΙΙ and Xba1 at the 3'-end is described in Example 1. Cloned fragments were used for sequential subcloning into the vector ρΒ1 ^^ Γίρΐ δΚΙΙ (+ ) between recognition sites of restriction endonucleases ΚρηΙ and Xba1 fragments of the 11ΤΕΡΤ gene promoter. cDNA of thymidine kinase and a fragment of the 3'-untranslated region of the ΌΝΜΤ1 transcript and obtaining the plasmid ρ! ΤΕΚΤ-ΤΚ-получения1 (Fig. 3) using well-known genetic engineering techniques.

Example 4. The creation of the plasmid ρ! ΤΕΚΤ-ΤΚ-ΤΘΡ2Α.

The plasmid ρ! ΤΕΚΤ-ΤΚ-ΤΘΡ2Α (Fig. 1) was created on the basis of the vector ρΒ1 ^^ ήρΐ δΚΙΙ (+) (Zyashdepe, USA) similarly to the plasmid ρ! ΤΕΚΤ-ΤΚ-ΌΝΜΤ1 (Example 3) and contains the simple virus thymidine kinase cDNA herpes type 1 under the control of a fragment of the 11ΤΕΡΤ gene promoter with a fragment of the 3'-untranslated region of the TOR2A transcript at the 3'-end of thymidine kinase cDNA (Fig. 3). The preparation of a fragment of the 3'-untranslated region of the TOR2A transcript with recognition sites for restriction endonucleases No. ίΙ at the 5'-end and Αί1ΙΙ and ХЫ at the 3'-end is described in Example 1.

Example 5. The creation of the plasmid ρ1ΤΕΡΤ-ΤΚ-δν40.

The plasmid ρ! ΤΕΚΤ-ΤΚ-ΤΘΡ2Α (Fig. 3) was created on the basis of the vector ρΒ1 ^^ τίρΐ δΚΙΙ (+) (δύΟη ^^. USA) and contains the herpes simplex virus type 1 thymidine kinase cDNA under the control of the 11ΤΕΡΤ gene promoter fragment with a DNA fragment. containing signals for terminating the transcription and polyadenylation of the virus δν40 at the 3'-end of the thymidine kinase cDNA (Fig. 3). DNA fragment. containing signals for the termination of transcription and polyadenylation of the virus δν40. were cut from the recognition sites of restriction endonucleases No. ίΙ and Αί1ΙΙ from the vector ρΕ0ΕΡ-Ν2 (C1oSeux. USA) and cloned instead of a fragment of the 3'-untranslated region of the TOR2A transcript into the plasmid ρ! ΤΕΚΤ-ΤΚ-ΤΘΡ2Α from the recognition sites of restriction endonucleases No. and obtaining plasmids ρ1ΤΕΚΤ-ΤΚ-δν40 (Fig. 3).

Example 6. Analysis of the transcriptional activity of the promoter of the 11ΤΕΡΤ gene (-206 - +37) in the cells of the ΝΟ-Η1299 line.

The transcript 11ΤΕΡΤ is present in the cells of the ΝΟ-Η1299 line (Lt δ.C .. L1 XV.C .. δΐιίΐι ί.Χν .. Nop§ Κ.Ε .. Ρap Υ.Ρ .. Lt 1.1. P1e! Ea ρο1уρйепок ΕССС амй ΕSS ge] ége55 ιιιΡΝΑ exx ^^ e88 ^ oot o Nitam 1e1 teegake geuegke ΐ ^ ap8c ^^ ΐΐa8e (1ΤΕΚΤ) ίη sagstota se11k. which suggests the ability of the 11ΤΕΡΤ gene promoter fragment in the recombinant expression construct to direct transgene transcription. To confirm the activity of the fragment of the promoter of the 11ΤΕΡΤ gene (206 - +37), an analysis of its transcriptional activity in cells of the Νί.Ί-Η1299 line was performed. The analysis was performed using a firefly luciferase reporter gene. For this, a fragment of the 11ΤΕΡΤ gene promoter (see Example 3) was cloned from the recognition sites of the VdSh and ΚρηΙ restriction endonucleases into the ρС ^ 3-Βа8 ^ s vector (Ggoted. USA) to obtain the plasmid ρΟΕ3-1ιΤΕΡΤ with firefly luciferase cDNA under the control of the 11ΤΕΡΤ gene promoter (-206 - +37). The plasmid ρС ^ 3-Са8 ^ s was used as a negative control. in which the firefly luciferase cDNA does not contain a promoter. As a positive control, the plasmid ρС ^ 3-Сопΐ ^ ο1 (Ggoteda, USA) was used. in which the transcription of firefly luciferase cDNA is driven by the promoter and enhancer of the δν40 virus. Линии, ΊН1299 cells were scattered into the wells of a 96-well plate and transfected with plasmid ρΟΕ3-1ΤΕΚΤ. ρΟΕ3Vays or ρС ^ 3-Сопί ^ ο1 together with the plasmid ρΡΕ-ί, ’Μν. encoding Kesh11a luciferase under the control of an early promoter of cytomegalovirus (Ggoted. USA). in a ratio of 50: 1 using a transfection reagent υηί№ΐίη-56 (ItRes! ΟΐΌΐιρ. Russia). Firefly and Kesh11a luciferase activity was analyzed 48 h after transfection using the Pia1-C1o® Lysyegake набораδ ^ -κ δу8ΐet kit (Tgoteda, USA). Firefly luciferase activity measurement results. normalized to luciferase activity Kesh11a. presented in FIG. 4. It is visible. that firefly luciferase activity in cells. transfected with plasmid ρΟΕ3-1ΤΕΚΤ. significantly higher. than in the cells. transfected

- 9 016223 non-motorized design pCb3-Ba81c. which indicates the activity of the promoter of the ETEWT gene (206 - +37) in the cells of the line N04-41299.

Example 7. Analysis of ganciclovir-dependent death of cells of the line Νί.Ί-Η1299. plasmid transfected with rETEBT-TK-ΌΝΜΤΙ. ryTEVT-TK-TOR2A or ryTEKT-TK-§U40.

Comparative efficiency of expression constructs with thymidine kinase under the control of the promoter of the ETEC.T. gene differing by Z'-untranslated regions. investigated on a model of cells of the line Νί.Ί-Η1299. in which the promoter of the ETECT gene (-206 - +37) is able to effectively activate transcription as part of the expression construct (example 6). Cells of the N0-41299 line were transiently transfected with plasmids pYE.Τ-ΤK- ^ NΜΤ1. r-TEKT-TK-TOR2A or r-TEKT-TK-§U40 under the same conditions (transfection was performed using transfection reagent IIES1SH-56 (IIIIES! Sgoyr. Russia)) and treated with ganciclovir at a concentration of 2 or 20 μM. After 6 days of incubation of the cells with ganciclovir, a quantitative assessment of cell viability was performed using the Ce11Tyeg-C1o® Bettex Ce11 U1a1iu Akkau kit (Rgoteda, USA). Ganciclovir treatment of cells. transfected with plasmid pB1expr1 §K ΙΙ (-). thymidine kinase-free cDNA (negative control). did not lead to a significant decrease in cell viability. as is the case with cells. transfected plasmids. containing thymidine kinase cDNAs under the control of the promoter of the ETEC.T gene (-206 - +37). but not treated with ganciclovir (Fig. 5). At the same time, transfection of cells with plasmids pYE-ΤK- ^ NΜΤ1. ryTEKT-TK-TOR2A or ryTEKT-TK8U40 with their subsequent treatment with ganciclovir led to a significant decrease in cell viability. Essentially. that the decrease in cell viability was approximately the same regardless of the 3'-untranslated region in the expression construct (Fig. 5). These data indicate a comparable efficiency of expression constructs and allow us to conclude. that the use of fragments of the 3'-untranslated region of the transcript ΌΝΜΏ or TOP2A instead of their traditional analogues does not reduce the efficiency of expression constructs. Essentially. that this fact was established in the model of ganciclovir-dependent death of tumor cells during the production of thymidine kinase in them under the control of the promoter of the 11TEKT gene (-206 - +37). which is a promising strategy for the development of gene therapeutic antitumor agents.

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List of sequences <110> ESTABLISHMENT OF THE RUSSIAN ACADEMY OF SCIENCES INSTITUTE OF MOLECULAR GENETICS OF THE RAS <120> METHOD AND GENE STRUCTURE FOR HIGHLY SPECIFIC INHIBITION OF UNDESIRABLE GROWTH OF CELLS <160 <2122102102102102102102102102102102102102102102102102102102102102102102102102102102102102102102102102102102102102102102121121021021110210210210210111021021011102102121021110210111210

<223> Fragment of 3'-untranslated region of the transcript BYMT1 (SepVapk ΝΜ_001379.1; nt 5108-5409) <400> 1 s6d666s6ddsassaddaa6ssssaasa6dsas6da6d66d6d66666aasabdbsaa6o 60 bdbssdbbsasabdbdbddbasabddbdbbbdbddssbbddsbdasabdaadsbdbTdbd 6dadd66sds66a6saas6aa6da666ad6da6saaa66d6dsad6as666d6dsabbs6 120 180 2 4 0 dda6bb6aaaadbbbbbbabbabdsab6a6a6saaabsbassaobdba6dad6ddaaa66 aadasb6ba6d6adb666ba6a6db6d6aa6a6b6s66saaabaaabsbsbss6a6aaas 300 ca 302 <210> 2 <211> 1033 <212 > OMA <213> Noto zar! Epz <220>

<223> Fragment of a 3’-untranslated $ my area of the TOR2A transcript {SepVapk ΝΤ ___ 010783.15; 381 994 917 nt 9-3818) <400> 2 dadbsadabdaadabdabsbdbbbaaaabdbdaddsdabbabbbbaadbaabbabsbba 60 ssaadsssaadasb ddb666aaad6bassbdaadsbs66aas6bss6ssss6sbdaa66b 120 sdbbsddddaaddbdbtsbsadbasaadasobsaaadb'daadb & aadsssaadbdsbsbb badsb666babaa6asbd6sbaaa6ad6dassabsbsabdddsabbd6666s66sbs6ds 180 240 300 666dbs6d6d6b66dad6s6ds666s6666d6s666aaaass6da66666aad66s66s6 daas6d6adaaa6ads6abs6da6sas6bsadsd6aaadsad6d6d66bab6aassabss as6aads6aaaasbadadsad666da6b6aaaad6d6sas6s66ss6ssb666s6as666 360 420 480 sadbadaba6dadabadadsa6aab6absbdS666a6s66ad6666a6asa6aa666ass a6sada6adaas6b6a6dd66sbad6asadabas6sbasbas sbsadss6s6ba6d6ds saad66b6bs66baadsaabdadaaabbdsbsabdb6s66sa6s66s6saaa6oa6sada 540 600 660 ddssdaadaaaaasas6b6dds6d6dbs6a6aas66dasasad6saabadaabdaadaaa abbadadbadbbabdbdabbabbbsadsbsbbdaosbdbssssbsbddsbdssbsbdadb s6daabsbsssaaadadadaaassaa666sbaadaddas6dda66dsadaadas6sdddd 720 780 840 asaasa66bdabssaadabs6baaabdbbaba66da6aassa6ds6sadsaabdads6ab 6adab6sab66bdddaaa6sbssa6aa666saa666dbaaas666d66aadass6d6s6a sa66d66ababdbdbd6das66dadbaa6db6a6saasd66666d6aaaba666asbabd 900 960 1020 bbbbbsbabbadsbaaabbssaasaabbb6d6asbb6aa6aaaa6d66s6aaasa66dsa asssabadaabds 1033 <210> 3 <211> 243 <2 12> OKA <213> Noto Zartepg

- 11 016223 <220 <223> Fragment of the NTEKT gene promoter (-206 - +37) (SepVapk ΆΕ128893; NT 110006-11248) <400 3 dsadasdssssaddassddsdssssssad ^ ddsddddddas ^ dddddssddddddasd

Rssdsssssassssadsssdssss-ssdsdsddassssdssssd-sssdasss s'sssdddssssddsssadsssss 120-ssss-ssdddsssFsssadssss ^ ssS'ssds 180 ddssssdsss sRssRsdsddsdsdad ^ ^ ^ bsaddsadsdsdsd ss.dsEdsdsasdrd DDA 240 243 <210> 4 <211> 29 <212> ϋΝΑ <213> Artificial sequence <220>

<223> oligonucleotide for amplifying a fragment area on March ¹ -netransliruemoy transcript 0ΝΜΤ1 and introducing restriction endonuclease recognition sites Νοί: Ι at its 5 -terminal ^t <400 ^ 4 adsddssdsd sSddsassaddaag 29 <210> 5 <211> 34 <212> RIA <213 > Artificial sequence <220 <223> Oligonucleotide for amplification of a fragment of the 3'-untranslated region of the transcript ΟΝΜΤ1 and the introduction of recognition sites for restriction endonucleases ΙΙ1 Х and Xa! at its 3'-end <400> 5 arsadadI: OadtsdOO OssadadatOa 34 <210 b <211> 32 <212> 0ΝΑ <213> Artificial sequence <220>

<223> Oligonucleotide for the amplification of a fragment of the 3'-untranslated region of the T0P2A transcript and the introduction of recognition sites for the restriction endonuclease ΝοϊΙ at its 5'-end <400 6 absddsddsdsdadBsadaRdaadaDdaBsBd'32 32 <210> 7 <211> 34 <212> Artificial sequence <220 <223> Oligonucleotide for amplification of a fragment of the 3'-untranslated region of the ΊΌΡ2Α transcript and the introduction of recognition sites for restriction endonucleases ΑίΙΙΙ and Xba1 at its 3'-end <400 7

- 12 016223 βίζ sadas1: aac | ^{sai, bc:,} sa.d'ds | 1 ^ 'b and ^4' ^^ B1 ^{^,} d'34 <210> 8 <211> 28 <212> ϋΝΆ <213 > Artificial sequence <220>

<223> Oligonucleotide for the amplification of herpes simplex virus thymidine kinase cDNA and the introduction of recognition sites for the restriction endonuclease Vd111 at its 5'-end <400> 8 аСддСассдассссдсааассд ^ саа 28 <210> 9 <211> 31 <212> ОКА <213> Artificial sequence 220 <223> Oligonucleotide for the amplification of the herpes simplex virus thymidine kinase cDNA and the introduction of restriction endonuclease recognition sites ΝοϊΙ at its 3'-end <400> 9 aldcdcdcdcdcdcbcccccdcdcc-bc 31 <210> 10 <211> OKA <213> Artificial sequence <220>

<223> Oligonucleotide for amplification of a fragment of the NTENT gene promoter (-206

- +37) <400> 10

1ddda11dsdsdddsasadasd 20 <210 11 <211> 19 <212> OMA <213> Artificial sequence <220>

<223> Oligonucleotide for amplification of a fragment of the promoter of the bTECT gene (-206

- +37) <400 11 dsadsassodsddd ^ ad'bd 19 <210> 12 <211> 36 <212> OMA <213> Artificial sequence <220>

<223> Oligonucleotide for reamplification of a fragment of the P1TEB.T gene promoter (206 - +37) for the introduction of recognition sites for restriction endonucleases ΚρηΙ and ΧΗο' at its 5'-end <400 12 ca £ ddaaos ± sdsassadasssdssaddassdds 36 <210> 13 <210> 13 <210> 13 <210> <212> ϋΚΑ <213> Artificial sequence <220>

<223> Oligonucleotide for reamplification of a fragment of the KTEB.T gene promoter (206 - +37) for the introduction of recognition sites for restriction endonucleases Wd111 and BATH! at its 3'-end <400> 13 a ^ dda ^ ssada ^ s ^ sssasd ^ dsdsadda 32

Claims (18)

1. A gene construct for inhibiting unwanted growth of target cells, containing: a) the 3'-untranslated region of the transcript of the gene selected from the human DNA topoisomerase II alpha (TOR2A) gene and the human DNA- (cytosine-5) methyltransferase 1 (ΌΝΜΤ1) gene; b) cDNA of at least one protein that inhibits cell growth; c) a promoter whose transcriptional activity is specific for target cells. 2. The gene construct according to claim 1, wherein the target cells are tumor cells. 3. The gene construct according to claim 1, wherein the target cells are non-tumor cells susceptible to abnormal proliferation. 4. The gene construct according to claim 1, wherein the cell growth inhibitory protein is a tumor growth suppressor. 5. The gene construct according to claim 1, wherein the cell growth inhibitory protein is an apoptosis inducer. 6. The gene construct according to claim 1, wherein the cell growth inhibitory protein is a cytotoxic protein. 7. The gene construct according to claim 6, wherein the cytotoxic protein is an enzyme that converts a substrate into a toxic compound that causes cell death. 8. The gene construct according to claim 7, wherein the cytotoxic protein is a herpes simplex virus thymidine kinase of the first type. 9. The gene construct according to claim 1, in which the promoter, the transcriptional activity of which is specific for target cells, is a promoter of the human telomerase reverse transcriptase gene (ETEW). 10. The gene construct according to claim 1, in which the promoter, the transcriptional activity of which is specific for target cells, is a hybrid promoter consisting of a promoter of the 11TET gene and a minimal early SMC promoter or synthetic TATA box. 11. The gene construct according to claim 1, in which the promoter, the transcriptional activity of which is specific for target cells, is a promoter of the human survivin gene (B1KS5). 12. A method for increasing the specificity of inhibiting undesired growth of target cells, comprising delivering the gene construct to the target cells according to any one of claims 1 to 11. 13. The method of claim 12, wherein the target cells are tumor cells. 14. The method of claim 12, wherein the target cells are non-tumor cells susceptible to abnormal proliferation. 15. A method of highly specific inhibition of unwanted growth of target cells, comprising: a) delivery to the target cells of the gene construct according to any one of claims 1 to 11; b) if necessary, for the implementation of a cytotoxic effect, delivery to the target cells of a substrate convertible by a protein encoded by the cDNA contained in the construct according to any one of claims 1-11 to a toxic compound that causes cell death. 16. The method according to clause 15, according to which the target cells are tumor cells. 17. The method according to clause 15, whereby the target cells are non-tumor cells susceptible to abnormal proliferation. 18. The method according to clause 15, according to which the protein encoded by the cDNA contained in the construct according to any one of claims 1 to 11, is a herpes simplex virus thymidine kinase of the first type, and the substrate convertible into a toxic compound is ganciclovir.