Ways to enhance and limitations of gene expression

 

The invention relates to gene therapy and for the strengthening and limitations of gene expression. The invention includes a set of vectors for supported and regulated expression, which contain a therapeutic gene products, expression of which is carried out by using inducible promoters, as well as the way to achieve localized and temporal expression under the control of the inducible heat promoter. The invention includes new vectors for gene therapy treatment of local and metastatic cancer of the breast, ovary and prostate. The advantage of the invention is to create new vectors, by which expression of the gene in target cells is enhanced and prolonged by using promoters that are induced by heat. 7 C. and 11 C.p. f-crystals, 10 ill.

Description text in facsimile form (see graphic part).

Claims

1. Recombinant vector pDATH-X (plasmid with the promoter of heat shock controlled by dominant-negative, antisense TET-ON), and the specified vector includes: (a) the 1st cassette, including TET-ON expressed under the control of heat shock promoter and tet-aprovecho repressor and the last C-terminal 130 amino acids of the activation domain of transcription protein VP16 of herpes simplex virus, moreover, the specified heat shock promoter consists of the elements of the response to heat shock (from -260 to 30) promoter gene protein heat shock 70-KD human, coupled with a minimal cytomegalovirus promoter (pCMV), and specified tet-operator consists of a 19-nucleotide inverted repeats operator 02 TN10, which is associated with the tet-repressor and TET-ON; (b) 2nd cassette comprising a cloning site of a therapeutic gene, located below the tetp promoter is a CMV, consisting of a tet operator linked with a minimal cytomegalovirus promoter (pCMV), moreover, the specified tet-operator consists of a 19-nucleotide inverted repeats operator 02 TN10, which is associated with the tet-repressor and TET-ON; (c) 3rd cassette comprising antisense TET-ON under control of the pCMV promoter, with the indicated antisense TET-ON consists of antisense sequences, complementary to the first 80 nucleotides sequence of the TET-ON, including the start codon ATG; (d) the 4th cassette comprising a dominant negative TET-ON under control of the pCMV promoter, moreover, the specified dominant negative TET-ON consists of a tet-repressor without TRANS-activation domain VP16.

2. The way to achieve localized and temporal expression of the gene under the control of the inducible heat p the Torah, includes the specified gene in the organism-owner; application of thermal energy to the location on the specified organism, the host, where the desired expression of a specified gene.

3. The method according to p. 2, characterized in that said body-the owner is the man.

4. Recombinant vector pDATE-X (expressing plasmid with the promoter of EGR is controlled by a dominant-negative, antisense TET-ON), and the specified vector includes: (a) the 1st cassette including the sequence of the TET-ON under control EGRp, the binding site of tetracycline operator and pCMV; (b) 2nd cassette comprising a therapeutic gene X under the control of the tetp promoter-pCMV; (c) 3rd cassette comprising antisense TET-ON under control of the pCMV promoter; (d) the 4th cassette, including dominant negative TET-ON under control of the pCMV promoter.

5. Recombinant expressing vector pRIBs-X (induced by the irradiation of a promoter specific for breast cancer), and the specified vector includes: (a) the 1st cassette, including Gal-DBD-mx, which is a chimeric open reading frame that encodes a N-terminal (amino acids 1-147) DNA-binding domain of the yeast GAL4 protein (Gal-DBD), chimericwanna on the main domain helix-loop-helix-lacinova zip” Max (amino acid is induced by irradiation promoter EDG-1; (b) 2nd cassette that includes a minimal CMV promoter, “antisense Gal-DBD-mx, which is an antisense construct, complementary to the sequence of Gal-DBD-mx internal website ribosome entry (IRES) and Gal-DBD, which competes with Gal-DBD-mx for the binding site pGAL; (c) 3rd cassette, including VP16-TA-mc”, which is a chimeric ORF, encoding the first N-terminal 11 amino acids of Gal4 (amino acids 1-147), with the subsequent nuclear localization signal large T-antigen of SV40, a 130-amino acid C-terminal transactivation domain protein VP16 of herpes simplex virus, the basic domain helix-loop-helix-lacinova lightning with-ICC (amino acids 350-439) and then poly(A) SV40, and educated in the chimeric gene VP16-TA-mc is under the control of the promoter of the C-rb2 “regu” before the first ATG; (d) the 4th cassette, including “Galp”, five copies of the 17-dimensional DNA-binding site for Gal4, and the sequence of the TET-ON placed under the control of the promoter GALp-pTET, a therapeutic gene X is connected with TET-ON through the IRES; (e) 5 cassette comprising antisense TET-ON, which is the sequence consisting of the sequence complementary to the first 80 nucleotides sequence of the TET-ON, including ATG, under the control of the pCMV promoter; (f) 6 ka is 7.

6. Recombinant vector according to p. 5, characterized in that the promoter perbB2 in the 3rd cassette is replaced by the promoter, whey acidic protein.

7. Recombinant vector according to p. 5, characterized in that the promoter perbB2 in the 3rd cassette is replaced by the promoter stromelysin-3.

8. Recombinant vector according to p. 5, characterized in that said gene X is the gene coding for the tumor necrosis factor alpha.

9. Recombinant ekspeditsiyasi vector pRIPs-X (induced by the irradiation of a promoter that is specific for prostate cancer), and the specified vector includes: (a) the 1st cassette, including Gal-DBD-mx, which is a chimeric open reading frame that encodes a N-terminal (amino acids 1-147) DNA-binding domain of the yeast GAL4 protein, chimericwanna on the main domain helix-loop-helix-lacinova zip” Max (amino acids 8-112) and then poly(A) SV40, and educated in the chimeric gene GAL-DBD-mx is under control induced by irradiation promoter Egr-1; (b) 2nd cassette that includes a minimal CMV promoter, antisense Gal-DBD-mx, which is an antisense construct, complementary to the sequence of Gal-DBD-mx, IRES, which is an internal site of entry of ribosomes, and Gal-DBD, which kankuamo reading encoding the first N-terminal 11 amino acids of Gal4, and subsequent nuclear localization signal large T-antigen of SV40, a 130-amino acid C-terminal transactivation domain protein VP16 of herpes simplex virus, the basic domain helix-loop-helix-lacinova lightning with-ICC (amino acids 350-439) and then poly(A) SV40, and educated in the chimeric gene VP16-TA-mc is under the control of the promoter of the gene probasin “pProbasin” before the first ATG; (d) the 4th cassette, including GALp, five copies of the 17-dimensional DNA-binding site for Gal4, and the sequence of the TET-ON placed under the control of the promoter GALp-ptet, a therapeutic gene X is connected with TET-ON through the internal website of the occurrence of a ribosome; and (e) 5 cassette comprising antisense TET-ON, which is the sequence consisting of the sequence complementary to the first 80 nucleotides sequence of the TET-ON, including ATG, under the control of the pCMV promoter; (f) the 6th cassette comprising a dominant negative TET-ON, consisting of the coding sequence for amino acids 1-207.

10. Recombinant vector according to p. 9, characterized in that the promoter probasin in the 3rd cassette is replaced by the promoter of the antigen that is specific for prostate cancer.

11. Recombinant violi.

12. Recombinant expressing vector pHIBs-X (induced by the warmth of a promoter specific for breast cancer), and the specified vector includes: (a) the 1st cassette, including Gal-DBD-mx, which is a chimeric open reading frame that encodes a N-terminal (amino acids 1-147) DNA-binding domain of the yeast GAL4 protein, chimericwanna on the main domain helix-loop-helix-lacinova zip” Max (amino acids 8-112) and then poly(A) SV40, and educated in the chimeric gene GAL-DBD-mx is under the control of inducible heat of the promoter of heat shock protein; (b) 2nd cassette that includes a minimal CMV promoter, antisense Gal-DBD-mx, which is the design, complementary to the sequence of Gal-DBD-mx internal website entry of ribosomes and Gal-DBD, which competes with Gal-DBD-mx for the binding site pGAL; (c) 3rd cassette, including VP16-TA-mc”, which is a chimeric open reading frame, encoding the first N-terminal 11 amino acids of Gal4, and subsequent nuclear localization signal large T-antigen of SV40, a 130-amino acid C-terminal transactivation domain protein VP16 of herpes simplex virus, the basic domain helix-loop-helix-lacinova lightning with-ICC (amino acids 350-439) and then “regu” before the first ATG; (d) the 4th cassette, including GALp, five copies of the 17-dimensional DNA-binding site for Gal4, and the sequence of the TET-ON placed under the control of the promoter GALp-ptet, a therapeutic gene X is connected with TET-ON through the internal website of the occurrence of a ribosome; and (e) 5 cassette comprising antisense TET-ON, which is the sequence consisting of the sequence complementary to the first 80 nucleotides sequence of the TET-ON, including ATG, under the control of the pCMV promoter; (f) 6-th cassette including dominant negative TET-ON, consisting of the coding sequences for amino acids 1-207.

13. Recombinant vector according to p. 12, characterized in that the promoter rb2 in the 3rd cassette is replaced by the promoter, whey acidic protein.

14. Recombinant vector according to p. 12, characterized in that the promoter rb2 in the 3rd cassette is replaced by the promoter stromelysin-3.

15. Recombinant vector according to p. 12, characterized in that therapeutic gene X is the gene factor-tumor necrosis.

16. Recombinant expressing vector pHIPs-X (induced by the warmth of a promoter that is specific for prostate cancer), and the specified vector includes: (a) the 1st cassette, including Gal-DBD-mx, which is a chimeric otkritochnoe on the main domain helix-loop-helix-lacinova zip” Max (amino acids 8-112) and then poly(A) SV40, and educated in the chimeric gene GAL-DBD-mx is under the control of inducible heat of the promoter of heat shock protein; (b) 2nd cassette that includes a minimal CMV promoter (mCMVp), antisense Gal-DBD-mx, which is the design, complementary to the sequence of Gal-DBD-mx internal website entry of ribosomes and Gal-DBD, which competes with Gal-DBD-mx for the binding site pGAL; (C) 3rd cassette, including VP16-TA-mc”, which is a chimeric open reading frame that encodes a first N-terminal 11 amino acids of Gal4, and subsequent nuclear localization signal large T-antigen of SV40, a 130-amino acid C-terminal transactivation domain protein VP16 of herpes simplex virus, the basic domain helix-loop-helix-lacinova lightning with-ICC (amino acids 350-439) and then poly(A) SV40, and educated in the chimeric gene VP16-TA-mc is under the control of the promoter of the gene probasin “pProbasin” before the first ATG; (d) 4th cassette, including GALp, five copies of the 17-dimensional DNA-binding site for Gal4, and the sequence of the TET-ON placed under the control of the promoter GALp-ptet, a therapeutic gene X is connected with TET-ON through the internal website of the occurrence of a ribosome; and (e) 5 cassette comprising antisense TET-ON, which is a serial is UCA ATG, under the control of the pCMV promoter; (f) the 6th cassette comprising a dominant negative TET-ON, consisting of the coding sequences for amino acids 1-207.

17. Recombinant vector according to p. 16, characterized in that the promoter probasin replaced by the promoter of the antigen that is specific for prostate cancer.

18. Recombinant vector according to p. 16, characterized in that therapeutic gene X is the gene factor-tumor necrosis.

 

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FIELD: biotechnology, veterinary science.

SUBSTANCE: invention relates to therapeutic vector used in therapy of infectious diseases in cats that comprises at least one foreign nucleic acid each of that (a) encodes protein taken among the group consisting of feline protein CD28 represented in SEQ ID NO:8 or its immunogenic moiety; feline protein CD80 represented in SEQ ID NO:2 or 3, or its immunogenic moiety; feline protein CD86 represented in SEQ ID NO:6 or its immunogenic moiety, or feline protein CTLA-4 represented in SEQ ID NO:10 or its immunogenic moiety; and (b) nucleic acid that is able to be expressed in insertion of vector in the corresponding host. Indicated therapeutic vector is used in effective dose as component of vaccine against infectious diseases in cats for their immunization and in methods for enhancement or inhibition of immune response in cats and reducing or eradication of tumor in cats. Invention provides stimulating the activation and proliferation of T cells and to enhance effectiveness of control of infectious diseases in cats.

EFFECT: valuable biological properties of recombinant virus.

41 cl, 13 dwg

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