Method for selective destruction of cells (options)

 

(57) Abstract:

The invention relates to medicine and can be used for selective destruction of cells infected with RNA of hepatitis C virus(HCV). The method includes option # 1: introduction to cells plasmids p-cDT containing DNA nucleotide sequence, privedennoi in Fig.1, coding for HCV pseudo <<->> RNA and capable of directing the synthesis of A-subunit of diphtheria toxin in infected cells, while pseudo HCV <<->> RNA does not kill healthy cells, uninfected HCV. 2nd option: introduction to cell pseudo HCV <<->> RNA encoded by DNA nucleotide sequence shown in Fig. 1, and capable of directing the synthesis of A-subunit of diphtheria toxin in infected cells. Pseudo HCV <<->> RNA does not kill healthy cells, uninfected HCV. Pseudo HCV <<->> RNA obtained by PCR using as template DNA with the nucleotide sequence shown in Fig.1, with subsequent transcription of RNA polymerase. The advantage of the invention lies in the selectivity of action above structure, namely in cells infected with hepatitis C virus (V). 2 S. and 2 C.p. f-crystals, 1 Il.

The invention relates to medicine and can betateam virus or RNA, the nature of chronic infection (Lemon SM, Brown EA. Hepatitis With virus and chronic liver disease. Curr. Clin. Top. Infect. Dis. 1994, v. 14, pp. 120-41). Virus or RNA capable of infecting not only the liver cells, but the cells of bone marrow and epithelial origin (Taglione E., Vatteroni ML, Martini P., Galluzzo E., F. Lombardini, Delle Sedie A., Bendinelli W., Pasero G., Bencivelli W , L. Riente With Hepatitis virus infection: prevalence in psoriasis and psorriatic arthritis. J. Rheumatol. 1999, v.26, N 2, p.p. 370-372; Jean-Pierre Bronowicki, Marie-Anne Loriot, Valeri Tiers, Yves Grignon, Anna Linda Zignero and Cristian Brechot. Hepatitis With Virus Persistence in Human Hematopoietic Cells Injected Into SCID Mice. Hepatology, 1998, v. 28, p.p. 211-218).

It is known that the genomic RNA of hepatitis C virus multiplies using complementary HCV-specific <<->> RNA (A. A. Khromykh, M. T. Kenney, E. G. Westaway. Trans-Complementation of Flavivirus RNA Polymerase NS5 Gene by Using Kunjin Virus Replicon-Expressing BHK Cells. J. Virology, 1998, v. 72, No. 9, p. p. 7270-7279). Both types of reactions replication leads HCV-specific RNA polymerase, which recognizes conservative 5'- and 3' sequences in the genomic and complementary RNA (Kolykhalov, A. A., S. M. Feinstone, Rice C. M. Identification of a hingly conserved sequence element at the 3 terminus of hepatitis With virus genome RNA. J. Virol. 1996, v. 70, p.p. 3363-3371).

All efforts to create DNA vaccines aimed at solving two problems: expressing or HCV-specific antigens, or the development of anti-HCV ribozymes. However, the variability of the gene is about invention is a plasmid, which can be used to selectively destroy cells infected with the genomic RNA of hepatitis C virus (HCV).

The problem is solved by creating a plasmid, called p-cDT, which when transcribed produces not translatable RNA. However, in the case of HCV infected cells the transcript from the 5'- and 3'-end of HCV-specific sequences can serve as a matrix for the synthesis of complementary RNA. This RNA will be mapped to the genomic RNA of HCV, but the broadcast is formed toxic to cells the enzyme ADP-ribosyl transferase datamining balance factor elongation, which is an inhibitor of protein synthesis.

Method of constructing plasmids is that in plasmid pSV3-neo between the restriction sites HindIII and EcoRI was embedded DNA, pre-composed of DNA fragments: 5'- and 3'-end of HCV-specific HindIII - Ncol and Xmal - EcoRI and Ncol - Xmal fragment encoding the enzyme. Sequence fragments, limited the necessary restriction sites, was obtained by polymerase chain reaction with appropriate primers and matrices. The DNA construct was synthesized by the combination of three parts: 300 nucleotides encoding the 5'-end portion of the HCV RNA, 103 nucleotide codereuse the th nucleotide sequence of the HindIII - EcoRI fragment shown in Fig.1.

To obtain RNA in vitro DNA construct amplified in PCR using accurate thermopolymers Pwo II, and then transcribable the resulting amplicon RNA polymerase of bacteriophage T7. The obtained RNA was mixed with infectious HCV RNA and were introduced by the method of electroporation in cells lines ner-2 or experiencing human hepatocytes. The effectiveness of anti-HCV action of DNA constructs was determined after its introduction into the retroviral vector and subsequent transformation of the received vector cell line ner-2. After selection of clones stably inherit the retroviral vector pSV3-neo with a DNA construct in cells by the electroporation method was introduced infectious HCV RNA.

In all cases within 7 days of supervised education HCV RNA specific antigen and amplification of RNA in cells and in the environment.

The above genetic constructions lead to the death of cells infected with HCV.

Neither cells nor in the incubation medium is not detected infectious HCV RNA.

Thus, the present invention has the following advantages: firstly, there is the selective destruction of cells infected genomic RNA virus hepatitis is a their design into the genome of cells and its expression. Thirdly, pseudo HCV <<->> RNA unable to broadcast.

The invention is illustrated in the following examples.

Example 1 using the plasmid p-cDT coding pseudo HCV <<->> RNA.

1. DNA plasmids p-cDT method of electroporation was introduced in cell lines ner-2.

2. Transformed cells were selected on medium RPMI 1640 with geneticin (1 mg/ml).

3. Clones resistant to geneticin (G-418) was used to determine HCV-specific antigens and RNA.

4. After determination of antigens and RNA in G418 cells by the electroporation method was introduced infectious HCV RNA.

5. 24 hours after electroporation in G418 cells began to identify HCV-specific antigens and RNA, as well as diphtheria toxin.

6. Controls showed that for 10 hours before the first morphological signs of cell death from the action of diphtheria toxin by the method of solid-phase ELISA determined the toxin.

7. Clear morphological signs of death G 418 cells were observed 48 hours after electroporation.

Example 2 using the plasmid p-cDT coding pseudo HCV <<->> RNA.

1. In PCR with thermopolymers Pwo II, two oligonucleotide primers (5' - what isovale for its transcription RNA polymerase of bacteriophage T7.

3. The obtained RNA electroporation method was introduced in cell lines ner-2.

4. 24 hours after electroporation the cells were begun to determine diphtheria toxin. The toxin was identified within 120 hours after electroporation.

5. The obtained RNA was mixed with infectious HCV RNA and the electroporation method was introduced in cell lines ner-2.

6. 6 hours after electroporation the cells were begun to identify HCV-specific antigens and RNA, as well as diphtheria toxin.

7. Controls showed that for 10 hours before the first morphological signs of cell death from the action of diphtheria toxin by the method of solid-phase ELISA determined the toxin.

8. Clear morphological signs of cell death was observed after 18 hours after electroporation.

The proposed genetically engineered design allows you to selectively destroy cells that are infected with hepatitis C.

1. Method for selective destruction of cells infected with hepatitis C virus (HCV) introducing into the cells a plasmid p-c DT, which is a DNA construct containing 300 nucleotides 5'end of the HCV RNA, 103 nucleotides to the 3'end of the RNA HCVC and 585 nucleotides encoding postroennuyu in plasmid p SV3-neo between restriction sites Hind 111 and EcoR1, encoding HCV pseudo <<->> RNA and capable of directing the synthesis of A-subunit of diphtheria toxin in infected cells.

2. The method according to p. 1, characterized in that the pseudo <<->> RNA does not kill healthy cells, uninfected HCV.

3. Method for selective destruction of cells infected with hepatitis C virus (HCV), providing for the introduction into cells of HCV pseudo <<->> RNA, obtained by PCR with thermopolymers Pwo 11 and oligonucleotide primers 5'-TAATACGACTCACTATAGGGAATTCGACAGCTGGGCGGA-3' and 5'TTCACGCAGAAAGCGTCTA-3' using as template DNA with the nucleotide sequence shown in Fig.1 and the subsequent transcription of RNA polymerase.

4. The method according to p. 3, characterized in that the pseudo HCV <<->> RNA does not kill healthy cells, uninfected HCV.

 

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