A plasmid for the expression of anti-mrna of the retrovirus hiv-1 (options)

 

(57) Abstract:

The invention relates to targeted blocking of the genetic information of the virus mRNA for replication of retroviruses such as HIV, in transfected hematopoietic cells, more particularly, to a plasmid for the expression of anti-mRNA of the retrovirus HIV-1. Plasmids encoding the sequence of anti-mRNA, provide complete inhibition of viral replication of HIV-1 in modified in respect of the genes in human cells. Plasmids suitable for somatic gene therapy of AIDS, because they protect hematopoietic cells from induced virus HIV-1 cytopathic effects. 2 S. p. f-crystals, 8 ill.

The invention relates to targeted blocking of the genetic information of the virus mRNA for replication of retroviruses, for example, HIV, transfected hematopoietic cells, more particularly, to a plasmid for the expression of anti-mRNA of the retrovirus HIV-1.

The invention relates to a plasmid coding sequence of anti-mRNA, which in contrast to the prior art provide a complete inhibition of viral replication of HIV-1 in modified in respect of the genes in human cells. Described in the framework of this proposal plasmids in particular godets the dummy virus HIV-1 cytopathic effects. Thus you can prevent immune deficiency in people infected with the HIV virus. The replication of the virus in the infected cell is controlled by genes of the viral genome. While these genes transcription and translation are formed regulatory gene products and structural proteins to build infectious viral particles, providing a replication of the virus. Playing a major role in this process, the product is mRNA, which gives information for the formation of regulatory proteins and structural proteins.

Highly specific blocking mRNA, based solely on the uniqueness of the molecular structure of viral mRNA, and it is specific to the virus sequence nucleosome this molecule may be carried out using complementary to ribonucleic acid sequences. In the specific sequence pairing nucleobase, as similarly described for deoxyribonucleic acid, single-stranded mRNA complexities in double-strand RNA, so that the genetic information sequence nucleosome for the implementation of the biosynthesis of proteins is not available. Thus the second stage expre-mRNA.

The mechanism of inhibition of gene expression in the interactions of anti-mRNA target mRNA is a complex and mostly not yet clarified. One explanation is the delay splicing and processing of RNA in the cell nucleus. But detailed studies in this regard have not yet been carried out. There is a publication on the exchange of adenosine to inosine in double-strand RNA, which results in a delayed broadcast. It is possible that zatrudnieniu transfer RNA through perinuclear the membrane in place of protein biosynthesis and masking double-strand RNA-Oh interaction between ribosomes and RNA can further contribute to the inhibition of gene expression caused by anti-mRNA.

Known delay metabolism the influence of anti-mRNA in natural biological systems, such as, for example, bacteria and some eukaryotic multicellular organisms [see K. M. Takayama and M. Inouye, Critical Reviews in Biochemistry and Molecular Biology, 25, 155 (1990)], as well as in artificially created by gene transfer of transgenic organisms [see Th. The moth and other FEBS Letters 268, page 427 (1900)].

There and publications relating to the creation in plants and animal cells resistant to virus organisms due to the inclusion of the gene exprimarea is to see the authors in these publications, compelling scientific rationale for the relationship between the expression of anti-mRNA and resistance to viruses still failed. For example, in particular, in the case microengineering, exprimarea anti-mRNA plasmid with temporal expression of the anti-virus action is weak or causes beyond the reasonable assessment of the results of [(K. Rittner and others, Nucleic Acids Resarch 19, page 1421 (1991)] . In the literature there is information about exprimarea anti-retroviral mRNA vectors for the formation of resistant viruses cells (see P.-L. , and others in Gene Regulation: Biology of Antisense RNA and DNA. ed. R. P. Erickson, and I. Isant Equal Press Ltd. New York 1992]. But these retroviral vectors can cause the growth of malignant tumors, as they contain such oncogenes gene sequence. The dependence of carcinogenesis from retroviruses are widely known.

The objective of the invention is the development of plasmids for high-efficiency expression of anti-mRNA of the retrovirus HIV-1.

The problem is solved by the proposed plasmid pSXK1 for the expression of anti-mRNA of the retrovirus HIV-1, including the following elements:

the gene fragment of the HIV-1 681 - 3825;

- promoter cytomegaly;

- early promoter of SV40;
<- resistance gene ampicillin;

- possible host E. coli.

The task is also solved by the proposed plasmid pSXK2 for the expression of anti-mRNA of the retrovirus HIV-1, including the following elements:

the gene fragment of the HIV-1 4158 - 5786;

hybrid promoter consisting of the nucleotide 209 - 865 promoter cytomegaly and nucleotides 150 - 369 metallothionein promoter;

- restriction site Hind III;

- gene resistance to ampicillin;

- possible host E. coli.

Relevant for viral replication of HIV-1 cells human hosts plasmids according to the invention show anti-viral action, which provides full protection against replication of the retrovirus HIV-1 in human cells for more than 60 days.

The invention is further illustrated by the following examples.

Example 1

Plasmid pSXK1

Plasmid pSXK1 for the expression of anti-mRNA of the retrovirus HIV-1 has been designed in order to show the expression of anti-mRNA under the control of a strong constitutive exprimarea promoter, in this case, the promoter of cytomegaly virus. In the restriction site of Hind III at nucleotide 891 (vector pRc/CMV, N V750-20 in the company's catalog Invitrogen, San Diego, CA 92121, USA) inserted in the orientation of the 3'- - > 5' gene fragment of HIV-1, watoga cattle. However plasmid also includes neomicina selection marker and the DNA fragment of pBR322 for replication and selection in E. coli cells.

Restriction map of the plasmid pSXK1 shown in Fig. 1.

Example 2

Plasmid pSXK 2

Plasmid pSXK 2 for the expression of anti-mRNA retrovirus HIV differs from plasmid pSXK 1 that is obtained by combining the promoter sequence comprising nucleotides 209 - 865 CMV promoter (see example 1) and nucleotides 150 - 369 metallothionein promoter (UWGCG GenEMBL case: MUSMETI. RO). Thus, it also includes a hybrid promoter. In addition, including nucleotides 4158 - 5786 proviral DNA sequence is placed in the orientation of the 3'- - > 5' as a matrix for the expression of anti-mRNA under the control of the hybrid promoter CMV and metallothionein. Restriction map of the plasmid pSXK2 shown in Fig. 2.

Example 3

Transfection and cloning of human cells offer plasmids for the expression of anti-mRNA

As examples transfected hematopoietic cell lines of human, which are resistant to the effects of HIV-1 endogenous exprimarea anti-mRNA-Oh, choose growing without adhesion with the substrate, p is aricanli collection of ATSS (12301 Parklawn Drive, , Rockville, MD 20852 - 1776, USA).

Cell lines were cultured in the recommended ATS environment. Transfection of plasmids carry out the following methods, of which the electroporation gives the best results.

1) a Method using calcium phosphate (similar to Wigglers and others, 1977, CELL 11, page 223).

2) Method with application containing diethylaminoethylamine group dextran.

The day before transfection cells adjusted to a density 3-4105/ml of culture medium. 2-4 hours prior to transfection, the cells centrifuged and served in a fresh environment. 0.5 to 20 µg DNA mixed with 1070 μl of PBS buffer and to the resulting mixture add 120 μl of a solution containing 10 mg/ml solution containing diethylaminoethylamine group of dextran and thus obtained solution was used for transfection. Before transfection 40 ml of cells centrifuged, served in 10 ml of PBS buffer and again centrifuged. Received this centrifugal suspended in 1.2 ml of PBS buffer and the above-mentioned dextran and incubated for 30 minutes, after which the cells add 0.8 ml of cold 20% aqueous solution of DMSO in TBS buffer. After three minutes, add 10 ml of TBS and centrifuged. Cells are washed with RPMI medium, posusje cells (1107cells/ml), centrifuged and washed with RPMI medium, then add 2 ml of suspension protoplasts of E. coli (2103cells/ml in 10% sucrose and 10 mm magnesium chloride). The protoplasts with an employee for transfection by plasmid receive according to the method of Sanary-Galdino and others After 8 minutes of incubation, centrifuged and carefully add in within two minutes of a 45% solution of polyethylene glycol with a molecular weight of 400 in RPMI medium. After a one-minute incubation, add within 7 minutes 10 ml of RPMI medium, which does not contain fetal calf serum. Cells are centrifuged, washed with RPMI medium and served in 10 ml of the same medium.

4) Electroporation

For the implementation of the electroporation apparatus used Gene Pulser Apparatus and Capacitance Extender firm Bio Rad. Thus logarithmically growing cells was adjusted to a density of 1107cells/ml, washed with PBS buffer and re-suspended in RPMI medium without fetal calf serum containing 10 mm dextrose and 0.1mm of dithiothreitol. 0.4 ml of cell suspension are served in the designed for electroporation cuvette and add 10 to 20 µg DNA. When this DNA is dissolved in a ratio of 1 mcg/μl of buffer TE. Electroporation is carried out in the following conditions: capacity: 260 - 960 F; voltage: 150 - 3 for 10 minutes at room temperature, then served in 10 ml of RPMI medium containing 10% fetal calf serum.

Product selection transfection

Product selection transfection is carried out in each case taking into account the resistance to the antibiotic G418.

1) Selection in viscous environments.

Transfected cells are first incubated in containing the antibiotic G-418 medium RPMI up until growth can no longer be detected. The cells are then sown in a viscous environment. For hardening the environment is either agar or methylcellulose. Pre-selected cells are sown in a plate size of 10 cm and add 10 ml of RPMI medium containing 1 mg/ml of the antibiotic G-418, and 2 ml of 2% aqueous solution of liquefied agar. After cooling the agar carry out incubation at a temperature of 37oC in thermostat. Additional experience agar substituted methylcellulose.

2) Selection and subsequent cloning in the cell type Transwell cell culture chamber TM company Costar.

48 hours after transfection the cells are sown in the supplied 96 microwells plate in the amount of 11050.2 ml of the cell. In the case of U937 cells to the antibiotic G418 added at a concentration of 0.7 mg/ml, and in the case of cells of HUT 78 is at a concentration of 1.0 mg/ml 1/2 obika G418. Incubation is carried out before the appearance of the individual cells of resistant cells. These mixed clones are grown to a larger size and then clone. To do this, use a special camera growth firms Costar (type: Transwell TM, No. directory: 3425, company address: 205 Broadway, Cambridge, MA 02139, USA.

Cells low density (50 to 200 cells / 6 ml) sown in soft arape with the antibiotic G-418 (see selection in a viscous medium) and 6 ml of medium served in the Central chamber, which membrane is separated from a very densely growing liquid culture of cells U 937 and HUT 78, respectively (200 000 cells/ml). After consumption of the liquid medium partially replaced, in order to ensure further growth of cells. Once in soft agar will appear resistant to the antibiotic G-418 colonies, they emit with a pipette and grow.

Approximately 10 independent transfected cell line U 937 and HUT 78 clone as above described plasmids pSXK1 and pSXK2, respectively, and further treated in order to prove the expression of anti-mRNA and resistance to retrovirus HIV-1.

Example 4

Evidence of integration of the intact expression vector constructs and proof of the expression of anti-mRNA.

After processing transferir the receiving cesium chloride, then DNA and RNA extracted from these cell lines by known methods [see L. G. Davis and others, Basic Methods in Molecular Biology, ed. Elsvier, New York (1986)]. Evidence of integration of intact plasmids for expresii anti-mRNA is carried out using a coupling reaction using a polymerase and oligonucleotide anticipated that amplified overlapping DNA fragments of promoter sequences, inserts, and end. Then amplified DNA identified by analysis according Sotero using gene probes insert DNA. The results are shown in Fig. 3 - 5. For the proposed plasmids used flanking a dose of 5'-CTT TCC AAA ATG TCG TAA CAA CTCC-C' of the promoter sequence and 5'-ATT TAG GTG ACA CTA TAG AAT-C' of the sequence end. In box (sequence HIV) select seed, which, depending on their pairs give the reaction products of approximately 500 - 2100 base pairs.

Proof the expression of anti-mRNA. performed by analysis according to Northern RNA cloned transfected cell lines.

The explanations referred to in the example of Fig. 3 - 5.

Fig. 3

Analysis according Sotero chromosomal DNA from product transfect the reduction in chromosomal DNA. The size of the fragments amplification and hybridization results gene probe HIV, which does not contain the priming sequence of the amplification product, indicate the integrity of the sequence of the promoter and insertion. Figure 1 shows the control experience, 5'- overhang fragment length 887 base pairs (the abbreviation "kb" stands for "kilobar reason"), figure 2 - control experience, the 3' overhang fragment length 775 base pairs, number 3 - chromosoma DNA, 5'-overhang fragment length 887 base pairs, figure 4 - chromosoma DNA 3'-overhang fragment length 775 base pairs.

Fig. 4

The proof of the gene fragment of the HIV-1 chromosomal DNA from product transfection of the cloned cell line HUT 78 after the above amplification. The product of amplification is divided into agar gel and after dyeing bromide, ativa removed under UV light. The reaction product has a length of 561 couple of grounds and meets comprising the nucleotide 699 - 1305 gene fragment of HIV-1, which is also a component plasmids pSXK 1.

This fragment is indicated by the arrow. The letter M denotes a DNA standard length, marked size 516 base pairs and 1018 base pairs. Shown in Fig. 4 digits are followed by what I DNA from cells U 937 (wild type)

3 = a chromosomal DNA of cells of HUT 78 (wild type)

4 = negative control, the reaction of the amplification is not available DNA

5 = positive control

6 = the chromosomal DNA of the products of transformation of HUT 78 (SSHUT 6/1 107 - 9/1)

7 = the chromosomal DNA of the products of transformation of HUT 78 (SSHUT 6/1 107 - 9/1)

8 = the chromosomal DNA of the products of transformation of HUT 78 (SSHUT 6/1 107 - 9/1)

9 = the chromosomal DNA of the products of transformation of HUT 78 (SSHUT 6/1 107 - 9/1).

Fig. 5 (a, b)

Proof includes nucleotides 4158 - 5786 gene fragment of the HIV-1 chromosomal DNA from monocytic cells U 937 and T - limfozitah cells of HUT 78 after the above amplification. Cells pre-transfererror the plasmid pSXK2. The reaction products are divided into agar gel after staining with ethidium bromide is removed under UV light. The product includes a common gene fragment of the HIV-1 plasmid pSXK2 (nucleotides 4158 - 5786) and has the length 1811 base pairs. This product is indicated by the arrow. The letter M denotes a DNA standard length, marked standards 1635 base pairs and 2036 base pairs. The figures have the following meanings:

1 = negative control, the reaction of the amplification is not available DNA

2 = chromosomal DNA from cells U 937 (wild type)

3 = chrome 5 = the chromosomal DNA of the products of transformation of U 937 (SSUK 3/1 107 -12/1).

Example 5

Evidence of resistance to viruses transfected cloned hematopoietic cell lines

Cloned, exprimarea anti-mRNA cell line HUT 78 and U 937 examples 3 and 4 infected with HIV-1 [see L. Ratner and others, Nature 313, page 277 (1985)] or fragment D 194 isolated from HIV-2 [X Kuehnel and others, Nucleic Acids Research (1990) 18 / 6142], compare the degree of resistance to viruses is taken as a control corresponding to the original cell line HUT 78 and U 937, infected by the same viruses.

1. Infection of cells of HUT 78 and U 937 by the retrovirus HIV-1 (control).

Implementation experience

10 ml of cell suspension is mixed with varying degrees of dilution of the supernatant liquid containing HUT 78/HIV-1 (approximately 104standard units/ml in the cultures of PBL, the reverse transcriptase activity of the frozen core solution: 650 000 counts per minute/ml) for 60 minutes. Then the cells are removed by centrifugation and washed three times to remove unbound virus particles and viral proteins. In conclusion, the cells re-suspended in 10 ml of nutrient medium and cultured in an incubator. Every 3 - 4 days 200 ál free from cell supernatant idcore replaced with 5 ml of fresh nutrient medium.

2. Infection exprimarea anti-mRNA of the cells of HUT 78 and U 937 by the retrovirus HIV-1

Implementation experience

10 ml of cell suspension (approximately 4105cells/ml) is mixed with the diluted supernatant fluid HUT 78/HIV-1 (approximately 104standard units/ml in the cultures of PBL, the reverse transcriptase activity of the frozen core solution: 650 000 counts per minute/ml) for 60 minutes. In the case of cells of HUT 78 final dilution of the primary virus solution is 1 : 1000, and in the case of cells U 937 - 1:50. Cells are removed by centrifugation and thoroughly washed four times to remove unbound virus particles and viral proteins. In conclusion, the cells re-suspended in 10 ml of culture medium (RPMI medium 1640 containing 16% fetal calf serum) and cultured in an incubator for 60 days. Every 3 - 4 days 160 ál free from cell supernatant examined for the presence of HIV antigens known trick. To supply the cells every 3 to 4 days away 5 ml suspension, which is replaced by 5 ml of fresh nutrient medium.

In the experiments used two products transfection HUT 78 (SSHUTK16/2-SXK1, SSHUTKI/1-SXK2; see Fig. 6) and one product transfection of cells U 937 (SSUK3/1-SXK2; see Fig. 7). As PTI-mRNA cell lines with plasmids pSXKI, pSXK2 after infection during the incubation period up to 60 days show no signs of reproduction of the retrovirus HIV. In contrast to the experiments according to the invention in the control cell line is determined by explicit multiplication of the retrovirus HIV. The results of the experiments shown in Fig. 6 and 7 are given the following explanation.

Fig. 6

Presents the process of formation of the antigen of HIV-1 within 60 days after infection (day 0) cell line HUT 78 fragment LAV/BRU allocated from the retrovirus HIV-1. On the ordinate plotted the optical density at 450 nm (OD 450 nm), the resulting immunofermentnogo solid-phase analysis of the supernatant of the cell culture. When the optical density greater than 2.0 supernatant is diluted and the specified value of optical density is calculated on the diluted solution. The top value on the ordinate represents the limit of detection of HIV antigen.

As can be seen in exprimarea anti-mRNA cell lines SSHUT K16/2-SXKI and SSHUT K1/1-SXK2 replication of the retrovirus HIV is fully retained within 60 days.

Fig. 7

Presents the process of education of the retrovirus HIV-1 within 60 days after infection (day 0) cell line U 937 fragment LAV/BRU selected from [retr] the aqueous solid-phase analysis of the supernatant of the cell culture. When the optical density greater than 2.0 supernatant is diluted and the value specified for the optical density calculated according to the dilution. The top value represents the limit of detection of the antigen of HIV-1. In exprimarea anti-mRNA cell lines SSUK3/1-SXK2 replication of the retrovirus HIV is fully retained within 60 days,

The fact of infection of HIV-1 in cell lines SSUK3/1-SXK2, SSHUT K16/2 - SXK1 and SSHUT K1/1-SXK2 that after incubation for 60 days do not show any activity of the reverse transcriptase or the presence of the antigen of HIV-1, prove, for example, using the above-mentioned reaction. In incubated during 60 days the cells SSHUT K16/2-SXK1 and SSHUT K1/1-SXK2 you can prove TAT sequence and contained in the envelope sequence of HIV-1 (see Fig. 8). In this reaction, apply a dose of 5'-ATG GAG CCA GTA GAT CCT-3' and 5'-TCT ACC ATG TCAT-3' for fragment length 690 base pairs. Plasmids pSXK1 and pSXK2 not contain these sequences and therefore they cannot give a wrong positive product. In Fig. 8 stained with ethidium bromide agar gel after electrophoresis shows the products obtained in the above-mentioned reaction of the chromosomal DNA following cell lines. For comparison with achiev the culture.

Mark M: fragment with standard molecular weight, size 600 base pairs marked.

Mark 1: SSHUT K16/2-SXK1 702 base pairs is positive (the experiment for the determination of antigen: negative).

Track 2: the same product, and on the trail 1 (parallel experience).

Track 3: the same product, and on the trail 1 (parallel experience).

Mark 4: SSHUT K1/1-SXK2 702 base pairs, positive (experiment for the determination of antigen: negative).

Track 5: the same product, and track 4 (parallel experience).

Track 6: the same product, and track 4 (parallel experience).

Mark 7: HUT 78, the positive control.

Tracks 8-10: culture SSUK3/1-SXK2: product 703 base pairs cannot be proven (the experiment for the determination of antigen: negative).

Example 6

Control experiments

Transfection of the cell line U 937 and HUT 78 plasmids of example 1, 2 without inserts proviral DNA of a retrovirus of the HIV-1 anti-m-orientation does not lead to the fact that viral replication in these cell lines zariwala. Control experiments using plasmids of examples 1 and 2, which have the insertion of proviral DNA of a retrovirus of the HIV-1 m-orientation, was not carried out, since the corresponding m-herbal delay the replication of the virus [see G. Th. Graham and other PNAS 87, page 5817 (1990)]. Therefore, control experiments of this kind have no meaning.

Summary of the results of experiments

Plasmids pSXK 1, pSXK 2 provide endogenous formation of anti-mRNA in respect of the retrovirus HIV-1 (see example 4) in human cell lines blood after transfection and cloning of transfected cell lines in example 3. In addition, according to example 5 proven their antiviral activity.

Unexpected is the fact that in transfected cell lines can prove a pronounced delay in the replication of the retrovirus HIV-1. In the case of applications specially adapted to transfected cell lines virus HIV-1 could equally prove resistance to virus exprimarea anti-mRNA cells (see Fig. 6, 7 ).

These results indicate that suitable promoters for the expression of anti-mRNA and, of course, the type of sequences of anti-mRNA ensure the genetically modified cells resistance to infection by the retrovirus. Example 4 shows that the result is unknown so far splicing of RNA transcripts (+) strands of proviral HIV DNA in the orientation of the 3'- - > 5' are formed nezianya in examples 1 - 6, show that the plasmid comprising encoding anti-mRNA DNA fragments from retrovirus, ensure the development of antiviral properties in cells that protect against infection by the retrovirus.

In Fig. 6 to 8 shows that 60 days after infection by the retrovirus HIV cell line SSHUT 16/2-SXK 1 and SSHUT K1/1-SXK2 show a positive reaction in the determination of proviral DNA HIV, while the definition of gene HIV is negative. For this result we can conclude that after the transmission of HIV 1 in the first stage of the replication cycle of HIV is still formed of proviral DNA in transfected cell lines HUT 78, however, the transcription and translation of proviral DNA is not permitted anti-mRNA, so that protein for HIV viral replication is not formed in proving quantities.

60 days after infection with HIV-1 in DNA of cell lines SSUK3/1-SXK2 it was impossible to prove in all experiments the formation of proteins HIV and proviral DNA HIV. For this result we can conclude that formed in cell lines SSUK3/1-SXK2 anti-mRNA is not only delays the first stage of the replication cycle of HIV, but does not allow carrying out the second stage of this cycle (the transcription and translation of proviral DNA).

The formation of proviral DNA from the genomic (+) litewave anti-mRNA is already assumptions in the literature (see R. I. L. and P. E. Neiman in Gene Regulation: Biology of Antisense RNA and DNA, ed. P. P. Erickson & Th.G. Sent, Revent Press Ltd, new York, 1992), but it still could not appear due to lack of appropriate high-efficiency plasmid.

1. Plasmid pSXK1 for the expression of anti-mRNA of the retrovirus HIV-1, including the following elements: gene fragment of the HIV-1 681 - 3825; promoter cytomegaly; early SV40 promoter; the polyadenylation signal; neomicina selection marker; the restriction site Hind III; the gene of resistance to ampicillin; possible owner of E. coli.

2. Plasmid pSXK2 for the expression of anti-mRNA of the retrovirus HIV-1, including the following elements: gene fragment of the HIV-1 4158 - 5786; a hybrid promoter consisting of the nucleotide 209 - 865 promoter cytomegaly and nucleotides 150 - 369 metallothionein promoter; the restriction site Hind III; the gene of resistance to ampicillin; possible owner of E. coli.

 

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