HU214684B - Method for the preparation of expression vectors and pharmaceutical compositions containing them - Google Patents

Abstract

Field of the Invention The present invention relates to a method for expressing an antisense RNA to an expression vector in an ancillary manner, preferably by incorporating a primer sequence into an HIV-1 viral DNA antisense isolation survival probe 7514 -> 474.5786 -> 474, 2096 -> 474, 3825 ->. 681, 5786-> 4158, 5746-> 4648.2369 -> 1635, 3226-> 3003, 2099-> 678, 3829 -> 2099, 4647 -> 4157.4157 -> 3830, 605 -> 455, 670 -> 440, 825 to 535, 6070 to 5800, 5640 to 5020, 5600 to 5040, 8690 to 8300, or 9160 to 8800. The invention relates to the preparation of transfected cell and pharmaceutical compositions comprising the above vector. ŕ

Description

The invention relates to the production of transfected cells and pharmaceutical compositions containing the above vector.

The present invention relates to the targeted blocking of viral messenger ribonucleic acid (mRNA) genetic information required for the replication of viruses (e.g., HIV) in transfected hematopoietic cells using an appropriate expression system for expression of complement antisense RNA.

More particularly, the invention relates to the production of expression vectors encoding an antisense RNA sequence which, contrary to the state of the art, completely inhibit HTV-lik replication and significantly reduce HIV-2 replication in genetically engineered human cells. The genetic units of the described expression vectors are particularly suitable for somatic gene therapy for HIV / AIDS diseases, since they protect hematopoietic cells from HIV-induced cytopathological effects. This allows people with HIV to avoid immune deficiency.

The growth of the virus in the infected cell is regulated by the genes of the virus genome. In doing so, transcription and translation produce regulatory gene products and structural proteins that serve to construct infectious virus particles necessary for viral growth. An essential intermediate of this process is messenger ribonucleic acid (mRNA), which mediates the information required for the formation of regulatory and structural proteins.

The base sequence complementary to the ribonucleic acid allows specific blocking of the messenger ribonucleic acid based solely on the specificity of the molecular structure of the viral mRNA, i.e., on the specific nucleotide base sequence of the molecule. Sequence-specific nucleotide base pairing, which is analogous to DNA, initially complexes with single-stranded mRNA into double-stranded RNA, and genetic information carried by the nucleotide base sequence becomes inaccessible to protein biosynthesis. This inhibits the second step of gene expression, translation. Complementary ribonucleic acid is called antisense RNA.

The mechanism by which the interaction between antisense RNA and target mRNA inhibits gene expression is completely unknown. One explanation is the inhibition of RNA cleavage and RNA processing in the nucleus, but detailed studies are not known. In double-stranded RNA, adenosine is known to be exchanged for inosine, and this exchange inhibits translation. Further data are provided on inhibiting gene expression by antisense RNA, inhibiting transport of RNA across the perinuclear membrane to the site of protein biosynthesis, and masking the ribosome / RNA interaction with double-stranded RNA.

Inhibition of metabolic processes by antisense RNA is known in biological systems of natural origin, such as bacteria and some eukaryotic multicellular organisms [K. M. Takayama and M. Inouye, Critical Reviews in Biochemistry and Molecular Biology, 1990, 25, 155] and in transgenic organisms produced by artificial gene transfer [J. Mole. et al., FEBS Letters, 268, 427 (1990)].

Experiments involving plants [M. Cuozzo et al., Biotechnology, 6, 549 (1988)] and in animal cells [T. Rüden and E. Gilboa, J. Virol. 63, 677 (1989) and European Patent 252,940] have attempted to establish a virus-resistant organism by introducing a gene expressing antisense RNA. As the authors themselves explain, there is no convincing and scientifically proven relationship between antisense RNA expression and virus resistance. Especially with plasmids expressing microinjected antisense RNA, the antiviral effect achieved by the transient expression is poor or difficult to evaluate [K. Rittner et al., Nucleic Acids Research, 19, 1421 (1991). About retroviral vectors expressing antisense RNA [R. Y-L. To et al., Gene Regulation: Biology of Antisense RNA and DNA, published by R.P. Erickson and J.G. Izant, Raven Press Ltd. New York (1992), are known to be useful in the production of virus resistant cells. The risk of such retroviral vectors is that they may induce malignant tumor growth because they contain gene sequences similar to oncogenes and the retrovirus-dependent oncogenesis is repeatedly covered.

Contrary to published work on regulating viral replication by antisense RNA, the present invention relates to an expression construct which combines a specific promoter construct with appropriately selected and antisense orientation sections of viral DNA to provide antiviral activity of sufficient quality and magnitude. is considered to be substantially higher than the state of the art. A combination of expression vector elements consisting of a 5'-flanking DNA sequence, a promoter, an insert / antisense DNA and a 3'-processing signal sequence, is detectable for the first time in retroviral infections and expresses transfected antisense RNA with distinct viral resistance. cells. In contrast, known solutions using other vector systems for endogenous antisense RNA expression and retroviral resistance provide only weak, often transient, viral activity. See, e.g., T. Rüden and E. Gilboa, J. Virol. 63: 677 (1989); European Patent Publication No. 252940; G. Sczakiel et al., Biochem. Biophys, Slit. Com., 169, 643 (1990); O.I.

Mirosknichenko et al., Gene 84: 83 (1989); T. Shimada et al., 1991, Antiviral Chemistry and Chemotherapy 2: 133; G. Sczakiel et al., J. Virol. 66, 5576 (1992)], or involve the aforementioned dangers (retroviral gene transfer) or require microinjection and are therefore not applicable in practice.

Thus, the present invention relates to a method of producing an expression vector construct capable of expressing antisense RNA

By inserting a promoter sequence and an HIV-1 proviral DNA in an antisense orientation 7514 -> 474, 5786 -> 474, 2096 -> 474, 3825 -> 681, 5786 - into a suitable expression vector. > 4158, 5746 -> 4648, 2369 -> 1635, 3226 -> 3003, 2099 -> 678, 3829 -> 2099, 4647 -> 4157, 4157 -> 3830, 605 -> 455, 670 -> 440, 825 - > 535, 6070-> 5800, 5640-> 5020, 5600-> 5040, 8690-> 8300 or 9160-> 8800.

The expression vector construct of the present invention contains a hybrid promoter gene sequence and exhibits strong constitutive or pathogenic infection-inducible promoter activity.

These are, for example, expression vector constructs containing the subgenomic gene sequence of a pathogenic virus in the 3 '-> 5' orientation (in the antisense orientation to the (+) - DNA strand) regulated by the promoter for expression of the antisense RNA.

Preference is given to expression vector constructs which, in antisense orientation, have the proviral DNA 7514-> 474 or 5786-> 474 or 2096-> 474 or 3825-> 681 or 5786-> 4158 or 5746-> 4648 or 2369 of the HIV-1 proviral DNA. > 1635 or 3226 -> 3003 or 2099 -> 678 or 3829 -> 2099 or 4647 -> 4157 or 4157 ->

3830 nucleotides.

Further, expression vector constructs containing a subgenic proviral gene sequence or an oncogenic sequence in the 3 'to 5' orientation can be used to express shorter antisense RNA transcripts.

In this regard, expression vector constructs that have 605 -> 455 or 670 -> 440 or 825 -> 535 or 6070 -> 5800 or 5640 -> 5020 or 5600 -> 5040 or 8690 -> of the proviral gene sequence of HIV-1 are preferred. 8300 or 9160-> 8800 nucleotides.

Preferred are expression vector constructs containing two, three or more subgenomic gene sequences from one or more viruses.

The invention also provides for the production of transfected cells by transfecting cells with known expression vector constructs of the invention in a known manner.

The invention further relates to the preparation of a pharmaceutical composition comprising an expression vector construct according to the invention.

The expression vector constructs disclosed herein exhibit anti-viral activity in human host cells relevant to HIV replication, which provides complete protection against HIV-1 replication in human cells for 60 days, and significantly inhibits HIV-2 replication.

Thus, the present invention provides the construction and composition of expression vector constructs that express messenger ribonucleic acid transcripts in transfected cells that are complementary to the mRNA sequence of unwanted viral genes. Examples of unwanted viral genes include the GAG, POL, VIF, RÉV, TAT and VPU genes of the human immunodeficiency virus HIV-1 [W. A. Haseltine et al., Scientific American USA, 34-42 (1988)]. In addition, the antisense RNA sequence disclosed herein exhibits appropriate homology to HIV-2 (e.g., HIV-2 Dl94 isolate, Human Retroviruses and AIDS, Myers et al., Los Alamos, National Foot., 1992); EMBL Accession Number X52223)] for the GAG, POL and VIF genes and for HIV-2 isolates. The expression vector construct contains a selected promoter sequence in a specific antisense orientation (3 '-> 5') and a hybrid promoter sequence (e.g., CMV-IE / Metallothionein I) with subgenomic HIV-1 DNA fragments constitutively or inducibly by multiple RNA cleavage. They allow transcription of RNA transcripts. The antisense RNA transcripts thus produced have surprisingly drastically or completely inhibited the replication of HIV-1 in human cells (e.g., COLD 78 or U 937 cells). Significant inhibition of HIV-2 replication is also found. HIV-2 exhibits appropriate homology to the RNA sequence in the described expression vector constructs, which inhibits viral replication through antisense RNA. The invention therefore provides a method that allows inhibition of replication of retroviruses, such as HIV-1 and HIV-2, thereby protecting the hematopoietic cells, and thereby important cells of human cell-mediated immune responses (e.g., Cells and Monocytes). cell-damaging and cell-destroying cells, to which antisense RNA with antiviral activity is expressed in a specific manner.

In order to protect the cells of the immune system by expressing antisense RNA with antiviral activity, the following steps are taken to protect against the cellular damage of retroviral reproduction, such as known HIV-1 and HIV-2 infection:

promoters:

Expression vectors containing promoters that are constitutively and strongly expressed in hematopoietic cells, such as the cytomegalovirus IEpromoter (CMV-IE), are prepared. In addition, fusion of nucleic acid sequences produces promoters that elicit increased expression upon viral infection. An important component of such hybrid promoters is the CMV-IE metallothionein promoter fragment and nucleotides 289-474 or 289-532 of the HIV-1 LTR nucleic acid fragment (numbered as follows: UWGCG GENEMBL DATA BANK Fill: HIVHXB2CG 1987). September 25, which exhibits enhanced, optionally transactivable complementary promoter activity).

Antisense nucleic acid fragments:

The vector construct, under the control of the promoters described, contains subgenomic, proviral DNA fragments in the antisense orientation, i.e., the positive strand of the DNA is transcribed in the 3 'to 5' direction.

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For safety reasons, not the entire proviral genomic DNA fragment of a retrovirus is used. However, longer subgenomic proviral DNA fragments are preferable to short subgenomic proviral DNA fragments, since longer RNA transcripts tend to have RNA cleavage and thus more antisense RNA transcripts may be expected from the proviral DNA fragment of the present invention, which is practically They justified. (The type and amount of expected fully processed antisense mRNA transcripts in the antisense orientation of the HIV sequence used cannot be predicted since the exact consensus sequence for mRNA cleavage is not known.)

Due to the increasing number of antisense RNA transcripts, due to their intrinsic properties, such as their exit from the nucleus or their stability in the cytoplasm, the likelihood of antisense RNA transcripts with potential antiviral activity is increased.

The invention further encompasses expression vector constructs comprising a fused gene fragment capable of transcribing antisense RNA under the control of a strong promoter. The fused gene fragment consists of two or more subgenomic DNA fragments of different pathogenic viruses such as HIV-1 and HIV-2. The above fusion gene fragments encoding an antisense RNA transcript containing a sequence of antisense RNA sequences complementary to a plurality of mRNA sequences of different viruses allow the production of a broader spectrum of antiviral expression vectors.

The validity of the above principle is demonstrated in Examples 1, 4, 5, 10 and 12. For example, expression vector constructs KTX11, pSXK1 and pSXK2 contain HIV-1 LAV / BRU-derived antisense DNA fragments which themselves contain shorter segments that are complementary only to mRNA from HIV-2 Dl94 (e.g., 620-660 or 837-899 or 4868-5044).

In this way, the antisense RNAs of KTX11, pSXK1 and pSXK2 also exhibit antiviral activity against HIV-2.

The antisense RNA coding sequence of the above vectors can also be fused to a fragment of the proviral DNA of other viruses (e.g., HIV-2 isolate or other HIV-1 isolate) in an antisense orientation, resulting in simultaneous expression vector structure due to its optimal complementarity with other viral sequences. virus can be better or completely inhibited.

Termination of the antisense RNA transcript is performed with a polyadenylation signal (e.g., a signal from SV 40 or bovine growth hormone) to obtain mRNA of sufficient stability. The vectors also contain the complete operon required to express the appropriate selection marker. In the examples given, TNR 5 neo (neomycin, geneticin, G 418, kanamycin) antibiotic resistance was used to allow antisensic activity following transfection.

Cells containing a gene structure that provides RNA expression.

This selection marker is applicable to human hematopoietic cells. However, other selectable markers can be used, such as puromycin or hygromycin or methotrexate in combination with transfection of a gene expressing dihydrofolic acid reductase.

Particularly preferred antisense nucleotide sequences in the POL / VIF region of the HVV are those between 2100 and 5800 nucleotides. In particular, the antisense nucleotide sequence between nucleotides 5786 to 4158 provides excellent antiviral activity. However, other fragments can be obtained from this gene region, for example, between nucleotides 5746 to 4648 or 4647 to 4157 or 5880 to 3880 or 5850 to 4158 or 5150 to 3200.

To verify the antiviral activity of the antisense RNA, such as HIV-1, a human cell line infectious with HIV-1 is transfected with the above expression vector construct. In a series of different cell types of human hematopoietic cells, it must be demonstrated that expression of endogenous antisense RNA in the cell inhibits viral replication, which primarily influences cellular immune responses. The breakdown of immune system cells in the final phase of AIDS disease is causally related to the increased incidence of subsequent infections, which are often fatal. It was an object of the invention to demonstrate viral resistance in hematopoietic cells and, first, in permanent cell lines, such as HUT 78, MOLT 4, U 937, Jurkat, CEM-CM3 (all found in the ATCC US collection) by transfection with the vector. after cell cloning and genetic characterization and experimental infection with appropriate HIV-1 and HIV-2 isolates.

The fact that the expression of antisense RNA with antiviral activity can be demonstrated in such cells supports the therapeutic utility of expression constructs in hematopoietic cells and, ultimately, in the transformation of hematopoietic bone cells. Introduction of genes into peripheral blood lymphocytes and bone marrow cells for therapeutic purposes is known [K. W. Culver et al., Transplant. Proc. 23: 170-177 (1991)] and in principle can be used in the antisense RNA expression systems described.

Example I

KTX11 Expression Vector Construction [Nucleotide numbers below refer to the sequence of human immunodeficiency virus type 1 (HXB2), unless otherwise noted, UWGCG, GENEMBL DATA BANK, Phi: HIVHXB2CG, VIRAL, September 25, 1987]

For expression of antigenic antisense RNA after transfection of HIV-1 infectious cells, the following expression vector was constructed:

Mouse metallothienine I promoter [1600 bp EcoRI-BglII fragment; N. Glanville et al., Natur, 292,

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267-269 (1982)] after ligation at the SalI site (5786 nucleotides) from the 3 'region of the proviral HIV-1 DNA sequence with a BglII-XhoI adapter, thereby converting the BglII, XhoI and SalI sites.

The BglII site (474 nucleotides) is ligated from the 5 'region of the proviral HIV-1 DNA to the BamHI site to which the SV 40 polyadenylation signal sequence is linked (nucleotides 2564-4713 with the deleted sequence from SVBLOXX of the UBLGCG geneEMBL). The same SV 40 polyadenylation sequence is used in the 3 'to> 5' direction to terminate TRN5NEO [neomycin selection marker (phosphotransacetylase), nucleotides 1-1286 TRN5NEO BACTERIA, UW GCG gene EMBL]. Expression of the neomycin gene was initiated from the SV 40 early promoter (nucleotides 5171-371, UWGCG gene EMBL SV 4OXX). In addition, the vector also contains bacterial PBR 322 DNA (nucleotides 2067-4363, UWGCG geneEMBL strand PBR 322) and an ampicillin resistance gene for use as a shuttle vector in E. coli and animal cells. A map of the KTX11 vector gene is shown in Figure 1.

Example 2

KTX12 expression vector construct

The KTX 12 vector for expression of HIV-1 antisense RNA is constructed in the same manner as the KTX 11 vector (Example 1), but contains an inducible hybrid promoter and the HIV-1 template DNA strand is located 3 'to 5' of the hybrid. promoter at nucleotides 7514 to 474.

The hybrid promoter is fused at nucleotides 289-532 of the HIV-1 LTR promoter fragment at 368 nucleotides (UWGCG site MUSMETI.RO). A restriction map of the KTX 12 vector is shown in Figure 2.

Example 3

KTX 15 expression vector construct

The KTX 15 vector suitable for expressing HIV-1 antisense RNA is constructed in a manner similar to the KTX 12 vector (Example 2), but the DNA fragment of the HTV-Ι template strand (in the 3 'to 5' orientation) is substantially truncated from 2096 nucleotides. It spans 474 nucleotides and thus encompasses the GAG sequence, the Leader sequence, the U5 sequence, and part of the R sequence. Another important feature of the KTX 15 vector is the deletion of the TAR sequence in the HIV-LTR hybrid promoter by deleting nucleotides 474-532. Thus, the hybrid promoter remains stimulable in HIV infection but TAR-encoded termination of transcription [S. Y. Kao et al., Natura 330: 489 (1987)] do not occur in the absence of the HTV-Ι TAT protein. Thus, this hybrid promoter is distinguished by an extremely high constitutive transcription under the control of the HIV-1 LTR promoter sequence. A restriction map of the KTX 15 vector is shown in Figure 3.

Example 4 pSXK1 expression vector construct

The vector pSXK1 for expressing HIV-1 antisense RNA is designed to be antisense

Expression of RNA is under the control of another highly constitutively expressed promoter, in this case the cytomegalovirus promoter. The HindIII restriction site at nucleotide 891 (pRc / CMV vector, catalog number V 750-20, Invitrogen, San Diego, Califomia, 92121 USA) is inserted at nucleotide 681-3825 of the HIV-1 gene fragment in the 3 'to 5' orientation. The polyadenylation signal is derived from the bovine growth hormone (BGH) gene.

In other respects, the vector was constructed in the same manner as the neomycin selection marker and the pBR322 DNA fag fragment required for replication and selection in E. coli as a commuting vector (Example 1). The cleavage map of vector pSXK 1 is shown in Figure 4.

Example 5 pSXK 2 expression vector construct

The pSXK2 vector suitable for expression of HIV-1 antisense RNA differs from the pSXK1 vector (Example 4) in that it contains a fused promoter sequence consisting of nucleotides 209-865 of the CMV promoter (see Example 4) for which the metallothionine promoter (UWGCG geneEEMBL branch MUSMETI.RO) is bound to nucleotides 150-369 and thus forms a hybrid promoter. In addition, the proviral DNA sequence of nucleotides 4158-5786 was inserted in the 3 'to 5' orientation as a template to express the antisense RNA under the control of the CMV / metallothionine hybrid promoter. A restriction map of vector pSXK 2 is shown in Figure 5.

Example 6 Expression vector constructs pHTT 1, 4, 6, 9, 10, 12 and 15

Vectors 1.4, 6, 9, 10, 12 and 15 suitable for expression of HIV-1 antisense RNA were prepared in a manner similar to vector pSXK1 (Example 4, Figure 4). All HIV-1 gene fragments incorporated in pHTT vectors were cloned in the 3 'to 5' orientation of pRc / CMV (Invitrogen, San Diego, Califomia 92121, USA, Cat. No. V 750-20 (1991)) under the control of the constitutively expressing cytomegalovirus promoter. under.

The pHTT vectors are specified below:

(a) pHTT1: The HIV-1 gene fragment (nucleotides 4648-5746) was inserted at the NotI site (966 nucleotides) of the pRc / CMV vector.

Ifi) pHTT 4: The HIV-1 gene fragment (nucleotides 1635-2369) was inserted into the XbaI site (985 nucleotides) of the pRc / CMV vector.

fi) pHTT 6: The HIV-1 gene fragment (nucleotides 3003-3227) is inserted at the Xba I site mentioned under (b).

(d) pHTT 9: The HIV-1 gene fragment (nucleotides 678-2099) is inserted at the Xba I site mentioned under (b).

21 (e) ρΗΤΤ 10: The HIV-1 gene fragment (nucleotides 2099-3829) is inserted into the Xba I site mentioned under (b).

(f) pHTT12: The HIV-1 gene fragment (nucleotides 4157-4647) is inserted at the Xba I site mentioned under (b).

(g) pHTT 15: The HIV-1 gene fragment (nucleotides 3830-4157) was inserted into the Xba I site mentioned under (b).

1-6. An overview of HIV-1 gene fragments incorporated in antisense orientation (3 '-> 5') into the vectors of Example 1 is shown in Figure 6.

Example 7

Transfection and cloning of human lymphocytes with antisense RNA expression vectors

Examples of transfected human hematopoietic cell lines exhibiting viral resistance to endogenous expressed antisense RNA against HIV-1 are the non-adherent, monocyte-like U 937 cell line (ATCC No. CRL 1593) and the non-adherent-developed CTO 78 T-lymphocyte cell line. ATCC No. TIB 161).

Cell lines are cultured in ATCC-recommended media. Vector transfection is performed by various methods, electroporation showing the best results:

1. Calcium phosphate method:

The procedure was carried out by the method of Wigler et al., Cell, 11, 223 (1977), and calcium precipitated DNA was used for transfection. Various amounts of DNA in the range 4.05 to 20 pg are dissolved in 0.25 ml of 250 mM calcium chloride solution and pipetted slowly to 0.25 ml of 2 X HEBS buffer. Transfection was done by adding the resulting precipitated mixture to 10 ml of cell culture. After 15 hours, the medium is aspirated and the cells are lysed with DMSO.

To the culture was added 3 ml of 25% DMSO in 25% sterile PBS. After 4 minutes, it is filtered off and washed with 5 ml of PBS. The cells are then resuspended in 10 ml of DMEM.

2. DEAE-dextran

On the day of transfection, the cells were adjusted 3-4xl0 ⁵ cells / ml of medium density. Two to four hours prior to transfection, the cells were centrifuged and resuspended. 0.5 to 20 µg of DNA were mixed with 1070 µl of PBS and 120 µl of 10 mg / ml DEAE-dextran solution (transfection solution) was added. Prior to transfection, 40 ml of cells were centrifuged, taken up in 10 ml of PBS and centrifuged again.

The cell pellet was resuspended in 1.2 ml PBS-DEAE-dextran solution and incubated for 30 minutes. Cells were then disrupted by addition of 0.8 mL of cold DMSO solution (25% concentration in TBS). After 3 minutes, TBS (10 mL) was added and centrifuged. The cells were washed in RPMI growth medium and resuspended in 40 ml of the above medium.

Fusion 3 (Sanari-Goldin method)

Logarithmically growing cells (1x10 ⁷ cells / ml) were centrifuged and washed once with RPMI medium.

To the cell pellet was added 2 ml of E. coli protoplast suspension (2x10 ³ cells / ml in 10% sucrose / 10 mM magnesium chloride). The protoplast, together with the plasmid to be transfected, was prepared according to the method of Sanari-Goldin et al. After 8 minutes incubation, centrifuge and dilute carefully with PEG solution (polyethylene glycol 400; 45% concentration in RPMI) for 2 minutes. After incubation for 1 minute, 10 ml of FCS-free RPMI medium is added over 7 minutes. The cells were centrifuged, washed with RPMI medium and resuspended in 10 ml medium.

4. Electroporation

Electroporation (EP) is performed on a Gene Pulser Apparatus and Capacitance Extender (Bio Rad). Logarithmically growing cells were adjusted to a cell density of 1x10 ⁷ cells / ml, washed once in PBS and resuspended in FCS-free RPMI medium supplemented with 10 mM dextrose and 0.1 mM dithiothreitol.

0.4 ml of cell suspension was transferred to EP cuvette and 10-20 pg DNA added. DNA is dissolved in 1 µl TE per mg.

The conditions of electroporation are modified as follows:

Capacity: 260-960 pF;

Voltage: 150-300 V;

Electrode distance: 0.2 / 0.4 cm;

Resistance: 100- = ° Ohm.

Before and after electroporation, the cells were incubated for 10 minutes at room temperature and then taken up in 10 ml of growth medium and 10% FCS.

Selection of transfectants:

Transfectants are selected for G 418 resistance in all cases:

1. Selection in a viscous medium

The transfected cells are first incubated in growth medium containing G 418 until further growth can be detected. It is then spread in a viscous medium. Agar (Gibco) or methylcellulose is used to solidify the medium. The pre-selected cells are plated on tissue culture plates (10 cm), to which 10 ml of selection medium (RPMI + 1 mg / ml G 418) and 2 ml of 2% liquid agar solution are added. After cooling the agar, incubate at 37 ° C in a hatcher. In one version of the experiment, methyl cellulose is used instead of agar.

2. Selection by subsequent cloning in Transwell cell culture chamber (Transwell TM, Costar)

48 hours after transfection, cells are transferred to a 96-well titration plate by adding 1 ^x 10 ⁵ cells to 0.2 ml wells. G 418 is added at a concentration of 0.7 mg / ml for U 937 and at a concentration of 1.0 mg / ml for COLD 78. New media amounting to half the volume of the wells was added after the use of the original medium, and for U 937 an additional 0.5 mg / ml G 418 was added.

Incubation is performed until resistant cells develop at each well. This mixed clone is expanded to a larger volume and then

HU 214 684 Β. Costar's Special Growth Chamber (Transwell TM, Cat. No. 3425, Cambridge, Massachusetts, USA) is used.

Cells are transferred to soft agar containing G 418 (see below, Selection in Viscous Medium) at low cell density (50-200 cells / 6 ml) and added to a central compartment (Upper compartment). The chamber is separated by a membrane from the rapidly growing liquid culture (200,000 cells / ml) of U 937 and HEAT 78 (Lower compartment). After use, the liquid medium is partially changed to ensure further cell growth. When G 418 resistant colonies appear on soft agar medium, they are isolated with a Pasteur pipette and expanded.

In this way, approximately ten independent U 937 and HUT 78 transfected cell lines were each cloned with the KTX 11, KTX 12, KTX 15, pSXK 1 and pSXK 2 vectors and further processed to demonstrate antisense RNA expression and HIV-1 resistance. In addition, the pHTT1, pHTT4, pHTT9 and pHTT10 vector generates a COLD 78 cell line and assays for HTV infection.

Example 8

Transformation of bone marrow cells with KTX15 expression vector

Basic experiments demonstrating the transformation of primary bone marrow cells were performed in a mouse model as follows:

Primary bone marrow cells were removed from the upper femur of 6 to 10 week old C57 mice. Cells were harvested in McCoy's medium (Flow) at 2.5 µg / ml with interleukin-3 and GM-CSF protein (granulocytamonocyte colony stimulating factor; Genzyme) and with a final concentration of 0.3% agar (Bacto Agar, Difco). and mixed at a concentration of 1 x 10 ⁵ cells / ml in the presence or absence of the toxic antibiotic G 418 (neomycin) in Petric. Transfection was performed immediately after bone marrow cell removal before loading onto agar. Cells were then cultured for several days in a 37 ° C incubator in the presence of 7.5% CO ² . The number of transformed colonies (at least 50 cells) and nodes (10-50 cells) were determined under a microscope at different times. Under the conditions described, most colonies were mixed granulocyte / monocyte or granulocyte colony, with rare erythroid eruptions and once megakaryocytes. Cell types were identified by May-Grünewald staining within the colony.

The transfection conditions are optimized. Effective transfection of primary bone marrow cells can be performed in the following mixture:

800 μΐ bone marrow cell suspension (5 × 10 ⁶ cells in electroporation medium of Example 7), μg KTX 15, electroporation: 300 V, 100 Ω, 250 FD, 6 seconds

After electroporation (Gene Pulser Apparatus and Capacitance Extender, Bio Rad), cells were selected in the above medium in the presence of 500 µg / ml G 418 (neomycin). Resistance G 418 exhibits effective transfection on proliferating primary bone marrow cells.

Example 9

Demonstration of integration of intact expression vector structure and expression of antisense RNA

After digestion of U 937 and HUT 78 cells transfected by density gradient centrifugation of cesium chloride, DNA and RNA from the cell line were recovered by standard method [L. G. Davis et al., Basic Methods Molecular Biology, Elsevier, New York (1986). Integration of the intact vector structure required for expression of antisense RNA was confirmed by polymerase chain reaction (POR Technology Principles and Application tor DNA Amplification, ed. Henry A. Erlich, M. Stockton Press, New York, USA, 1989) using an oligonucleotide primer and overlapping DNA fragments of the promoter sequence, the insert sequence and the termination sequence are used. The processed DNA is then identified by Southern blot analysis (Davis et al., Cited above) by inserting a DNA gene probe. As an example, the analysis data shown in Figures 7, 8 and 9 is given. For example, KTX 11, KTX 12 and KTX 15 are derived from the promoter sequence

5'-CAA ACC CTT TGC GCC CG-3 'or

5'-ACT CGT CCA ACG ACT AT-3 'Primer and derived from Polyadenylation Sequence

5'-TTT TTT CAC TGC ATT CTA CTT-3 'sequence was used. Expression vectors containing the pRc-CMV vector include, for example, the ₅ ' -Ctt TCC AAA ATG TCG TAA CAA CTCC-3 '

5, .AT TAG GTG ACA CTA TAG AAT-3 'is used. In the insert (HIV sequence), a primary sequence is selected which results in a PCR product of about 500-2100 bp depending on the primer pair.

Expression of antisense RNA was performed by Northern blot analysis (Davis et al., Cited above) on the RNA of the cloned transfected cell line. Analysis of HIV-1 antisense RNA in a transfected U 937 cell line is shown in Figure 10.

Example 10

Demonstration of virus resistance in transfected cloned hematopoietic cell lines

An infection study comparing cloned antisense RNA expressing cell lines produced and characterized according to Examples 7 and 9 with HIV-1 virus [L. Ratner et al., Natura, 313, 277 (1985)] or HTV-2 with Dl94 virus [H. Kühnel et al., Nucleic Acids Research 18: 6142 (1990)] as a control using the starting CELL 78 and U 937 cell lines.

1. Infection of COLD 78 and U 938 cells with HIV-1 virus (control)

10-10 ml of cell suspension is heated for 60 minutes

Mix various dilutions with the supernatant of COLD 78 / HIV-1 culture (about 10 ⁴ SFU / ml in PBL culture, frozen stock solution RT activity 650,000 cpm / ml). The cells are then centrifuged, washed three times to remove unbound virus particles and proteins. Finally, the cells were resuspended in 10-10 ml of culture medium and cultured in a hatcher. Every 3-4. day, 200 μΐ cell-free supernatant was assayed for HIV antigens. AntigenCapture-Assay (Organon Technique) is used. To supply the cells, 5-5 ml of cell suspension was removed and replaced with 5 ml of culture medium.

2. Infection of CELL 78 and U 937 cells expressing antisense RNA with HIV-1 virus

10-10 ml of cell suspension (approximately 4 x 10 ⁵ cells / ml) was mixed with the diluted supernatant of COLD 78 / HIV-1 culture (approximately 10 ⁴ SFU / ml PBL, frozen stock solution RT activity 650,000 cpm / ml) for 60 minutes. , final dilution of virus stock solution 1: 1000 for COLD 78 and 1:50 for U 937. The cells are then centrifuged and washed gently four times to remove unbound virus particles and proteins. Finally, the cells were resuspended in 10-10 ml of culture medium (RPMI 1640 + 16% FCS) and cultured in a hatcher for 60 days. 160 μΐ cell-free supernatants were assayed every 3-4 days for HIV antigens. For this, Antigen-Capture-Assay (Organon Technique) was used together with the accompanying microtiter plate photometer.

For cell supply, 5 ml of cell suspension is removed every 3-4 days and replaced with 5 ml of fresh culture medium.

Representative of other HIV antisense RNA expressing cell lines with three CUT 78 transfectants (CUT K14-KTX11, SSHUTK16 / 2-SXK1, SSHUTK1 / 1-SXK2) (see figure below) and three U 937 transfectants (ssUK 20/4-KTXlla). , ssUK 3/1-SXK2 and ssUK 20/15-KTX1 lb) (see Fig. 12), which express antisense RNA expressing HVV proliferation pSXKJ and pSXK2 expression vectors on the basis of 60 days of incubation. cell lines show no evidence of reverse transcriptase activity (RT activity) or HIV antigen. In contrast, HIV proliferation in control cell lines is clearly measurable. In addition, there is a delayed increase in viral replication in transfectants containing the KTX 11 expression vector, which provides poor expression. The results are shown in Figures 11 and 12.

For example, SSHUT K16 / 2- incubated for 60 days incubated for 60 days incubation to check for successful HIV-1 infection of SSV K3 / 1-SXK2, SSHUT K16 / 2-SXK2 and SSHUT K1 / 1-SXK2 cell lines lacking HIV antigen and RT activity. Positive infection confirmed by HIV-1 TAT and envelop gene sequences detected by PCR in SXKI and SSHUT K1 / 1-SXK2 cells (Figure 13). In doing so, the

5'-ATG GAG CCA GTA GAT CCT-3 'and

5'-TCT ACC ATG TCAT-3 '

PCR primer generates 690 bp PCR fragments. The expression vectors pSXKI and pSXK2 do not contain these sequences and therefore cannot produce a false positive PCR product.

Example 11

Control experiments

If the U 937 and HUT 78 cell lines are any one of 1-6. transfected with an example vector which does not contain the antisense orientation insert of proviral DNA from HIV-1, no cell line in which viral replication is inhibited is obtained. However, no control experiment was performed in which the results of Figs. A vector containing the proviral HIV-1 DNA fragment in the sense orientation is used as the corresponding sense transcripts bind to HIV replication regulatory proteins, such as the TAT and RÉV proteins, and thus competitively inhibit viral replication [G. J. Graham et al., PNAS 87, 5817 (1990)], that is, not applicable as a control experiment.

Example 12

Infection of HIV-2 with HTJT 78 and U 937 expressing antisense RNA

An infection experiment was performed with HIV-2 Dl94 isolate [H. Kühnel et al., Nucleic Acids Slit. 18: 6142 (1990)]. The ^χ 2.14 10 ⁵ cpm / ml of RT activity and (1: 1000 dilution) OD 0.249 concentration (160 μΐ pattern) showing virus strain suspension was stored at -80 ° C for an antigen _{of 450} nm. 50 μΐ of this virus strain suspension was used to infect 2 ^× 10 ⁶ COLD 78 cells in 4 ml of culture medium and incubated for 3 hours. Incubated for 24 hours with 2 ^χ 10 ⁶ cell density of 4 ml of culture medium of U-937 cells 100 μΐ vírustöizs suspension. Unbound virus particles are then removed by washing the cell culture.

The culture medium is replenished every 3-4 days and the supernatant is sampled for quantitative determination of HIV antigen by HIV antigen-Capture-Assay (Organon Technique) using a dedicated microtiter plate photometer. The progression of HIV-2 D194 proliferation in transfected cell lines showing HIV-2 inhibition due to antisense RNA expression was compared to the HÜT 78 and U 937 control cell lines without the antisense RNA expression vector.

15.

Results: Expression vectors KTX 11, KTX 12, KTX 15, pSXK 1, pSXK 2, pHTT 1, pHTT 4, pHTT 6, pHTT 9, pHTT 10, pHTT 12 and pHTT 15 in human blood cell lines and after cloning of the transfected cell line (Example 7), antisense RNA (Example 9) is produced endogenously to HIV-1 virus (Example 10-12).

It is surprising that transfected cell lines show significantly enhanced inhibition of retroviral HIV-1 replication. Virus resistance of cells expressing antisense RNA a

It can also be detected in the same way as the HIV-1 virus adapted to the transfected cell line (Figures 11 and 12). The antisense RNA also shows significant anti-viral activity against HIV-2 virus (Figures 14, 15).

These results demonstrate, by way of example, that promoters capable of expressing antisense RNA molecules and, of course, the type of antisense RNA sequence in genetically altered cells lead to the development of resistance to retroviral infection. Example 9 confirms that cleavage of previously unknown RNA transcripts of HTV-1 RNA transcripts of the proviral HIV-1 positive strand results in several mRNA molecules in the 3 '-> 5' orientation which lead to antiviral activity.

1-12. The experimental work described in Examples 1 to 5 demonstrates that an appropriate expression vector construct containing an antisense RNA encoding a retroviral DNA fragment develops anti-retroviral properties in the cell, which protects against retroviral infection.

11-13. Figure 5A shows that the SSHUT K16 / 2-SXK1 and SSHUT K1 / 1-SXK2 cell lines give positive results for the detection of proviral HIV DNA by PCR analysis 60 days after HIV infection, but with negative results for HIV antigen detection. These results suggest that after infection of cells with HIV-1, in the first step of the HTV replication cycle, proviral DNA is formed in the transfected HTC 78 cell line, but transcription and translation of proviral DNA into antisense RNA is inhibited and the HV required for viral replication. no detectable amount of protein is formed.

In the DNA of the SSU K3 / 1-SXK2 cell line, 60 days after HIV-1 infection, no HIV-1 protein or proviral HIV DNA was formed in any of the tested mixtures using either antigen test or PCR analysis. These results imply that the antisense RNA produced in the SSU K3 / 1-SXK2 cell line inhibits not only the second step of the HIV replication cycle (transcription and translation of proviral DNA), but also the first step of the replication cycle.

Thus, it is obvious that antisense RNA can be used to prevent proviral DNA from reverse transcription of the extra strand of genomic RNA. The above effect of antisense RNA was speculatively inferred, but so far it has not been demonstrated [R. Y. L. To and P. E. Neiman, Gene Regulation: Biology of Antisense RNA and DNA, R. P. Erickson and J. G. Izant, Raven Press Ltd., New York, USA (1992)], because expression vector constructs with sufficient performance have not been found.

Legend:

Figure 1

KTX 11 expression vector suitable for expression of HIV-1 antisense RNA. Metallothionein promoter (MTIpromoter) promotes transcription of antisense RNA from 5786 to > 474 nucleotides of a proviral HIV-1 DNA fragment (numbered: UWGCG geneEMBL DATA BANK FILE

HXB2CG.VIRAL). Antisense RNA transcripts are down-regulated by SV 40 polyadenylation sequence. The TRN5neo gene conferring neomycin resistance is transcribed by the SV 40 early promoter. The commuter vector contains the pBR322 origin and the ampicillin gene (pBR322-amp). Further details are given in Example 1.

Figure 2

KTX12 expression vector suitable for expression of HIV-1 antisense RNA, regulated by HIV-1 LTR Metallothionein hybrid promoter. Details of the construction of the vector are given in Example 2.

Figure 3

KTX 15 expression vector suitable for expression of HTV-1 antisense RNA, regulated by HTV-1 LTR Metallothionein hybrid promoter, in which the HTV-1 TAR sequence is deleted in order to obtain maximum expression in the absence of the TAT transactivator protein. Details of the construction of the vector are given in Example 3.

Figure 4

An expression vector pSXK1 suitable for expression of HIV-1 antisense RNA under the control of the cytomegalovirus promoter (CMV). Details of the construction of the vector are given in Example 4.

Figure 5

An expression vector pSXK2 for expression of HIV-1 antisense RNA under the control of the cytomegalovirus promoter (CMV). The proviral HIV-1 DNA fragment from the middle genomic region of the HVV does not contain the amino acids 1-4 in this example. 5 ', but instead the sequence corresponding to the POL / VIF region. Details of the construction of the vector are given in Example 5.

Figure 6

Flowchart for the production of HTV-1 gene fragments which occur in the antisense RNA expression vectors in the 3 '-> 5' orientation (as indicated by the arrow) as indicated in column 1. The promoter of the expression vector is given in column 2, and the nucleotide numbering of the HTV sequence is shown in column 3 of the UWGCG gene EMBL DATA BANK: HIVHXB2CG.VIRAL.

Figure 7

Southern blot analysis of chromosomal DNA from cloned U 937 transfectant after PCR containing the pSXK1 expression vector incorporated into the chromosomal DNA. The fragment size of the PCR products and hybridization with the HTV gene probe which does not contain the primary sequence of the PCR product confirm the integrity of the promoter / insert sequence.

Control mixture # 1 vector, 5'-transition, 887 bp fragment;

Control mixture vector 2, 3 'transition, 775 bp fragment;

Chromosomal DNA # 3, 5'-transition, 887 bp fragment;

Chromosomal DNA # 4, 3'-transition, 775 bp fragment.

Further details are given in Example 9

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Figure 8

Detection of HIV-1 gene fragment in chromosomal DNA from cloned LUT 78 transfectants by PCR. The PCR reaction was resolved on an agarose TAE gel and photographed under UV light after staining with ethidium bromide. The PCR product is 561 bp in size and corresponds to the HIV-1 gene fragment (699-1305 nucleotides) that is also present in the pSXK1 vector. The 561 bp PCR product is indicated by an arrow.

M: standard DNA length, 516 bp and 1018 bp, respectively;

1: negative control, no DNA added to the PCR reaction;

2: chromosomal DNA from U 937 (wild type);

3: chromosomal DNA from COLD 78 cells (wild type); 4: negative control, no DNA added to the PCR reaction;

5: positive control;

6: chromosomal DNA from COLD 78 transformants (SSHUT 6 / 1-107-9 / 1)

7: chromosomal DNA from COLD 78 transformants (SSHUT 6 / 1-107-9 / 1)

8: chromosomal DNA from COLD 78 transformants (SSHUT 6 / 1-107-9 / 1)

9: chromosomal DNA from COLD 78 transformants (SSHUT 6 / 1-107-9 / 1)

Figure 9

Detection of HIV-1 gene fragment (nucleotides 4158-5,792) in chromosomal DNA from U 937 monocyte and HUT 78 T-lymphocyte cells by PCR. Cells were pre-transfected with pSXK2 vector. The PCR reaction was separated on an agarose TAE gel and photographed in ETV light after staining with ethidium bromide. The PCR product covers the entire HIV-1 gene fragment (nucleotides 4158-5792) in the pSXK2 vector, 1811 bp in length. The 1811 bp PCR product is indicated by an arrow.

M: DNA length standard, 1635 bp and 2036 bp separately;

1: negative control, no DNA added to the PCR reaction;

2: chromosomal DNA from U 937 (wild type);

3: chromosomal DNA from COLD 78 cells (wild type); 4: Chromosomal DNA from WT 78 Transformant (SSHUT 2 / 1-107-12 / 1)

5: Chromosomal DNA from the U 937 transformant (SSHUT 2 / 1-107-12 / 1)

Figure 10

Northern blot analysis to detect multiple antisense RNA transcripts from a transfected, cloned U 937 cell line containing the KTX11 expression vector in the SSUK 20/15-KTX11b cell line. At least three antisense RNA transcripts of 800 bp, 1200 bp and 3200 bp in the RNA of the viral resistant cell line (field 1) can be detected by the gene probe corresponding to the HIV-1 insert DNA of the K.TX11 vector. Field 2 contains RNA from an untransfected U 937 cell line (negative control), Field 3 contains an RNA molecular weight standard.

Figure 11

The process of HTV-1 antigen formation was performed for 60 days following infection of the CELL 78 cell line with HTV-Ι LAV / BRU isolate (day 0). The ordinate shows the optical density of the cell culture supernatant at 450 nm (OD 450 nm). For optical densities greater than 2.0, the supernatant is diluted and the OD values calculated are calculated from the dilution and the cut-off value represents the limit of detection of the HIV antigen.

In the SSHUT K16 / 2-SXK1 and SSHUT K1 / 1-SXK2 cell lines expressing antisense RNA, HTV replication is completely inhibited within 60 days.

In the CELL K14-KTX11 cell line, HIV replication is inhibited relative to the non-transfected wild-type CELL 78 cell line.

Figure 12

HTV-1 antigen formation process 60 days after infection of U 937 cell line with HTV-1 LAV / BRU isolate (day 0). The ordinate shows the optical density of the cell culture supernatant at 450 nm (OD 450 nm). For optical densities greater than 2.0, the supernatant is diluted and the OD value is calculated from the dilution. The cut-off value represents the limit of detection of the HIV antigen assay.

In the SSUK3 / 1-SXK2 cell line expressing antisense RNA, HTV replication is completely inhibited for 60 days, and in the other transfected cell line, inhibition of HIV replication is observed compared to the wild-type cell line (U 937).

Figure 13

PCR analysis for detecting a cell line containing HIV-1 DNA antisense RNA expression vector at day 60 post-HIV infection. Ethidium bromide-stained electrophoresis agarose gel shows PRC products derived from chromosomal DNA corresponding to a given cell line.

The 5'-ATG GAG CCA GTA GAT CCT-3 'and

5-TCT GT TCT ACC ATG TCAT-3 'primer pair was used to treat a 702 bp PCR fragment from the TAT-ENV gene region in the HIV infected cell line. For comparison of PCR results, the results of the HIV antigen test of the corresponding cell culture samples are given in brackets.

M: standard molecular weight, 600 bp indicated;

1: COLD K14-KTX11 720 bp positive (antigen test positive);

2: SSHUT K16 / 2-SXK1 702 bp positive (antigen test negative);

3: A parallel infection attempt of 2

Its PCR product;

4: A parallel infection attempt of 2

Its PCR product;

5: SSHUT K1 / 1-SXK2702 bp positive (antigen test negative);

6: A parallel infection assay of 5

Its PCR product;

7: A parallel infection assay of 5;

8: COLD 78, positive control;

9-11: SSUK3 / 1-SXK2 culture, 702 bp PCR product not detectable (antigen test negative).

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Figure 14

The course of HIV antigen formation in HIV-2 Dl94 isolate (day 0) in HUT 78 cell lines for 30 days. The ordinate shows the optical density of the cell culture supernatant at 450 nm (OD 450 nm). At 2.0, the supernatant is diluted at optical density and the OD value given is calculated by taking into account the dilution. The cut-off value represents the limit of detection of the HIV antigen assay.

In the SSHUT K1 / 1-SXK2 cell line expressing the antisense RNA, HIV replication is completely inhibited for 14 days, while the other transfected cell line shows impaired HIV replication compared to the wild-type cell line (HEAT 78).

Figure 75

The course of HIV antigen formation in HIV-2 Dl94 isolate (day 0) in the U 937 cell line for 30 days. The ordinate shows the optical density of the cell culture supernatant at 450 nm (OD 450 nm). For optical densities greater than 2.0, the supernatant is diluted and the OD value calculated is calculated taking into account the dilution. The out-off value represents the limit of detection of the HIV antigen assay.

In the SSHUT K3 / 1-SXK2 cell line expressing antisense RNA, HIV replication is completely inhibited for 30 days, while the other transfected cell line shows impaired HIV replication compared to the wild-type cell line (U 937).

Claims (7)

PATENT CLAIMS A method for producing an expression vector capable of expressing antisense RNA, comprising operably inserting a promoter sequence and a proviral DNA of HTV-1 into an appropriate expression vector in the antisense orientation 7514 -> 474, 5786 -> 474, 2096 -> 4725, 3825 -> 681, 5786 -> 4158, 5746 -> 4648, 2369 -> 1635, 3226> 3003, 2099 -> 678, 3829 -> 2099, 4647 -> 4157, 4157 -> 3830, 605 -> 455, 670 - > 440, 825-> 535, 6070-> 5800, 5640-> 5020, 5600-> 5040, 8690> 8300 or 9160-> 8800. 2. The method of claim 1, wherein said HIV-1 proviral DNA is in the antisense orientation of 7514 to 474, 5786 to 474, 2096 to 474, 3825 to 681, 5786 to 4158, 5746 to > 4648, 2369> 1635, 3226-> 3003, 2099-> 678, 3829-> 2099, 4647-> 4157 or 4157-> 3830 nucleotides. The method of claim 1 or 2, wherein the promoter is a hybrid promoter. 4. The method of claim 1, wherein the expression vector is pSXK1. 5. The method of claim 1, wherein the expression vector is pSXK2. 6. A method for producing transfected cells, characterized in that the cells are known in the art 1-5. Expression vector prepared according to any one of claims 1 to 6. 7. A process for the preparation of a medicament comprising the steps of 1-5. The expression vector produced according to any one of claims 1 to 6, in admixture with a pharmaceutical carrier and optionally other pharmaceutical excipients, and converting the mixture into a pharmaceutical composition.