PL175143B1 - Expression vectors and their application for generation of human cells resistant against hiv for therapeutic purposes - Google Patents

Abstract

1 . Constructs vector expressive down expression antisense RNA, significant this that contain and) sequences strong horse constitutive promoter chosen one out of promoter metallothioneins and promoter CMV or sequences hybrid promoter covering replaced strong promoters or one with listed strong promo tracks and promoter HIV-1 LTR and b) vortex Owls GOUT in orientation antisense.

Description

The invention relates to expression vector constructs for the expression of antisense RNA.

The expression vectors of the invention serve to generate HIV resistant human cells for therapeutic purposes. The invention relates to the targeted blocking of the genetic information and replication of a virus (e.g. HIV) in viral messenger ribonucleic acid (mRNA) in transfected hematopoietic cells with the aid of special expression systems that induce the expression of complementary antisense RNA.

In particular, the invention relates to expression vectors encoding antisense RNA sequences which, unlike the prior art, completely inhibit HIV-1 replication and markedly inhibit HIV-2 replication in genetically engineered human cells. The genetic units of the expression vectors described herein are especially intended for somatic gene therapy of HIV / AIDS disorders as they protect hematopoietic cells from HIV induced cytopathic effects. Thus, immune failure in HIV-infected individuals can be avoided.

Reproduction of the virus in an infected cell is controlled by genes of the viral genome. In this process, products are made from viral genes through transcription and translation

Regulatory and structural proteins to build infectious viral particles for virus multiplication. An important intermediary in this process is the messenger ribonucleic acid (mRNA), which transmits information about the structure of regulatory and structural proteins.

Blocking of messenger ribonucleic acid in a highly specific manner which relies entirely on the specificity of the molecular structure of the viral mRNA, namely the virus specific nucleobase sequence of this molecule, can be achieved with ribonucleic acid with a complementary base sequence. By sequence-specific nucleobase pairing as described for deoxyribonucleic acid, the primary single-stranded mRNA becomes complexed into double-stranded RNA and the genetic information contained in the nucleobase sequence becomes partially unavailable for protein biosynthesis. The second step in gene expression, translation, is thus inhibited. Complementary ribonucleic acid is referred to as antisense RNA.

The mechanism by which gene expression is inhibited by antisense RNA interacting with target mRNA is complex and largely unclear. One explanation is the inhibition of RNA cleavage and splicing and RNA processing in the cell nucleus, but exact studies are not known. An adenosine-inosine exchange in double-stranded RNA is described and translation inhibition is suggested to be a consequence of this exchange. Inhibition of RNA transport across the nuclear membrane to the protein biosynthesis site and masking the interaction of ribosomes and RNA by double-stranded RNA may be a further cause of antisense RNA inhibition of gene expression.

Inhibition of metabolic processes by antisense RNA in naturally occurring biological systems such as bacteria and some eukaryotic multicellular organisms has been described (KM Takayamai M. Inouye, Critical Reviews in Biochemistry and Molecular B iology 25,155 (1990), as well as in artificially derived organisms by gene transfer. transgenics (J. Mol. et al. FEBS Letters 268 (1990)).

Attempts to generate virus-resistant plant organisms (M.Cuozzo et al., Biotechnology 6,549 (1988) and animal organisms (T. Riiden and E. Golboa, J. Virol. 63, 677 (1989)) have also been reported after insertion of the gene encoding However, as the authors of these publications themselves state, there are no convincing, scientifically established relationships between antisense RNA expression and viral resistance, particularly in the case of plasmids introduced by microinjection, conditioning the expression of transiently expressed antisense RNA, antiviral activity is weak or undetectable (K. Rittner et al., Nucleic Acids Research 19, 1421 (1991)). The use of retroviral vectors conditioning antisense RNA expression to produce virus-resistant cells has also been reported (R. YL. , To et al. In: Gene Regulation: Biology of antisense RNA and DNA, RP Ericskon and JG Izant, Raben Press Ltd., New York 1992) These retroviral vectors carry the risk of inducing malignant tumor growth as they contain oncogen-like gene sequences and there is ample evidence of retrovirus-dependent oncogenesis.

Contrary to published work on antisense RNA in the regulation of viral replication, the present invention describes expression constructs that exhibit a particular type of promoter design in combination with specially selected regions of the viral DNA in antisense orientation, and thus exhibit antiviral activities that qualitatively and quantitatively are surprising and are well beyond the heretofore known range. The combination of the expression vector components: 5 'DNA flanking sequence, promoter, insert / antisense DNA and 3' processing signal sequences for the first time in retroviral infections gave reliable and clearly expressed resistance to virus in transfected cells expressing antisense RNA. In contrast, in the prior art, weak, only slight, transient antiviral effects are associated with other vector systems for endogenous expression of antisense RNA (e.g. T. Riiden and E. Gilboa, J. Virol., 63, 677 (1989)), EP 252 940, G. Sczakiel et al., Biochem. Biophys. Res. Com. 169, 643 (1990), O.i. Mirosknichenko et al., Gene 84, 83 (1989), T. Shimada et al., Antiviral Chemistry and Chemotherapy 2, (1991), G. Sczakiel et al. J. Virol. 66, 5576 (1992) or included above

1715 143 of these risks (retroviral gene transfer), or are impractical because they require microinjection.

The invention relates to expression vector constructs for the expression of antisense DNA, which contain a) a strong constitutive promoter sequences selected from the metallothionein promoter and the CMV promoter, or hybrid promoter sequences comprising the said strong promoters or one of the strong promoters mentioned and the HIV-1 LTR promoter and b) viral DNA in antisense orientation.

They are preferably such expression vector constructs which contain pathogenic virus subgenomic gene sequences in the 3 '-> 5' orientation (antisense orientation) under the control of an antisense RNA expression promoter whose nucleobase sequence is complementary to pathogenic virus mRNA.

Constructs that contain proviral subgenomic DNA in antisense orientation from retroviruses such as HIV-1 or HIV-2, oncoviruses such as HTLV-I, HTLV-II, or oncogenes such as abl, erb A, erb B are very preferred. , fms, fos, myb, wash, ras, sis, src.

Expression vector constructs that contain promoter or hybrid promoter sequences and HIV-1 proviral DNA in the antisense orientation of nucleotides 7514 474, or 5786 -> 474, or 2096 474, or 3925 681, or 5786 -> 4158, or 5746 4648, are preferred. , or 2369 -> 1635, or 3227 3003, or 2099 -> 678, or 3829 2099, or 4647 -> ^ 1 ^ 7, or 4157 3830.

Expression vector constructs that contain subgenic proviral gene sequences or oncogenic sequences in the 3 '-> 5' orientation are also preferred for the expression of shorter antisense RNA transcripts.

Also preferred are expression vector constructs which contain HIV-1 proviral gene sequences, nucleotides 605 155, or 670 -> 440, or 825 535, or 6070 -> 5800, or 5640 -> 5020, or 5600 -> 5040, or 8690 -> 8300 or 9160 8800.

Expression vector constructs that contain two, three or more subgenomic sequences from one virus or from several viruses are preferred.

Cells can be transfected with a construct of the invention. The expression vector constructs are also suitable for the production of medicaments in which the active ingredient is one or more constructs of the invention.

Using the expression vector constructs described herein in appropriate human host cells, an antiviral activity against HIV-1 replication was demonstrated, which provided complete protection against HIV-1 replication in human cells for more than 60 days and significantly inhibited HIV-2 replication. .

The expression vector constructs of the invention produce in the transfected cells as a messenger transcript ribonucleic acid that is complementary to the mRNA sequence of unwanted viral genes, for example the gAg, POL, VIF, REV, TAT and VPU genes of the human immunodeficiency virus, HIV-1 (WA Haseltine et al. Scientific American USA, 1988, 34 to 42). In addition, the described antisense RNA sequence is sufficiently homologous to HIV-2 (e.g., HIV-2 isolate D194, in Human retroviruses and AIDS, Myers et al., Los Alamos National Lab., 1992, and EMBL Deposit No. X52223) in range of the GAG, POL and VIF genes to exert antiviral activity also on HIV-2 isolates. The expression vector constructs according to the invention are distinguished by a particular arrangement of selected promoter sequences and hybrid promoter sequences (e.g. CMV-IE / metallothionein I), with HIV-1 subgenic DNA fragments in antisense (3 '- 5') orientation, resulting in of several transcripts, antisense RNA, by constitutive or induced cleavage and RNA splicing. The antisense RNA transcripts so formed show surprisingly drastic or complete inhibition of HIV-1 replication in human cells (HUT 78, U 937, for example). There was also a clear inhibition of HIV-2 replication. HIV-2 exhibits sufficient RNA sequence homology in the expression vector constructs described herein such that antisense RNA inhibits viral replication. Thus, using the constructs according to the invention, the replication of retroviruses, e.g. HIV-1 and HIV-2, is inhibited, thus protecting the hematopoietic cells - and therefore important cells of the human cellular immune response (e.g. T cells,

175 143 monocytes) - against the harmful and cell-damaging multiplication of the virus, by a specific expression of antiviral antisense RNA.

The following experimental procedure was performed to render immune cells resistant to the damaging proliferative effects of retroviruses known to infect HIV-1 and HIV-2 by expression of antiviral antisense RNA.

Promoters:

Expression vectors were constructed containing promoters which should provide constitutive and efficient expression in hematopoietic cells, such as the cytomegalovirus IE promoter (CMVEE). In addition, promoters were also constructed by fusing nucleic acid sequences, which should additionally induce amplified expression in the case of viral invasion. In particular, a component of these hybrid promoters is the CMV-IE-metallothionein promoter fragment and the HTV-1 LTR nucleic acid fragment from nucleotides 289 to 474 or 289 to 532 (nucleotide numbering: UWGCG GENEMBL Data Bank, File: HIVHXB2CG. VIRAL September 25, 1987), which contributes to the amplified, possibly transactivable, additional promoter activity.

Antisense nucleic acid fragments.

Additionally, the vector constructs contain, under the control of the described promoters, subgenomic proviral DNA fragments in antisense orientation, i.e. the (+) strand of DNA is transcribed in the 3 '- 5' direction.

For safety reasons, the complete proviral genomic DNA fragment of the retrovirus is not used. However, longer subgenomic proviral DNA fragments are used rather than short subgenic proviral DNA fragments, mainly because longer RNA transcripts are more likely to be cleaved and spliced and thus more antisense RNA transcripts can be expected from proviral DNA fragments, according to invention, as actually found in the examples mentioned. (The type and number of fully processed antisense mRNA transcripts that can be expected for the HIV sequences used in antisense orientation is unpredictable as there are not enough consensus sequences for mRNA splicing to allow for robust prediction).

As the number of antisense RNA transcripts increases, with traits associated with them, such as nuclear export or cytoplasmic stability, the likelihood of producing an antisense RNA transcript with potent antiviral activity also increases.

The invention relates to expression vector constructs that contain a fused gene fragment that is transcribed into an antisense RNA under the control of a strong promoter. The fused gene fragment consists of subgenomic DNA fragments from two or more different pathogenic viruses, for example HIV-1 and HIV-2. By using such fused gene fragments that encode an RnA transcript containing sequentially several regions of antisense RNA sequence complementary to mRNA sequence regions from different viruses, expression vectors with broader antiviral activity can be constructed.

The validity of this principle is confirmed by Examples I, IV, V, X and XII. For example, the constructs of the expression vector KTX 11, pSXK1 and pSXK2 contain HIV-1 LAV / BRU antisense DNA fragments, which in turn contain shorter stretches that are also complementary to HIV-2 D194 mRNA (e.g. nucleotides 620-660 or 837 -899 or 4868-5044).

Thus, the antisense RNA from KTX11, pSXK1 and pSXK2 is antiviral also against HIV-2.

The antisense RNA coding sequence of these vectors can now be additionally fused with proviral DNA fragments from other viruses in antisense orientation (e.g. HIV-2 isolates, other HIV-1 isolates) such that as a result of segmentally optimized complementarity of the antisense RNA to the sequences of other viruses, better or complete inhibition of different viruses can be achieved simultaneously with one expression vector construct.

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Termination of antisense RNA transcripts is achieved with a polyadenylation signal (e.g. from SV 40 or bovine growth hormone) to generate a highly stable mRNA. Furthermore, the vectors contain a complete operon for the expression of an appropriate selectable marker. In these examples, resistance to the neo TNR antibiotics (neomycin, geneticin, G 418, kanamycin) was used to select cells which, after transfection, contain a genetic construct for expression of antisense DNA.

This selectable marker has proven useful for human hematopoietic cells. Other selectable markers, such as for example puromycin or hygromycin or methotrexate, could also be used in conjunction with the transfection of the gene for dihydrofolate reductase.

Particularly useful as antisense nucleotide sequences are sequences from the POL / VIF region of the HIV gene between nucleotides 2100-5800. Especially the antisense sequences of nucleotides 5786-4158 showed outstanding antiviral activity. Other fragments from this region can also be used, for example 5746-> 4648 or 4647-> 4157, or 5880-> 3880, or 5850-> 4158, or 5150-> 3200.

In order to demonstrate the antiviral activity of the antisense RNA, for example against HIV-1, human cell lines which could be infected with HIV-1 were transfected with the above-mentioned expression vector constructs. The aim was to demonstrate on a wide variety of human hematopoietic cells that endogenous expression of antisense RNA leads to inhibition of viral replication in cells that are specifically involved in the cellular immune response. This destruction of immune cells in the end stage of AIDS is causally linked to the increased incidence of opportunistic infections, which are then often fatal. Therefore, the aim of work with hematopoietic cells and first continuous cell lines, such as HUT 78, MOLT 4, U 937, Jurkat, CEM-CM3 (all according to the American Type Culture Collection ATCC, Bethesda, Maryland, USA) after vector transfection, cloning and In genetic characterization, in infection experiments with the appropriate HTV-1 and HIV-2 isolates, the virus resistance of these transfected cell lines was tested.

The demonstrated expression of antiviral-active antisense RNA in these cells indicates the utility of the expression constructs in hematopoietic cells and ultimately also their use in transforming bone marrow hematopoietic tissue cells for therapeutic purposes. The integration of genes into peripheral blood lymphocytes and bone marrow cells for therapeutic purposes has been described elsewhere (KW Culver et al., Transplant Proc. 23, 170-177 (1991), and should in principle be applicable to the antisense RNA expression constructs mentioned herein. .

Example I. KTX 11 expression vector construct. (All nucleotide numbering below refers to the human immunodeficiency virus type 1 (HXB2) sequence, WUGCG, GENENBLData Bank, file: HTVHXB2CG VIRAL, from September 25, 1987, unless otherwise indicated).

The following expression vector was constructed for the endogenous expression of an antiviral-functional antisense RNA after transfecting cells that could be infected with HTV-1.

The mouse metallothionein I promoter (1600 bp EcoRI Bgl II fragment (N. Glanville et al., Nature 292, 267-269 (1982)) was ligated at the Sal I cleavage site (nucleotide 5786) with the 3 'region of the HIV-1 proviral DNA sequence when ligated to adapter RNF Bgl Ii-Xho I, resulting in modification of the Bgl Π, Xho I and Sal I cleavage sites.

The 5 'region of HIV-1 proviral DNA at the Bgl II cleavage site (nucleotide 474) was ligated to a Bam HI cleavage site to which an SV 40 polyadenylation signal sequence (nucleotides 2564 to 4713 with a sequence deletion from UWGCG GenEMBL file Sv 40xX) was attached. The same polyadenylation sequence from SV 40 was used in the 3 '- 5' direction for TRN5NEO termination (neomycin selection marker (phosphotransacetylase), nucleotides 1-1286 TRN5NEO BACTERIA, UW GCG GenEMBL). Expression of the neomycin resistance gene is initiated by the SV 40 early promoter (nucleotides 5171-371), UWGCG GenEMBL SV 40XX). In another case, the vector also contains pBR 322 DNA (nucleotides 2067-4363, WUGCG, GenEMBL File PBR 322) of bacterial origin and an ampi175 143cillin resistance gene to be used as a shuttle vector for E. coli and animal cells. . The gene map of this KTX 11 vector is shown in Figure 1.

Example II. KTX 12 expression vector construct.

The KTX 12 expression vector of HIV-1 antisense RNA is constructed in the same way as the KTX 11 vector (Example I). However, it contains an inducible hybrid promoter and the strand of the HIV-1 template DNA extends in a 3 '- 5' orientation beyond this hybrid promoter, from nucleotides 7514 - 474.

To obtain a hybrid promoter, the HIV-1 LTR promoter fragment, nucleotides 289 to 532, was fused to nucleotide 368 of the metallothionein promoter (UW GCG File MYSMETI.RO). The restriction map of this KTX 12 vector is shown in Figure 2.

Example III. KTX 15 expression vector construct.

The KTX 15 expression vector of HIV-1 antisense RNA is constructed similar to the KTX 12 vector (Example II). However, the DNA fragment of the HIV-1 template strand (in the 3 '- 5' orientation) is significantly shortened and extends from nucleotide 2096 to nucleotide 474, thus including the GAG sequence, leader sequence, U5 sequence and part of the R sequence. A further important feature of the vector KTX is a deletion of the TAR sequence, with the deletion of nucleotides 474-532 of the HTV-jLTR hybrid promoter. In this way, it should still be possible to activate the hybrid promoter in case of HIV infection, but in the absence of the HIV-1 TAT protein, transcription termination, encoded by the TAR, should not occur (S.Y. Kao et al., Nature 330, 489 (1987)). This hybrid promoter is thus characterized by particularly high constitutive transcription under the control of the HIV-1 LTR promoter sequence. The restriction map of the KTX 15 vector is shown in Figure 3.

Example IV. The PSXK 1 expression vector of HIV-1 antisense RNA was prepared to demonstrate the expression of the antisense RNA under the control of another strong promoter for efficient and constitutive expression, in this example the cytomegalovirus promoter. The HIV-1 gene fragment, nucleotides 681-3825, was inserted in the 3 '- 5' orientation into the Hind III restriction site at nucleotide 891 (pRc / CMV vector, catalog number V 750-20, Invitrogen, San Diego CA 92121, USA) . The polyadenylation signal comes from the bovine growth hormone (BGH) gene.

In another instance, the vector is constructed as a shuttle vector, such as the KTX 11 vector (Example I) with a neomycin selectable marker and a pBR322 DNA fragment for replication and selection in E.coli. The restriction map of the pSXK 1 vector is shown in Figure 4.

Example 5. Construct of the expression vector pSXK 2.

The pSXK 2 expression vector of the HIV-1 antisense RNA differs from the pSXK 1 vector (Example IV) in that it has promoter sequences containing nucleotides 209-865 of the CMV promoter (see Example IV) fused to nucleotides 150-369 of the metallothiotionein promoter (UWgCg GenEMBL file MUSMETI .RO) and is therefore a hybrid promoter. In addition, the proviral DNA sequence of nucleotides 4158-5786 was inserted in the 3 '- 5' orientation as template for antisense RNA expression, under the control of the CMV / metallothionein hybrid promoter. The restriction map of the pSXK 2 vector is shown in Figure 5.

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

The expression vectors pHTT 1,4,6,9,10,12 and the HIV-1 antisense RNA were prepared in a similar manner to the vector pSXK 1 (Example IV, Fig. 4). All HIV-1 gene fragments inserted into pHTT vectors were cloned in the 3 '- 5' orientation into the pRc / CMV vector (Invitrogen, San Diego, CA 92121, catalog number V 750-20, 1991), under the control of the cytomegalovirus promoter necessary for constitutive expression. The pHTT vectors are given below:

(a) pHTT 1: the HIV-1 gene fragment (nucleotides 4648-5746) was inserted into the Not I cleavage site (nucleotide 966) of the pRc / CMV vector.

(b) pHTT 4: the HIV-1 gene fragment (nucleotides 1635-2639) was inserted into the Xba I cleavage site (nucleotide 985) of the pRc / CMV vector.

(c) pHTT 6: the HIV-1 gene fragment (nucleotides 3003-3227) was inserted into the cleavage site

Xba I as described in (b). , (d) pHTT 9: the HIV-1 gene fragment (nucleotides 678-2099) was inserted into an Xba I cleavage site as described under (b).

(E) pHTT 10: the HIV-1 gene fragment (nucleotides 2099-3829) was inserted into an Xba I cleavage site as described under (b).

(f) pHTT 12: the HIV-1 gene fragment (nucleotides 4157-4647) was inserted into an Xba I cleavage site as described under (b).

(g) pHTT 15: the HTV-1 gene fragment (nucleotides 3830-4157) was inserted into an Xba I cleavage site as described under (b).

A general diagram of the HIV-1 gene fragments that are inserted in the 3 '- 5' antisense orientation in the vectors of Examples I to VI is provided in Figure 6.

Example VII. Transfection and cloning of human lymphocytes with vectors for antisense RNA expression.

As examples of transfected human hematopoietic cell lines that show resistance to the virus by endogenous expression of anti-HIV-1 antisense RNA, the suspension growing monocytic cell line U 937 and the same growing T lymphocyte line HUT 78 were selected. Both cell lines were obtained from American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852-1776, USA.

Cell lines were grown in the medium recommended by the ATCC. Vector transfection was carried out in various ways, with the best results being obtained by electroporation:

1. Calcium phosphate method:

Modification of the method of Wigler et al., 1977, Cell 11, 223.

Transfection is performed with DNA calcium precipitates. Various amounts of DNA from 0.05 to 20 gg were dissolved in 0.25 ml in 250 mM CaCl ₂ and slowly added dropwise to 0.25 ml of HEPES X2 buffer. Transfection was obtained by adding the entire precipitate mixture to 10 ml of cell culture. 15 hours later, the substrate was removed prior to aspiration. Cells were shocked with DMSO. 3 ml of 25% DMSO solution in sterile PBS were added per 10 ml of culture. After 4 minutes, the fluid was aspirated and the cells were washed with 5 ml PBS. The cells were then resuspended in 10 ml of DMEM medium.

2. DEAE dextran

The day before transfection, cells were suspended at a density of 3-4 x 10 ⁵ per ml of growth medium. 2-4 hours before transfection, cells were centrifuged and resuspended in a fresh aliquot of medium. 0.5 to 20 g of DNA was mixed with 1070 g of PBD and 120 g of DEAE solution (transfection solution) was added. Before transfection, 40 ml of cell suspension was centrifuged, resuspended in 10 ml PBS and centrifuged again.

The cell pellet was suspended in pBS-DEAE dextran solution and incubated for 30 minutes. The cells were then shocked by adding 0.8 ml of cold DMSO solution (25% TBS). After 3 minutes, 10 ml TBS was added and centrifuged. The cells were washed with RPMI growth medium and resuspended in 40 ml of this medium

3. Fusion (Sanari-Goldin method)

Exponentially growing cells (1 x 10 ⁷ / ml) were centrifuged and washed once with RPMI medium. 2 ml of a suspension of E. coli protoplasts (2 x 10 ³ / ml) in 10% sucrose in 10 mM MgCl ₂ was added to the cell pellet. Protoplasts with transfecting plasmid were prepared according to Sanari-Goldin et al. After 8 minutes of incubation, centrifugation was carried out and a PEG solution (polyethylene glycol 4000, 45% solution in RPMI) was carefully added for 2 minutes. After 1 minute incubation for 7 minutes, 10 ml RPMI medium without FCS was added. The cells were centrifuged, washed with RPMI medium and resuspended in 10 ml.

4. Electroporation

Electroporation (EP) was performed on a Gene Pulser and Capacitance Extender, Bio Rad. Exponential growth cells were adjusted to a density of 1 x 107 / ml, washed once in PBS and resuspended in RPMI medium without FCS with 10 mM dextrose and 0.1 mM dithiothreitol. 0.4 ml of the cell suspension was placed in an EP cuvette and 10-20 Lg of DNA was added. DNA was dissolved to a concentration of 1 gg per 1 g of TE. The electroporation conditions ranged as follows:

Capacity: 260-960 gF; voltage: 150-300 V; electrode distance 0.2 / 0.4 cm; resistance: 100 - °° ohms.

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Before and after electroporation, cells were incubated for 10 minutes at room temperature, then resuspended in 10 ml of growth medium + PCS.

Selection of transfectants:

The selection of transfectants was performed in each case on the basis of the resistance to G 418.

1. Selection on a semi-liquid medium

The transfected cells were first incubated in growth medium containing G 418 until no further growth was detected. Then they were sown on a semi-liquid substrate. Agar (Gibco) or methylcellulose was used to solidify the medium. The pre-selected cells were plated on a tissue culture plate (10 cm) and 10 ml of selection medium (RPMI plus 1 mg / ml G 418) and 2 ml of a 2% liquefied agar solution were added. After the agar had cooled down, it was incubated in an incubator at 37 ° C. In a variant of this experiment, the agar was replaced with methyl cellulose.

2. Selection and cloning in a Costar Transwell TM cell culture chamber. Hours after transfection, cells were plated in a 96-well microtiter plate at a density of 1 x 10 ⁵ cells per well of 0.2 ml. G 418 was added to a concentration of 0.7 mg / ml for U 937 cells and 1.0 mg / ml for HUT 78. As soon as the medium was consumed, 1/2 volume of well and medium were added; in the case of U 937 only 0.5 mg / ml of G 418 was added.

Incubation was continued until resistant cells had grown up in individual wells. These mixed clones were made up to a larger volume and then cloned. For this purpose, special rearing chambers from Costar (Transwell TM, Catalog No. 3425, 205, Broadway, Cambridge, MA 02139, USA) were used.

The cells in the low-density suspension (50 to 200 cells per 6 ml) were plated on soft agar containing G 418 (see slurry selection) and 6 ml was placed in the central chamber (upper compartment). The chamber is separated by a membrane from the very densely growing liquid feed culture of U 937 or HUT 78 (20,000 cells / ml) in the lower compartment. The liquid medium was partially replaced as used up to allow further cell growth. When G 418 resistant colonies appeared in the soft agar, they were isolated with a Pasteur pipette and propagated.

Approximately 10 independent, transfected U 937 and HUT 78 cell lines were cloned each with the vectors KTX 11, KTX 12, KTX 15, pSXK 1 and pSXK 2 and tested further to demonstrate antisense RNA expression and HIV-1 resistance. In addition, HUT 78 cell lines were generated using the vectors pHTT1, pHTT4, pHTT9 and pHTT10 and tested for HIV infection.

Example VIII. Transformation of marrow cells with the expression vector KTX 15.

As an example, the basic procedure for studying transformation of primary bone marrow cells in a mouse model was as follows:

Primary bone marrow cells were obtained from the femurs of C57 mice six to ten weeks of age. Cells were harvested in McCoy's broth (from Flow) and plated in petri dishes after mixing with interleukin 3 and GM-CSF (Granulocyte-monocyte colony stimulating factor, Genzyme) at a concentration of 2.5 gg / ml , and with agar (Difco Bacto Agar, 0.3% final concentration) at a concentration of 1 x 10 ⁵ cells per ml, in the presence or absence of the toxic antibiotic G 418 (neomycin). Transfection was performed immediately after harvesting the marrow cells, prior to plating on agar. The cells were then cultured for several days in a greenhouse with CO2 (7.5%) flow at 37 ° C. At various time intervals, the number of transformed colonies (at least 50 cells) or cell clusters (10 to 50 cells) was counted under the microscope in each case. Under the given experimental conditions, most of the colonies were mixed granulocyte / monocyte or granulocyte colonies, outbreaks of erythroid cells were rarely observed, in one case megakaryocytes were found. The cell types in the colonies were identified by May-Griinewald staining.

The transfection conditions have been optimized. Successful transfection of primary cells marrow was obtained in the following manner: 800 gl suspension of bone marrow cells (5 x 10 ⁶ cells in the fluid to electroporation as described in Example VII) 5 gg KTX15, electroporation at 300 V, 100 Ω, 250 GFD 6 ms .

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After electroporation (Gene Pulser and Capacitance Extender by Bio Rad), cells were selected in the described culture medium in the presence of 500 µg / ml G 418 (neomycin). G-418 resistance indicated successful transfection of propagating primary bone marrow cells.

Example IX. Demonstration of integration of intact expression vector constructs and demonstration of antisense RNA expression.

The transfected U 937 and HUT 78 cell lines were disintegrated and DNA and RNA from these cell lines were isolated by standard methods using cesium chloride density gradient centrifugation (L.G. Davis et al., Basic Methods in Molecular Biology, Elsevier, New York 1986). Evidence for the integration of intact antisense RNA expression vector constructs was performed by the polymerase chain reaction (PCR Technology Principles and Application of DNA Amplification, Henry A. Erlich Ed., M. Stockton Press, New York 1989) using oligonucleotide primers that amplified overlapping DNA fragments of the promoter sequence, insert sequence and termination sequence. Then, the amplified DNA was identified by Southern Blot analysis (Davis et al., Supra) using gene probes for the DNA inserts. Examples of the results of the analysis are shown in Figures 7, 8 and 9. For the vectors KTX 11, KTX 12 and KTX 15, for example, the flanking primers 5'-CAA ACC CTT TGC GCC CG-3 'or 5'-ACT CGT CCA were used. ACG ACT AT-3 'from the promoter sequence and 5'-TTT TTT CAC TGC ATT CTA CTT-3' from the polyadenylation sequence. In the case of expression vectors with the pRc-CMV vector for example, the flanking primer 5'CTT TCC AAA ATG TCG TAA CAA CTCC-3 'for the promoter sequence and 5'-ATT TAG GTG ACA CTA TAG AAT-3' for the termination sequence were used. Primer sequences were selected in the insert (HIV sequence) which, depending on the primer pair selected, resulted in PCR products of approximately 500 to 2100 bp.

Expression of antisense RNA was demonstrated by Northern Blot analysis (Davis et al., Supra) of RNA from cloned, transfected cell lines. An example of the result of the analysis showing HIV-1 antisense RNA in the transfected U 937 cell line is shown in Figure 10.

Example X. Demonstration of virus resistance of transfected cloned hematopoietic cell lines.

Cloned cell lines expressing antisense RNA obtained and characterized according to Examples 7 and 9 were tested in comparative HIV-1 (L. Ratner et al., Nature 313, 277, 1985) or HIV-2 194 (H Kuhnei et al., NucleicAcids Research 1990, 18, 6142) together with the original HUT 78 and U 937 cell lines as controls, for expression of antiviral resistance.

1. Infection of HUT 78 and U 937 with HIV-1 (control)

Execution

Aliquots of 10 ml of cell suspension is treated for 60 minutes with various dilutions of liquid cultures HUT 78 (HUT-1) of about 10 ⁴ PFU / ml in PBL cultures, RT activity of the frozen stock solution 650 000 cpm / ml). The cells were then centrifuged and washed three times to remove unbound virus particles in viral proteins. Finally, in each case, the cells were resuspended in 10 ml of medium and grown in an oven.

Every 30-40 days, 200 µl of cell-free culture fluid was tested for HIV antigens. The antigen capture test by Organon Technika was used.

In order to maintain the cells, 5 ml of the cell suspension were removed each time and replaced with 5 ml of medium.

2. HIV-1 infection of HUT 78 and U 937 cells expressing antisense RNA.

In each case, 10 ml of cell suspension (approximately 4 × 10 ⁵ cells per ml were treated for 60 minutes with diluted HUT 78 / HIV-1 culture fluid (approximately 10 ⁴ PFU / ml in PBL cultures, RT activity of the frozen stock 650,000 cpm / ml); the final dilution of the virus stock solution was 1: 1000 for HUT 78 cells and 1:50 for U 937 cells. The cells were then centrifuged and washed four times carefully to remove unbound virus particles and viral proteins.

Each case was suspended in 10 ml of medium (RPMI 1640 + 16% FCS) and cultured for 60 days in an incubator. Every 3-4 days, 160 µl of cell-free culture fluid was tested for the presence of HIV antigens using the Organon Technika antigen capture assay with an appropriate microtiter plate reader.

To maintain the cells, 5 ml of the cell suspension were removed every 3-4 days in each case and replaced with 5 ml of fresh medium.

Two series of experiments with three HUT 78 transfectants (HUT K14-KTX11, SSHUTTK16 / 2-SXK1, SSHUTK1 / 1-SXK2, see Fig. 11) and three U 937 transfectants ( suction unit 20/4-KTX1 la, suction unit 3/1-SXK2 and suction unit 20/15-KTX1 1b, see Fig. 12). Following infection for an incubation period of up to 60 days, both in the measurements of reverse transcriptase (RT) activity as well as in the VIF antigen assays, these cell cultures expressing the antisense RNA and containing the expression vectors pSCK1 and pSXK2 showed no signs of HI V. in a control cell line, HIV proliferation could be clearly determined. Moreover, a delayed increase in viral replication was found in transfectants with weaker expression of the KTX11 vector. The result is shown in Figures 11 and 12.

To verify that HIV-1 infection has occurred in sSu K3 / 1-SXK2, SSHUT K16 / 2-SXK1 and SSHUT K1 / 1-SXK2 cell lines, which showed no HIV-1 antigen or RT activity after 60 days of incubation, positive for example, infection was demonstrated by PCR of the HIV-1 TAT gene sequence and the envelope gene sequence in the 60 days incubated SSHUT K16 / 2-SXK1 and SSHUT K1 / 1-SXK2 cell lines (Figure 13). For this purpose, the PCR primer 5'-ATG GAG CCA GTA GAT CCT-3 'and 5'-TCT ACC ATG TCAT-3' were used to generate a PCR fragment with a length of 690 bp. The expression vectors pSXK1 and pSXK2 do not contain these sequences and therefore cannot give a false positive PCR product.

Example XI. Control experiences.

The transfection of the vectors of Examples I to VI, without the HIV-1 proviral DNA inserts in the antisense orientation, did not produce U 937 and HUT 78 cells in lines in which viral replication was inhibited. However, control experiments with the vectors of Examples I to VI in which there are insertions of the proviral HTV-1 DNA fragment in the sense orientation have not been performed because the corresponding sense transcripts may bind HIV replication regulatory proteins such as TAT and REV and therefore may competitively inhibit virus replication (GJ Graham et al. PNAS 87, 5817, 1990) and therefore prove to be unsuitable for control experiments.

Example XII. HIV-2 infection of HUT 78 and U 937 cell lines expressing antisense RNA.

Infection trials were performed with the HIV-2 D194 / H isolate. Kuhnle et al., Nucleic Acids Res. 18, 6142,1990 /. A suspension of virus RT activity of 2.14 x 10 ⁵ cpm / ml and the antigen concentration (dilution 1: 1000) of 0.249 OD450 nm (Sample D 160) were stored at -80 ° C. In the suspension of the virus to infect 2 x 10 ⁶ HUT 78 cells was collected 50 gl added to 4 ml of medium and incubated for 3 hours. U 937 cells were infected (2 × 10 ⁶ ) with 100 µl of a virus suspension in 4 ml of medium and incubated for 24 hours. Then, unbound virus particles were removed in the culture by washing.

At intervals of 3-4 days, the cell culture was maintained and the supernatant was removed for quantification of HIV antigen using Organon Technika's HIV Antigen Capture Assay in conjunction with an appropriate microtiter plate photometer. The replication course of HIV-2 D194 in control HUT 78 and U 937 cell lines that do not contain antisense RNA expression vectors is compared in Figures 14 and 15 with transfected cell lines that exhibit delayed proliferation of HIV-2 by expression of the antisense RNA.

Results

Using the 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 (Examples I to VI) in human blood cell lines after gene transfection and cloning of the transfected cell lines (Example VII) it was found 12

The endogenous production of anti-HIV-1 antisense RNA (Example IX) has been demonstrated and its antiviral activity has been demonstrated (Examples X, XII).

Surprisingly, there was a clear inhibition of HIV-1 retrovirus replication in the transfected cell lines. Similarly, by using HIV-1 specially adapted to the transfected cells, it was possible to demonstrate viral resistance of cells expressing antisense RNA (Figs. 11, 12). Clear antiviral features of the anti-HIV-2 antisense RNA were also observed (Figures 14, 15).

These results exemplify that the respective promoters of the expression of antisense RNA molecules, and of course the type of antisense RNA sequence, lead to the development of resistance to retrovirus infection in genetically altered cells. Example 9 shows that due to the hitherto unknown cleavage and splicing of HTV-1 RNA, transcripts of the +/- strand of proviral DNA in the 3 '- 5' orientation, several mRNA molecules were produced which lead to an antiviral effect.

The experimental work described in Examples I to XII has shown that by using appropriate expression vector constructs with retroviral DNA fragments encoding antisense RNA, antiretroviral properties can be obtained in cells that protect against retroviral infection. It can be seen from Figures 11 to 13 that the SSHUT K16 / 2-SXK1 and SSHUT K1 / 1-SXK2 cell lines 60 days after HIV infection are positive by PCR analysis of HTV proviral DNA, although the determination of HTV antigen is negative. This result allows the conclusion that after infection of HIV-1 cells, although in the first stage of the HIV replication cycle, proviral DNA is still formed in the transfected HUT 78 cell lines, but antisense RNA prevents transcription / translation of proviral DNA and HIV protein is not produced for viral replication in quantifiable amounts.

In the DNA of the SSU K3 / 1-SXK2 cell line, 60 days after HIV-1 infection, the production of HIV proteins or HIV proviral DNA could not be detected in all test samples by antigen test or PCR analysis. This result may mean that in the SSU K3 / 1-SXK2 cell line the produced antisense RNA not only prevents the second stage of the HIV replication cycle (transcription and translation of proviral DNA) but also inhibits the first stage of the replication cycle.

Antisense RNA can apparently prevent proviral DNA from being formed from the genomic / + / RNA strand by a reverse translation process. This effect of antisense RNA has been speculatively assumed, but has not yet been demonstrated (RYL To and PE Neiman, Gene Regulation, Biology of antisense RNA and DNA, RP Erickson and JG Izant, Raven Press Ltd., New York 1992) since No sufficiently efficient expression vector constructs were found.

Descriptions of figures

Figure 1

KTX 11 expression vector, for expression of HIV-1 antisense RNA. The metallothionein promoter (MTI) triggers the transcription of antisense RNA from the HIV-1 proviral DNA fragment spanning nucleotides 5786 - 474 (numbering UWGCG GENEMBL DATA BANK File; HIV HXB2CG.VIRAL). Termination of antisense RNA transcripts occurs by the SV 40 polyadenylation sequence. Transcription of the TRN5neo neomycin resistance gene is initiated by the SV 40 early promoter. The shuttle vector also contains the pBR322 start and the ampicillin gene (pBr 322-amp). Further details are described in Example I.

Figure 2

KTX 12 expression vector, for expression of HIV-1 antisense RNA under the control of the HTV-1 LTR hybrid metallothionein promoter. Details of the vector structure are described in example 2.

Figure 3

KTX 15 expression vector, for expression of HIV-1 antisense RNA under the control of the HIV-1 LTR hybrid metallothionein promoter, in which there is a deletion of the TAR sequence

175 143

HIV-1 for maximal expression in the absence of TAT transactivator protein. Details of vector construction are described in Example 3.

Figure 4

PSXK 1 expression vector, for expression of HIV-1 antisense RNA under the control of the cytomegalovirus (CMV) promoter. The details of the vector structure are described in Example 4.

Figure 5

PSXK 2 expression vector, for expression of HIV-1 antisense RNA under the control of the cytomegalovirus (CMV) promoter. The HIV-1 proviral DNA fragment from the middle region of the HIV genome comprises in this example not sequences from the 5 'region as in Examples I to IV, but from the POL / VIF region. Details of vector construction are described in Example 5.

Figure 6

Indicative digram for the preparation of HIV-1 gene fragments which, in the 3 '- 5' antisense orientation (arrow direction), are included in the antisense RNA expression vectors, with the markings given in column 1. The promoter of the corresponding expression vector is given in column 2, column 3 nucleotide numbering of HTV sequence according to UWGCG GENEMBL DATA BANK file: HIVHXB2CG.VIRAL.

Figure 7

Southern Blot analysis, after PCR amplification, of chromosomal DNA from the cloned U 937 transfectant which contains the pSXK1 expression vector integrated into the chromosomal DNA. The fragment size of the PCR products, and hybridization with the HIV gene probe that does not contain the primer sequence of the PCR products, indicate the intact nature of the promoter / insert sequence.

1. Vector control, 5 'combination, 887 bp fragment

2. Vector control, 3 'combination, 775 bp fragment

3. Chromosomal DNA, 5 'junction, 887 bp fragment

4. Chromosomal DNA, 3 'junction, 775 bp fragment.

Further details are given in Example IX.

Figure 8

Detection of HIV-1 gene fragment in chromosomal DNA from cloned HUT 78 transfectants by PCR amplification. The PCR reaction was separated on a TAE agarose gel and, after staining with ethidium bromide, photographed under UV light. The PCR product covers 561 bp and corresponds to the HIV-1 gene fragment (nucleotides 699-1305) which is also a component of the pSXK1 vector. The 561 bp PCR product is shown by the arrow.

M = DNA length standard, 516 bp and 1018 are shown = negative control, DNA not added to PCR = chromosomal DNA from U 937 cells (wild type) = chromosomal DNA from HUT 78 cells (wild type) = negative control, DNA not added to PCR reaction = positive control = chromosomal DNA from HUT 78 transformants (SSHUT 6/1 107-9 / 1) = chromosomal DNA from HUT 78 transformants (SSHUT 6/1 107-9 / 1) = chromosomal DNA from HUT 78 transformants (SSHUT 6/1 107-9 / 1) = chromosomal DNA from HUT 78 transformants (SSHUT 6/1 107-9 / 1)

Figure 9

Detection of HIV-1 gene fragment (nucleotides 4158-5792) in chromosomal DNA in U 937 monocytes and HUT 78 T lymphocytes by PCR amplification. Cells were previously transfected with the pSXK2 vector. The PCR reaction was separated on a TAE agarose gel and, after staining with ethidium bromide, photographed under UV light. The PCR product covers the entire fragment of the HIV-1 gene in pSXK2 (nucleotides 4158-5792) and is 1811 bp in length. The arrow shows the 1811 bp PCR product.

M = DNA length standard, 1635 bp and 2036 bp are shown = negative control, DNA not added PCR

175 143 = chromosomal DNA from U 937 cells (wild type) = chromosomal DNA from HUT cells 78 (wild type) = chromosomal DNA from HUT transformants 78 (SSUK 3/1) = chromosomal DNA from transformants U 937 (SSUK 3/1 107 -12/1)

Figure 10

Northern Blot analysis to determine a series of antisense RNA transcripts in the transfected cloned U 937 cell line, which contains the KTX 11 expression vector in the SSUK 20/15-KTX1 1b cell line. With the help of the gene probe corresponding to the insert DNA of the KTX 11 vector, at least three antisense RNA transcripts of 800, 1200 and 3200 bp can be found in the RNA of the virus resistant cell line (lane 1). Lane 2 contains RNA from the non-transfected U 937 cell line (negative control). Band 3 contains the molecular weight standards.

Figure 11

The course of HIV-1 antigen production within 60 days after infection (on day zero) of the HUT 78 cell line with the HIV-1 LAV / BRU isolate. The ordinate shows the optical densities at 450 nm (OD 450 nm) of HIV ELISA results with cell culture fluid. At an optical density> 2.0, the culture fluid was diluted and the reported value was calculated from the dilution. The value of the abscissa represents the detection limit of the HIV antigen assay. In the SSHUT K16 / 2-SXK1 and SSHUT K1 / 1-SXK2 cell lines expressing antisense RNA, HIV replication is completely inhibited over a 60 day period. In the HUT K14-KTX11 cell line, the origin of HIV replication is delayed compared to the untransfected wild-type cell line HUT 78.

Figure 12

The course of HIV-1 antigen production within 60 days after infection (on day zero) of the U 937 cell line with HIV-1 LAV / BRU isolate. The ordinate shows the optical density at 450 nm (OD 450nm) of HIV ELISA results with cell culture fluid. At an optical density> 2.0, the culture fluid was diluted and the reported value was calculated from the dilution. The value of the abscissa represents the detection limit of the HIV antigen assay. In the SSUK 3/1-SXK2 cell line expressing the antisense RNA, HIV replication is completely inhibited within 60 days, the remaining transfected cell lines show a delay in HIV replication compared to the wild-type cell line (U 937).

Figure 13 ,,

PCR analysis to determine HIV-1 DNA in cell lines that contain antisense RNA expression vectors and were tested 60 days after HIV infection. The ethidium bromide stained agarose gel electrophoresis shows the PCR products of chromosomal DNA from the following cell lines. Using the primer pair 5'-ATG GAG CCA GTA GAT CCT-3 'and 5'-TCT GT TCT ACC ATG TCAT, a 702 bp PCR fragment from the TAT-ENV gene region was amplified in HIV infected cells. For comparison with the PCR result, the result of the HIV antigen test of the respective cell culture samples is given in parentheses.

M band: molecular weight standard, 600 bp is marked

Lane 1: HUT K14-KTX11, 702 bp positive (antigen test positive)

Lane 2: SSHUT K16 / 2-SXK1,702 positive (antigen test negative)

Lane 3: PCR product from a parallel infection experiment as in PiSime 2

Lane 4: PCR product from a parallel infection experiment as in lane 2

Lane 5: SSHUT K1 / 1-SXK2, 702 dodatai (negative antigen test)

Lane 6: PCR product of the parallel infection experiment as in lane 5

Lane 7: Parallel experiment with infection as in lane 5 Lane 8: HUT 78, positive control

Lanes 9-11: SSUK3 / 1-SXK2 cultures - PCR product 702 bp undetectable (antigen test negative)

175 143

Figure 14

The course of HIV antigen production within 30 days after infection (on day zero) of the HUT 78 cell line with the HIV-2 isolate D194. The ordinate shows the optical density at 450 nm (OD 450nm) of the results of the HIV ELIS A assay with cell culture fluid. At an optical density> 2.0, the culture fluid was diluted and the reported OD value was calculated from the dilution. The value of the abscissa represents the detection limit of the HIV antigen assay. In the SSHUT K1 / 1-SXK2 cell line, which expresses the antisense RNA, HIV replication is completely inhibited within 14 days, the remaining transfected cell lines show a delay in HIV replication compared to the wild-type cell line (HUT 78).

Figure 15

The course of HIV antigen production within 30 days after infection (on day zero) of the U 937 cell line with HIV-2 isolate D194. The ordinate shows the optical density at 450 nm (OD 450nm) of the results of the HIV ELIS A assay with cell culture fluid. At an optical density> 2.0, the culture fluid was diluted and the reported OD value was calculated from the dilution. The value of the abscissa represents the detection limit of the HIV antigen assay. In the SSHUT K3 / 1-SXK2 cell line, which expresses the antisense RNA, HIV replication is completely inhibited within 30 days, the remaining transfected cell line shows a delay in HIV replication compared to the wild-type cell line (U 937).

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Claims (7)

Patent claims 1. Expression vector constructs for the expression of antisense RNA, characterized in that they contain a) a strong constitutive promoter sequences selected from the metallothionein promoter and the CMV promoter, or hybrid promoter sequences comprising the mentioned strong promoters or one of the strong promoters mentioned and the HIV-1 LTR promoter and b ) viral DNA in antisense orientation. 2. Constructs according to claim The method of claim 1, comprising pathogenic virus subgenomic gene sequences in the 3 - 5 'orientation (antisense orientation), under the control of an antisense RNA expression promoter whose nucleobase sequence is complementary to pathogenic virus mRNA. 3. Constructs according to claim Antisense-oriented subgenomic proviral DNA from retroviruses such as HIV-1 or HTLV-2, oncoviruses such as HTLV-I, HTLV-II, or oncogens such as abl, erb A, erb B, fms, moat, myb, wash, ras, sis, src. 4. Constructs according to claim Claim 1, characterized in that they contain promoter or hybrid promoter sequences and HlV-1 proviral DNA in antisense orientation of nucleotides 7514 474, or 5786 -> 474, or 2096 -> 474, or 3825 -> 681, or 5786 -> 4158, either 5746 -> 4648, or 2369 -> 1635, or 3227 -> 3003, or 2099 -> 678, or 3829 -> 2099, or 4647 - + 4157, or 4157 -> 3830. Constructs according to claim 1 The method of claim 1, comprising subgenic proviral gene sequences or oncogenic sequences in the 3 '- 5' orientation for expression of shorter antisense RNA transcripts. Constructs according to claim 5, characterized in that they contain proviral gene sequences from HIV-1, nucleotides 605 - 155, or 670 - 440, or 825 - 535, or 6070 -> 5800, or 5640 -> 5020, or 5600 -> 5040, or 8690 -> 8300 or 9160 -> 8800. Constructs according to claim 1 The method of claim 1, comprising two, three or more subgenomic sequences from one virus or from several viruses.