JP2746480B2 - Vectors with multiple target response factors affecting gene expression - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for causing inhibition of a virus by a DNA sequence encoding a plurality of target response elements, and a composition suitable for use in the method. In particular, the present invention relates to a method for inhibiting human immunodeficiency virus (HIV virus).

BACKGROUND OF THE INVENTION The HIV tat protein transactivates viral gene expression and is essential for virus production [Arya et al, Science 229: 69-73 (1985); Sodroak
i et al, Science 229: 74-77 (1985); Dayton et al, Cel
l 44: 941-947 (1986); Fisher et al, Nature 320: 367-3.
71 (1986)]. The tat activity response element (the so-called TAR) was located in a region of the first 44 nucleotides downstream of the transcription start site [Chen, C., and Okayama, H., Mol. Cell. Bio.
l.7: 2745 (1987); Rosen et al, Cell 41: 813-823 (198
5); Tong-starksen et al, Proc. Natl. Acad. Sci., USA 8
4: 6845-6849 (1987); Hauber et al, J. Virol. 62: 673-6.
79 (1988)].

This region is present in all HIV-1 transcripts and forms an unusually stable slam loop structure [Okamoto, T., and
Wong-Staal, F. Cell 47: 29-35 (1986)], some evidence suggests that the transcriptional effect of tat is mediated through its interaction with the TAR region of viral RNA [ Sharp et al, Cell 59: 229-230 (1989); Viscidi et a
l, Science 246; 1606-1608 (1989); Berkhout et al, Cel
l 59: 273-282 (1989); Garcia et al, EMBO J. 8: 765-778.
(1989); Feng, S. and Holland, ECNature 334: 165-167
(1988); Southgate et al, Nature 345: 640-642 (199
0)].

Binding to the TAR RNA sequence has been demonstrated [Rappaport, Je
t al, Cold Sprin Harbor, New York (1989b); Dingwall
et al, Proc. Natl. Acad. Sci USA 86: 6925-6929 (198
9)], The sequence required for tat to bind is not sufficient to explain the sequence and structural requirements required for transactivation. Cellular factors also appear to play a role in tat-mediated transactivation, which confer additional specificity [Marciniak et al. Pro.
c. Natl. Acad. Sci. 87: 3624-3628 (1990)]. tat appears to function poorly in non-primate cells, and studies with cross-specific hybrids suggest that transactivation ability is associated with the 12th presence of the human chromosome [Hart er al, Science 246: 488-49
1]. Several cellular TAR RNAs have been identified as well as TAR DNA binding proteins [Gaynor, RBEMBO J. 8: 765-778.
(1989); Gatignol et al, Proc. Natl. Acad, Sci. USA 86:
7828-7832 (1989); Wu et al, EMBO J. 7: 2117-2129 (198
9); Jones et al, Science 232: 755-758 (1986); Garcia
et al, EMBO J. 8: 765-778 (1989); Marciniak et al, Pr
oc. Natl. Acad. Sci. 87: 3624-3628 (1990)], however, the role of these proteins in tat-mediated activation from trans remains unclear.

In vitro, the tat protein can be released and taken up by cells [Frankel, AD, and Pabo,
COCell 55: 1189-1193 (1988)], in addition to its role in HIV promoter activation, has a biological effect on the control of cell growth. Recent studies have shown that tat inhibits antigens that induce lymphocyte proliferation [Viscidi et al, Scie
nce 246: 1606-1608 (1989)], which has an activity to promote the growth of cells derived from a Kaposi's sarcoma lesion of an AIDS patient [Ensolie
tal, Nature 340: 84-86 (1990)]. Conversely, tat does not cause a significant decrease in lymphocyte proliferation in response to mitogens. If HIV-Itat is associated with the cause of HIV associated with the disease, interfering with tat function may be of therapeutic importance. Trans-dominant mutants for HIV proteins have been reported [Malim et al, Cell 58: 205-214 (1989); Trono et
al, Cell 59: 113-120 (1989); Marciniak et al, Proc. N
atl.Acad.Sci.87: 3624-3628 (1990)]. These proteins are produced structurally from strong promoters,
HIV-I growth can be stopped and thus used to create "immune" cell lines against viral infection.

TAR RNA directly interacts with the tat protein [Southgate et al, Na
Nature 345: 640-642 (1990)] or through the combined activation of cellular factors [Maroniak et al, Proc. Natl. Acad.
Sci. USA 87: 3624-3628 (1990)], and applicants hypothesized that TAR RNA produced in large quantities is acting as a competitive inhibitor of tat function. The results of the examples described below show that overproduction of TAR RNA
It is shown that transactivation via is suppressed to a low level. Attempts to biologically control the expression of high levels of target RNA factors that sequester viral or cellular transactivators have general applicability in the generation of new classes of antiviral reagents . Po illustrated here
Inducible transcripts of ly-TAR can bind to coding sequences whose trans-dominant shootant confers a mutually exclusive effect on intracellular immunity.

SUMMARY OF THE INVENTION It is an object of the present invention to provide an HIV-1 LTR that directs gene expression.
Is to provide a way to down regulate

Another object of the present invention is to provide competitive inhibitors of tat function.

It is a further object of the present invention to provide a class of antiviral reagents for intracellular immunity.

Various other objects and advantages will be apparent from the detailed description and drawings of the present invention.

In one embodiment, the invention is directed to a DNA construct comprising a vector and a promoter operably linked to at least two target response elements transcribed in tandem. The construct may further comprise a DNA segment encoding a ribozyme specific for viral DNA or a DNA segment encoding a trans-dominant negative mutant of a viral protein.

In another embodiment, the invention is directed to a method of treating a viral infection. This method
Obtaining cells from a patient infected with the virus and transforming the cells with a composition according to the invention. The cells are then reintroduced into the patient.

In a further embodiment, the invention comprises introducing a composition of the invention into a cell infected with a virus,
The present invention relates to a method for inhibiting virus replication. The products of the virus regulate the transcripts of the constituent factors so that the inhibition is effective.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the hypothesis of the effect on the intracellular inhibition of HIV gene expression using HIV-LTR-driven multiple TAR factors.

According to this model, tat expressed from the viral LTR
Activates LTR-driven transcription of multiple TAR elements. Multiple TAR
RNA competes with tat binding. Thus, viral gene expression decreases with tat expression until an equilibrium is reached that depends on the number of TAR factors in the construct.

FIG. 2A shows the composition of the multiple TAR elements and the expected secondary structure of the transcript.

This figure shows an annealed oligonucleotide containing a complete wild-type TAR element with Dra I and Sma I half palindrome sequences. Arrows indicate the direction of transcription. The predicted secondary structure of multiple TAR RAN elements is also shown.

B shows the general structure of the plasmid.
The plasmid used in this experiment contains CD7-LTR,
This CD7-LTR has been obtained from the wild-type HIV-1-LTR by deletion of the negative regulator (NRE). All constructs include the C-terminal portion of the bacterial CAT gene (downstream of the NcoI site) and SV-40 splicing and polyadenylation signals.

FIG. 3 (ad) shows that multiple TAR elements down-regulate transactivation.

Figures 3a and 3b: Cos cells contain 4.4 μg of pnismid
Different poly-TAR, 1 μg RSV-luciferase, 2.2
μg LTR-CAT and 0.48 μg LTR-tat as indicated (FIG. 3)
a) or 0.48 μg pSV _L -tat (FIG. 3b) with the plasmid. + And-
In co-transfection analysis, this indicates whether a particular plasmid is present or absent, respectively. 48 hours after transfection, cell lysates were analyzed for CAT expression and luciferase activity.

FIG. 3C: Total RNA was prepared from Kos cells transfected with different plasmids as shown (13).
+ And-indicate the presence or absence, respectively, of the particular plasmid in the co-transfection analysis. 10 μg RNA, Northern blot
Analyzed by hybridization. As FIG. 2 shows, the construct used for co-transfection contains the short C-terminal part of the CAT gene. Nick-translated CAT fragment (Pharmacia) was used as a ³² P-labeled probe.

FIG.3d: Cos cells show 4.4 μg, 1 μg RSV-luciferase, 2.2 μg of different indicated poly-TARs containing the expression plasmid.
μg HTLV-I-LTR-CAT, 0.48 μg HTLV-I-LTR-TAX
(13) Co-transfected with 0.48 μg LTR-tat as indicated. 48 hours after transfection, cell lysates were analyzed for CAT expression and luciferase activity.

FIG. 4 (ac) shows that transactivation inhibition is due to TAR RNA.
This shows that it depends on the amount of the transcript.

FIG. 4a: Kos cells were co-transfected with 2.2 μg LTR-CAT, 0.48 μg LTRtat and increasing amounts of LTR-5STAR. Reduced LTR-0TAR (X represents 0.22 μg) was used to keep the promoter concentration constant. + And-indicate the presence or absence of each particular plasmid in the co-transfection analysis. 48 hours after transfection, cell lysates were analyzed for CAT expression.

Figures 4b and 4c: Total RNA was prepared from Kos cells transfected with different plasmids as shown. 10 μg RNA was analyzed by Northern blot hybridization using a nick-translated ³² P-labeled CAT DNA fragment (FIG. 4 b). The CAT probe anneals to all the components represented in FIG. ³² P-labeled ta
The tDNA probe is a tat mRNA expressed under different conditions.
(Figure 4c).

FIG. 5 shows the change in the amount of transactivation inhibition depending on the amount of TAR RNA transcript. 0TAR to 50TA
The inhibition caused by the construct containing the R factor is compared.

FIG. 6 shows ribozyme-inhibition of viral replication inhibition.
It shows an example of a poly-TAR construct.

FIG. 7 shows examples of GAG-poly-TAR constructs for inhibiting replication of HIV.

DETAILED DESCRIPTION OF THE INVENTION A particular purpose of the work leading to the present invention was to establish a transduction vector system that could be activated by the activity of the tat protein and simultaneously inhibit tat activity. The inventor's attempt was to inhibit the function of tat by overproduction of the tat activity response (TAR) factor. FIG. 1 depicts this attempt. The protected cell contains at least one copy of the composition according to the invention and, after infection, one copy of the integrated provirus. If the HIV-LTR is activated, the tat protein is made from the proviral genome as the first gene product. Some of this tat protein activates viral gene expression and some activates transcripts of multiplexed TAR factors from the construct. Multiple TAR R
As NA competes with tat binding, viral gene expression is reduced. Since expression itself depends on the presence of tat, its expression decreases with all LTR-directed gene expression until reaching an equilibrium depending on the number of TAR factors in the multimer. This is the first time that a proposed composition for gene therapy has been placed under the control of biological regulation. If the cells become infected and tat is made, only the Protective gene product will be expressed. Nevertheless, the construct is inactive in the genome.

Inhibition of viral replication with multiple TAR factors is effective against all isolated HIV, as it is a functional inhibition. The problem encountered by most HIV vaccines is the high mutation rate of the virus.
The constructs of the present invention are not limited to retroviral mutations.

Accordingly, the present invention provides a method for preparing at least one copy of
Preferably it relates to a DNA construct encoding 5 to 50 copies, most preferably 20 copies or more. A composition according to the invention comprises a DNA segment comprising a multi-target response element such as a TAR element, a promoter such as LTR-HIV and a vector such as pCD7. Multiple activation response elements must be in tandem or, if separated, their transcription must not be hampered by distant sequences. The present invention is such that a further increase in the number of TAR factors does not increase inhibition.
It was found that until TAR RNA was included, inhibition of HIV increased with increasing TAR factors. Decision tests were performed by transient expression analysis, and in the case of stable integration, the added TAR provided a further increase in inhibition.

In the compositions of the present invention, a promoter is operably linked to multiple TAR DNA segments to control the amount of TAR RNA produced. In the embodiments described below, HIV-
Although the LTR promoter is used, multiple TAR elements can be transcribed by various other promoters. For example, tat
A promoter that can be activated by a protein is used. Other promoters (CMV, SV40 or tRNA promoters) can be used, but these promoters produce constitutive expression of the gene product. The advantage of using promoters like the HIV-LTR is that they are driven by the virus. This means
More specific than any promoter. Furthermore, tissue-specific promoters are useful for these constructs.

The vectors used in the constructs of the present invention must ensure high efficiency of gene transfer into target cells in vivo (eg, retroviral vectors).
Vectors suitable for use in the present invention include vectors comprising an origin of replication and a selectable marker for prokaryotic cell growth. Vectors may include one or more promoters. Further, the vector of the construct of the present invention can contain a sequence that allows site-specific integration of the construct into the chromosome without eliciting cellular functions. Vectors can also include "helper virus" sequences that allow the constituent genes to target cells and promote propagation of the vector upon further infection.

Using the constructs of the present invention, the inventors have identified HIV-1
It has been shown that the degree of down-regulation of gene expression depends on the number of TAR factors in the construct. This indicates that the use of some excess target nucleotide sequence can be used to down regulate the activation of unwanted genes.

TAR factors can be tandemly linked to other factors that act by inhibiting viral expression or inducing protective proteins that act against the disruptive effects of viral proteins. For example, the constructs of FIGS. 6 and 7 made by the methods of the present invention can be used to down regulate the activity of an undesired gene. Examples of factors suitable for inclusion in the construct include trans-dominant regulatory proteins, antisense sequences,
Coding sequences for antiviral factors, such as interferons or factors that stimulate the immune system. The components are
It may also include response agents of different activities or inhibitions.

The inhibitory activity of the constructs of the invention as described above may be enhanced by including in the construct a trans-dominant negative mutant of the viral protein (eg mutant GAG, or ribozyme command for HIV mRNA such as GAGNAM). Can be. The construct may also include a rev response factor (RRE). The constituent RRE elements function to transport RNA made from the constituents from the nucleus to the cytoplasm.

The combination of a ribozyme and TAR against the viral mRNA in a single construct gives two different types of inhibition. TAR is HIV by isolating tat
Inhibiting the expression of -1 directed genes, ribozymes can inhibit protein translation by hybridizing to and cleaving target RNA. The connection of these two inhibitory functions increases the likelihood of overall inhibition. The ribozyme used in the examples described below, GAGNAM, is directed against GAG mRNA, a particularly good target, since it is conserved in American HIV isolates.

Constructs containing trans-dominant mutants of HIV proteins, such as TAR and GAG, inhibit both HIV gene expression and viral assembly. The combination of TAR and mutant GAGs provides a purely functional inhibition that the virus cannot overcome by mutation.

The compositions of the present invention can be made by any suitable method known in the art. Multiple target response elements can be prepared using purified response elements that can be ligated by methods of adding sequences in tandem to provide multiple target response elements. Although constructs containing multiple activation response elements have been exemplified, it should be confirmed that the constructs can also contain multiple inhibitory response elements.

The use of multiple inhibitory sequences is expected to suggest to one of skill in the art to stimulate the activity of the desired promoter. Such constructs containing a tandem multiple inhibitory response element can be used to increase the production of a desired product, for example, by stimulating a promoter responsible for the expression of the desired protein.

The composition of the present invention can be used for gene therapy by a known method. David Baltimore (Nature 335: 39
5-396 (1988)) and a method known as "intracellular immunity" can be used. For example, the compositions of the present invention can be introduced into bone marrow cells, including all hematopoietic stem cells. Blood cells can be any of a homogeneous population, such as a lymphocyte, where the mixed population is. Using the illustrated construct, cells of an HIV-infected solid are used. After introducing the gene, the cells are injected back into the patient. The bone marrow will be partially removed by irradiation or by drug therapy before the modified cells are infused to make room for the growth of the transplanted cells.

Further, the vector of the present invention can be introduced into blood cells from a patient. The cells that have preserved the composition are re-introduced into the patient. In treating HIV-infected individuals, the component is CD4
+ Must be introduced into cells. Since the replacement of these cells is relatively fast, reintroduction of protective cells is necessary as long as viral infection is present. Repeated introduction of such cells may be necessary.

It is believed that the multiple TAR constructs and / or TAR + ribozyme constructs according to the invention produce only inhibitory RNAs, not protein products that are important in gene therapy strategies.

The use of multiple TAR factor constructs of the present invention is highly advantageous. For example, this construct provides for specific inhibition of HIV-directed gene expression. The expression of the defense gene product is biologically regulated and can be performed in vivo using normal cellular methods.
Since the structural expression of transcripts including TAR is harmful,
Significantly advantageous. The usefulness of the composition is different HI
It is not limited by the diversity between the V. In addition, the use of the construct can be extended by downstream insertion of sequences such as either ribozymes or trans-dominant mutants of HIV proteins.

Examples The following non-limiting examples are provided to further illustrate the invention. Although the present invention is illustrated using the HIV system and TAR factors, those skilled in the art will recognize that using the methods of the present invention can be expected to result in inhibition of other viruses.

Plasmid Constructs Plasmids containing different numbers of single directional TAR elements under the control of the HIV-LTR were constructed (see FIG. 2).
The "multiplexed" TAR sequence has a 5 'HIV-1-LTR deletion mutant CD7 (i.e., lacks the negative regulator (NRE) and has a higher level of expression as compared to the wild-type HIV-I-LTR. [Siekevitz et al, Science 238: 1575-1578 (198
7)]. Plasmid pCD7 containing a portion of the HIV-1 LTR (-278- + 63) (kindly provided by Stepen Josephs) was digested with restriction enzymes and ligated into a multiple tandem TAR element. L
TR-1TAR, LTR-4TAR and LTR-5TAR are each 1
One, four, and five copies of the TAR element were included.

To construct plasmids LTR-5TAR and LTR-4TAR, include the sequence of the complete TAR element of HIV-I (+1 to +63) located next to half the palindromic sequence for the DraI and SmaI restriction oxygen recognition sites. Were synthesized. The oligonucleotide was purified, phosphorylated and ligated. Ligated DNA (TAR)
Is ligated in the presence of DraI and SmaI and allows only tandem (directed) ligation of TAR elements. HIV
-Plasmid CD containing part of -I-LTR (-278 to +63)
7-CAT [Siekevitz et al, Science 238: 1575-1578 (198
7)] was digested with the restriction enzymes HindIII and NcoI, and the ends were filled with multiple tandem TAR elements and ligated. Of the several strains of E. coli tested, only one,
That ^{Bj 5183: F -, recBC,} endo I, gal, met, str, thi, b
io, hsd (kindly provided by F. Lacroute) was able to maintain these plasmids without transfer.

LTR-1-TAR was constructed by digesting CD7-CAT with HindIII and NcoI, the ends filled and religated.

Two control plasmids were created: 5TAR lacking the upstream promoter sequence (the TAR sequence is present but not transcribed) and LTR-0TAR without the TAR sequence but containing the upstream promoter sequence (Figure 2). . LTR-0-TAR is Pvu
CD7-CAT plasmid [Siekevitz using II and NcoI
et al, Science 238: 1575-1578 (1987)] and made blunt ends and ligated.

The 5TAR plasmid can be constructed by deleting the 5 'portion of the HIV-LTR from plasmid LTR-5TAR by digestion with XbaI and PvuII, the ends of which were filled and ligated. LTR-tat is available for Sal I and Bam HI
In pSV _L -tat [Rappaport et al, the New Bilogist 1: 101
-110 (1989a)]: 350BPtat containing the fragment was isolated and blunt-end ligation was performed.
This was done with the vector CD7-CAT between the HindIII and NcoI sites.

All constructs were confirmed by restriction maps and sequences.

Cloning strategies allow directionality, but TA
Reverse repetition of the R factor is not allowed. The reason is,
This is because the reverse repeat is cleaved by the Sma1 and Dra1 enzymes during ligation. The correct orientation and secondary structure of each factor is probably important for the desired effect, because TAR functions for transactivation only in a position-dependent manner. [Peterlin e
tal, Proc. Natl. Acad. Sci. USA 83: 9734-9738 (1986)]
Down-regulation of transactivation, depending on TAR factor transcription.

To determine the effect of multiple TAR factors on HIV-LTR-directed gene expression, the TAR expression plasmid was tested for LTR-C
AT [Siekevitz et al ;, Science 238: 1575-1578 (198
7)] and LTR-tat or pSVL-tat in Kos cells
[Rappaport et al, The New Biologist 1: 101-110 (1989
a)].

COS-1 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (GIBCO). 2 × 10 ⁵ cells were transplanted one day prior to transfection into 3 ml of medium in 6-well tissue culture medium. 25 μg of total plasmid was used in three wells. The amounts of the different plasmids are as shown in the figure. Plasmid pBR322
Was used as a carrier for DNA. Transfection was performed by the calcium phosphate method [Ch
en, C., and Okayama, K. Mol. Cell. 7: 27-45 (1987)]. 48 hours after transfection, cells are harvested (SI
GMA cell removal reagent), a crude cell extract was made in PBS.

Plasmid RSV-Lugiferase was included as an internal control to detect transfection efficiency. The amount of CAT protein and the relative level of luciferase activity were determined from extracts of infected cells. CAT protein was analyzed using a 5-prime 3-prime ELISA kit according to the product instructions; luciferase activity was determined by PEStanley and SGWilliams [Stanley, PEa
nd Willams, SGAnal. Bichem 29: 381 (1969)] and the activity was expressed in arbitrary units (ARU).

Multiple T transcribed from HIV-LTR, as shown in FIG.
AR factor inhibits HIV-LTR-directed gene expression in the presence of tat, and the observed down-regulation is
It is proportional to the number of R factors. LTR-4TAR and LTR-5TAR inhibited transactivation on average by 70% and 80%, respectively. LT
R-1TAR also has an important configuration that results in down regulation of up to 40%. This decrease is due to CAT and tat
The cumulative effect of both inhibitions of the expression of This is because both gene products are under the control of the HIV-I-LTR in this experiment. Multiple TAR elements can suppress transactivation when tat is constitutively expressed from the SV40 late promoter despite reduced levels (FIG. 3). FIGS. 2A and B show that when tat is expressed from the HIV-1 LTR, the expression of CAT is reduced to 82% compared to the 50% reduction observed with constitutively expressed tat. ing.

Transcription of multiplexed TAR sequences requires efficient down-regulation, and accumulation of steady-state competing RNA is
Only occurs in the presence of at (see 3C). LTR-0TAR plasmid without TAR sequence or 5TAR plasmid with deletion of upstream promoter sequence does not produce significant effect on HIV-LTR directed gene expression (see FIGS. 3A, 3B, 3C).

Cotransfection is another human retrovirus to determine the specificity of the effect of multiplexed TAR RNA
Made in LTR. HTLV-I-LTRCAT (kindly provided by M. Nerenberg) was used as the reporter gene, and the HTLV-I-LTR-TAR plasmid was included as a transactivator of HTLV-ILTR [Sodeoski et al. , Sci
225: 381-385 (1984); Felber et al, Science 229: 6.
75-679 (1985)]. LTR-tat is also a multiplexed TAR
Used for transcription of factors. The results presented in FIG.3D show that expression of multiple TAR RNA factors does not affect HTLV-I promoter activity, suggesting that down-regulation of gene expression is specific for the HIV-LTR. I have.

That multiple TAR RNA factors can specifically inhibit transactivation of HIV gene expression,
This is demonstrated in FIG. 3, where the RSV promoter was linked to the luciferase reporter gene. Using this construct, it was demonstrated that the specificity of the multiple TAR RNA elements did not affect the use of foreign promoters. RS
An important difference in V promoter activity is that TAR RNA or DNA
Not detected using the factor. This means that HIV-LT
The specific activity of the R versus the promoter of the RSV promoter is shown as the ratio of CAT and luciferase expression. Inhibition of transactivation is equivalent to the amount of TAR transcript.

LTR-CAT and LTR-tat were co-transfected with increasing amounts of LTR-5TAR plasmid and the effect of different amounts of AR transcript on transactivation was determined. RNA is
It was isolated using the CINNA / BIOTECX RNAzol reagent according to the manufacturer's protocol. For Northern blot analysis,
RNA was electrophoresed on a 1% formaldehyde / agarose gel. RNA was transferred to nitrocellulose paper and hybridized with a nick-translated ³² P-labeled probe as described previously [Maniatis et al, Cold Sp.
ring Harbor, New York: Cold Spring Harbor Laboratory
(1982)].

FIG. 4A shows that increasing the amount of LTR-5TAR plasmid results in a proportional decrease (up to 97%) in CAT expression. Inhibition was due to HIV-level because the amount of upstream sequence of the infected LTR was kept constant in this experiment.
It is not due to competition limiting the transcription factors involved in the I-LTR. Increasing the amount of infected LTR-5TAR plasmid results in a similar increase in LTR-5TAR transcript (see FIG. 4B). These experiments show that down-regulation of HIV-1 LTR-directed gene expression depends on the relative amount of expression of plasmid DNA introduced into cells, in addition to the number of transcribed TAR elements contained in the expression plasmid. Is concluded.

Northern blots show that expression of tat and CAT is reduced in parallel by the multiplexed TAR RNA (FIG. 4).
C), it is expected that they are both driven by the HIV-LTR.

The data in FIGS. 4a-c support the teaching that down regulation of gene expression depends on the number of transcribed target activator nucleotide sequences. The evidence is
HIV-1 transactivation by tat was down-regulated to 97% using the 5TAR RAN factor in tandem, indicating that down-regulation is a function of the amount of TAR RNA transcript. The data suggests that constructs containing more than 5 TAR factors will give more effective down-regulation effects.

Constructs Containing Five or More TAR Elements Multiple TAR constructs were expanded and plasmids containing up to 50 TARs were constructed and tested (see FIG. 5).

The pSPI18-polyTAR construct containing between 15 and 45 TARs
It was constructed by cutting the SPT18 vector (Pharmacia) with XbaI. The ends were then blunt-ended by Klenow enzyme and dephosphorylated. The insert was prepared by cutting pLTR-5TAR with PvuII and SCaI and isolating a fragment containing 5TAR (455 Bp). .

The above vector and insert were then ligated together and used to transform E. coli. Plasmid DN
A was prepared from a single colony and clones were selected to contain the large insert using methods well known to those skilled in the art. The orientation of the insert was confirmed by restriction enzyme digestion with SspI and HindIII + SspI.

The following LTR-5TAR-CAT was cloned. The LTR-CAT vector was cut with XbaI + HindIII and a larger fragment was isolated. The insert, LTR-5TAR fragment, prepared by the polymerase chain reaction, was prepared by cutting the PCR fragment with XbaI + HindIII.
The vector and insert were ligated together and used to transform E. coli. A single colony was examined.

LTR-46TAR cuts LTR-CAT with HindIII + BamHI, LT
Prepared by isolating the large fragment containing R-1TAR + pBR322. PSPT prepared as described above
45TAR was cut with HindIII + BamHI and a large fragment containing 45TAR was isolated. The isolated fragments were ligated together and E. coli was transformed with the ligated product.

LTR-25TAR and LTR-50TAR have also been prepared. The vector LTR-5TAR-CAT is cut with SalI + BamHI and
A fragment containing R + pBR322 was isolated. pSPT-20TA
R or pSPT-45TAR is cut with SalI + BamHI and 20TAR
Alternatively, a fragment containing 45TAR was isolated. The vector and insert were then ligated together and E. coli was transformed with the ligated product.

The results featured in FIG. 5 indicate that inhibition of HIV-1-LTR directed gene expression increases with the number of TARs in the construct until 25 TAR is used. TAR number 2
Increasing above 5 does not increase inhibition. Therefore, the tat protein is considered to be saturated at this point.
It is also possible that this test is not sensitive enough to detect further increases in inhibition.

LTR-50TAR, DC vector [Hantzopaulos et al, Proc. N
Atl. Acad. Sci. USA 86: 3519 (1989)]. LTR-50TAR is X
It is cut with baI, filled with Klenow enzyme, and inserted into the SnaBI site of a DC vector for efficient gene transfer.
Other vectors that ensure high efficiency gene transfer can also be used in the present invention.

Construction of Ribozyme-Poly-TAR For the construction of pRRE-ribozyme, the vector pRRE containing RRE (rev-corresponding element under control of the T7 promoter) [D
aefler et al, Proc. Natl. Acad. Sci. USA 87: 4571-4575
(1990)] was cut with BamHI, dephosphorylated, and purified. As insert, 65 Bp located next to the BamHI site
Long ribozyme PCR fragment [Chang et al, Clini
cal Biotechnology 2: 23-31 (1990)] was digested with BamHI and purified. This ribozyme is directed against HIV-1 GAG mRNA and was disclosed by N. Sarver et al. In Science 247: 1222 (1990).

The vector and insert were ligated and a portion of the ligation mixture was used to transform E. coli. Plasmids were prepared from independent transformants and tested by restriction enzyme digestion. Eight clones were found to contain the insert. These clones were tested for biological activity in vitro and three out of eight clones were found to have the ability to cleave a synthetic substrate (a substrate provided by J. Rossi).

For the construction of the LTR-polyTAR-RRE-ribozyme (see FIG. 6), the vector LTR-46TAR is cut with SalI, its ends are filled with Klenow enzyme and then cut again with HindIII. The DNA is then purified. For the preparation of the insert, the pRRE-ribozyme is cut with HindIII and SmaI. The 314Bp fragment is isolated by gel electrophoresis. The vector and insert are then ligated,
It was used to transform E. coli. Each colony is examined.

LTR-polyTAR-RRE-ribozyme is a DC vector [Hantz
opaulos et al, Proc Natl. Acad. Sci. USA 86: 3519 (198
9)] and introduced by cutting with XbaI. The large XabI fragment was filled by the Klenow enzyme and the SnaB
Inserted into I. Placing the construct on a retroviral vector is necessary for high efficiency gene transfer, and DC vectors are preferred. However, any vector that ensures high efficiency gene transfer is suitable.

Construction of ΔGAG-Poly-TAR pRRE-ΔGAG was constructed by cutting the pRRE vector with BamHI, dephosphorizing the ends, and purifying the vector. Mutant virus DNAHT4 (VI-ΔE-dhfr)
The ΔGAG protein encoded in [Trono et al, Cell 59: 113-120 (1989)] can predominantly prevent HIV-1 replication. Plasmid DNA HT4 (VI-ΔE-dhf
r) was cut with BglII and the 1429 Bp fragment containing ΔGAG was isolated from a 1% agarose gel. The vector and insert were communicated and a portion of the communication mixture was used to transform E. coli. Plasmids are prepared from individual transformants and digested with restriction enzymes, EcoRI
+ SphI.

For the construction of LTR-plyTAR-RRE-ΔGAG (see FIG. 7), the vector LTR-46TAR is cut with SalI and its ends are filled with Klenow enzyme. The DNA is then purified.
For the preparation of the insert, pRRE-ΔGAG was used for EcoRV and SmaI.
Cut at The 1.6KB fragment is isolated by gel electrophoresis. The vector and insert are then ligated and used to transform E. coli. Individual colonies are examined. The construct is inserted into the DC vector described above.

Plasmid identified as LTR-5TAR (including 5TAR) was obtained from American T, Bethesda, MD.
Entered into the ype Culture Collection in E. coli and deposited on January 17, 1990 under accession number 68203. In addition, LTR-5
The 0TAR plasmid is available from Bethesd, MD
In the a) American Type Culture Collection, it was placed in Escherichia coli and deposited on October 12, 1990 under the accession number 68446. The plasmid was deposited under the Budapest Treaty.

All publications mentioned above are a part of the disclosure of this specification.

While the invention has been described in detail for purposes of clarity and understanding, it is understood that various modifications, both in form and in trivial aspects, are possible from reading this disclosure without departing from the true scope of the invention. It will be recognized by those skilled in the art.

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Claims (19)

(57) [Claims] 1. A DNA construct comprising a vector and a promoter operably linked to at least two HIVtat activation response (TAR) factors such that the factors are transcribed in tandem. 2. The DNA construct according to claim 1, wherein said promoter is a human immunodeficiency virus (HIV) LTR promoter. 3. The DNA construct according to claim 1, which has 5 to 50 of said factors. 4. The method of claim 2, wherein said factor has 25 of said factors.
DNA construct. 5. The DNA construct according to claim 1, wherein the transcription of said factor can be regulated by a viral expression product. 6. The DNA construct according to claim 4, wherein said promoter is HIV-1 LTR. 7. The DNA construct according to claim 1, wherein said vector is pCD7. 8. The DNA construct according to claim 1, wherein said vector is a retroviral vector. 9. An additional factor that encodes a molecule that inhibits viral replication and is transcribed with said factor by the same transcription, further comprising an additional factor operably linked to said factor. A DNA construct according to any one of claims 1 to 8. 10. The DNA construct according to claim 9, wherein said molecule is an RNA molecule. 11. The DNA construct according to claim 10, wherein said RNA molecule is a ribozyme specific to viral RNA. 12. The DNA construct according to claim 11, wherein the ribozyme is specific for HIV RNA. 13. The method according to claim 9, wherein said molecule is a protein.
The described DNA construct. 14. The DNA construct according to claim 13, wherein said protein is a mutant virus protein. 15. The DNA construct according to claim 14, wherein said mutant virus protein is a trans-predominant gag mutant protein. 16. The DNA construct according to claim 9, wherein said virus is HIV. 17. The DN according to any one of claims 1 to 16
A cell comprising the A construct. 18. The cell according to claim 17, wherein said cell is infected with a virus. 19. The cell according to claim 18, wherein said virus is HIV.