AU642959B2 - Vector with multiple target response elements affecting gene expression - Google Patents

Description

WO 91/10453 PCT/US91/00175 VECTOR WITH MULTIPLE TARGET RESPONSE ELEMENTS AFFECTING GENE EXPRESSION BACKGROUND OF THE INVENTION This application is a continuation-in-part of Lisziewicz application Serial No. 07/467,407 filed on January 18, 1990, the entire content of which is hereby incorporated by reference.

Field of the Invention The present invention relates to a method of effecting viral inhibition with DNA sequences encoding multiple target response elements, and to constructs suitable for use in same. In particular, the invention relates to a method of inhibiting the Human Immunodeficiency Virus (HIV).

Background Information The tat protein of HIV transactivates viral gene expression and is essential for virus production [Arya et al, Science 229:69-73 (1985); Sodroski et al, Science 229:74-77 (1985); Dayton et al, Cell 44:941-947 (1986); Fisher et al, Nature 320:367-371 (1986)]. The tat activation response element (termed TAR) has been localized withih the region of the first 44 nucleotides downstream of the transcription initiation site TChen, and Okayama, Hol. Cell. Biol. 7:2745 (1987); Rosen et al, Cell 41:813-823 (1985); Tong-Starksen et al, Proc.

Natl. Acad. Sci., USA 84:6845-6849 (1987); Hauber et al, J.

Virol. 62:673-679 (1988)]. This region, present in all HIV-1 transcripts, forms an unusually stable stem loop structure [Okamoto, and Wong-Staal, F. Cell 47:29-35 (1986)], and several lines of evidence suggest that the transcriptional effect of tat is mediated through interac on with TAR region of viral RNA [Sharp et al, WO 91/10453 PCT/US91/00175 2 Cell 59:229-230 (1989); Viscidi et al, Science 246:1606- 1608 (1989); Berkhout et al, Cell 59:273-282 (1989); Garcia et al, EMBO J. 8:765-778 (1989); Feng, S. and Holland, E. C. Nature 334:165-167 (1988); Southgate et al, Nature 345:640-642 (1990)].

While tat binding to TAR RNA sequences has been demonstrated [Rappaport, J. et al, Cold Spring Harbor, New York (1989b); Dingwall et al, Proc. Natl. Acad.

Sci USA 86:6925-6929 (1989)], the sequence requirements for tat binding are not sufficient to explain the sequence and structural requirements needed for transactivation. Cellular factors also appear to play a role in tat mediated transactivation which may confer additional specificity [Marciniak et al, Proc. Natl. Acad.

Sci. 87:3624-3628 (1990)]. Tat appears to function poorly in nonprimate cells and studies using interspecific hybrids suggest that transactivation potential is correlated with the presence of human chromosome 12 [Hart et al, Science 246:488-491].

Several cellular TAR RNA as well as TAR DNA binding proteins have been identified [Gaynor, R. B. EHBO J.

8:765-778 (1989); Gatignol et al, Proc. Narl. Acad. Sci. USA 86:7828-7832 (1989); Wu et al, EMBO J. 7:2117-2129 (1988); Jones et al, Science 232:755-758 (1986); Garcia et al, EMBO J. 8:765-778 (1989); Marciniak et al, Proc.

Natl. Acad. Sci. 87:3624-3628 (1990)] however the role of these proteins in tat mediated transactivation, however, is not yet clear.

In vitro, tat protein can be released and taken up by cells [Frankel, A. and Pabo, C. O. Cell 55:1189-1193 (1988)], and has biological effects on the WO 91/10453 PCT/US91/00175 regulation of cellular proliferation in addition to its role in HIV promoter activation. Recent studies indicate that tat inhibits the antigen induced lymphocyte proliferation [Viscidi et al, Science 246:1606-1608 (1989)] and has growth promoting activity on cells derived from Kaposi Sarcoma lesions of AIDS patients [Ensoli et al, Nature 340:84-86 (1990)]. In contrast, tat does not cause significant reduction of lymphocyte proliferation in response to mitogens.

Given the implication of HIV-I tat in the causation of HIV associated diseases, interference with the tat function might be therapeutically significant.

Transdominant mutations for HIV proteins have been reported [Malim et al, Cell 58:205-214 (1989); Torno et al, Cell 59:113-120 (1989); Marciniak et al, Proc. Natl. Acad. Sci. 87:3624-3628 (1990)]. These proteins, produced constitutively from a strong promoter, can antagonize the growth of HIV-I and therefore, can be used to create cell lines "immunized" to viral infection.

Since TAR RNA appears,to interact wit tat protein directly [Southgate et al, Nature 345: 640-642 (1990)] or through the combined activities of cellular factor(s) [Marciniak et al, Proc. Natl. Acad. Sci. USA 87: 3624-3628 (1990)], Applicant hypothesized that TAR RNA, produced in large amounts, might serve as a competitive inhibitor of tat function. The results presented in the Examples that follow indicate that overproduction of TAR RNA downregulates tat mediated transactivation in a dose dependent manner. The approach of biologically controlled expression of high levels of target RNA elements to sequester viral or cellular WO 91/10453 PCT/US91/00175 4 transactivators has general application in the generation of a novel class of anti-viral reagents.

Inducible transcripts of poly-TAR, exemplified herein, can be combinedwith coding sequences for transdominant mutants to provide a synergestic effect for intracellular immunization.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a means of down-regulating HIV-l LTR directed gene expression.

It is another object of the present invention to provide a competitive inhibitor of tat function.

It is a further object of the present invention to provide a class of anti-viral reagents for intracellular immunization.

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

In one embodiment, the present invention relates to a DNA construct comprising a vector and a promoter operably linked to at least two target response elements linked so that they are transcribed in tandem. The construct may further comprises a DNA segment encoding a ribozyme specific for a viral DNA or a DNA segment encoding a transdominant negative mutant of a viral protein.

In another embodiment, the present invention relates to a method of treating viral infection. The method involves obtaining cells from an viral infected patient and transforming the cells with a construct of the present invention. The cells are then introduced back into the patient.

"w WO 91/10453 PCT/US91/00175 In a further embodiment, the present invention relates to a method of inhibiting viral replication comprising introducing into a cell, infected with a virus, the construct of the present invention. The product of the virus regulates transcription of the elements of the construct so that inhibition is effected.

BRIEF DESCRIPTION OF THE DRAWINGS Fig 1. shows the working hypothesis for intracellular inhibition of HIV gene expression using HIV-LTR-driven multiple TAR elements.

According to this model, tat expressed from the viral LTR activates the LTR driven transcription of the multiple TAR elements. Multiple TAR RNA competes for tat binding. Therefore, viral gene expression together with tat expression decreases until an equilibrium is reached, which is dependent on the number of TAR elements in the construct, is reached.

Fig. 2A shows the construction of the multiple TAR elements and the predicted secondary structure of the transcript.

The figure shows the annealed oligonucleotide containing the entire wild type TAR element with the half palindromic sequence of Dral and Smal. Arrows represent the direction of transcription. The predicted secondary structure of the multiple TAR RNA elements is indicated as well.

B. shows the general structure of the plasmids Plasmids used for this experiments contain the CD7-LTR, which is derived from the wild type HIV-1- LTR by deletion of the negative regulatory element (NRE). All constructs contain the C-terminal part,9f 9 the bacterial CAT gene (downstream from the Ncol site) and SV-40 splice and polyadenylation signals.

Fig. 3(a-d) show transcription of multiple TAR elements downregulates transactivation.

Figs. 3a and 3b. COS cells were cotransfected with 4.4g .different poly-TAR containing plasmid, l1g RSV- LUCIFERASE, 2.2Ag LTR-CAT and 0.48ig LTR-tat (Fig. 3a) or 0.48pg pSV. tat (Fig. 3b) plasmids as indicated. and represent the presence and absence of the particular plasmids respectively in the cotransfection assay. 48 hrs. after transfection. cell lysates were analyzed for CAT expression and LUCIFERASE activity.

Fig. 3c. Total RNA was prepared (13) from COS cells transfected with different plasmids indicated on the figure. and represent the presence and absence of the particular plasmids respectively in the cotransfection assay. 10g RNA was analyzed by Northern blot hybridization. As Fig. 2 shows the constructs used for cotransfections contain a short C-terminal part of the CAT gene. A nick-translated CAT fragment (Pharmacia) was used as 32P-labelled probe.

Fig. 3d. COS cells were cotransfected with 4.4pg of the indicated different poly-TAR containing expression plasmid, 1lg RSV-LUCIFERASE, 2.2pg HTLV-I-LTR-CAT, 0.48g HTLV-I-LTR-TAX 0.48pg LTR-tat as indicated. and represent the presence and absence of each particular plasmids in the cotransfection assay. 48 hrs. after transfection, cell extracts were assayed for CAT expression and LUCIFERASE Activity.

30 Fig. 4(a-c) show inhibition of transactivation is dependent on the amount of TAR RNA transcripts.

"S Fig. 4a. COS cells were cotransfected with 2.2pg LTR-CAT, 0.48g LTR-tat and increasing amounts of LTR- Decreasing amounts of LTR-OTAR (x indicated 0.22pg) were used to keep the promoter concentration constant. and represent the presence and absence of each particular plasmids in the cotransfection assay.

48hrs after transfection cell lysates were analyzed for CAT expression.

Fig. 4b and 4c. Total RNA was prepared from COS cells transfected with different plasmids as indicated on the figure. 10g RNA was analyzed by Northern blot hybridization using a nick translated, 32 P-labelled CAT DNA fragment (Fig. 4b). The CAT probe anneals to all constructs illustrated in Fig. 1. A 32 P-labelled tat DNA probe was used to determine the amount of tat mRNA expressed under different conditions (Fig. 4c).

Fig. 5 shows the variation in the amount of inhibition of transactivation depending upon the amount of TAR RNA transcripts. The inhibition producted by construct comprising OTAR to 50 TAR elements is compared.

Figure 6 shows an example of a ribozyme-poly-TAR construct for inhibition of viral replication.

Figure 7 shows an example of a GAG-poly-TAR construct for inhibition of HIV replication.

S 25 DETAILED DESCRIPTION OF THE INVENTION The specific aim of the studies leading to the present invention was to establish an inducible vector system which is activated by the action of tat protein :and which can concomitantly inhibit tat activity. The 30 approach of the present inventor was to inhibit tat function by overproduction of the tat activation -o.l i WO 91/10453 PCT/US91/00175 8 responsive (TAR) elements. Figure 1 depicts this approach. Protected cells contain at least one copy of the construct of the present invention and, after infection, one copy of the integrated provirus. If the HIV-LTR is activated, tat protein is made from the proviral genome as an early gene product. Some of this tat protein activates viral gene expression and some activates the transcription of the multimerized TAR elements from the constr iot. As the multiple TAR RNA competes for tat binding, the viral gene expression decreases. Since tat expression itself depends on the presence of tat, its expression would slow down together with all LTR directed gene expression until a certain equilibrium, which is dependent on the number of TAR elements in the multimer, is reached. This is the first time that a construct, proposed for gene therapy use, is under the control of a biological regulation. The protective gene product will only be expressed, if the cell becomes infected and tat is made. Otherwise, the construct is silent in the genome.

Inhibition of viral replication using multiple TAR elements is effective against all HIV isolates because it is a functional inhibition. The problem most HIV vaccines encounter is the virus' high mutational rate. The present construct is not limited by retroviral mutations.

Accordingly, the present invention relates to a DNA construct encoding at least one copy, and preferably between 5 and 50 copies, and most preferably more than 20 copies, of the TAR element. Constructs to which the present invention relate comprise a DNA WO 91/10453 PCT/US91/00175 9 segment including multiple target response elements, such as TAR elements, a promoter, such as LTR-HIV, and a vector, such as pCD7. The multiple activation response elements must be in tandem or if separated, their transcription must not be interrupted by separating sequences. The present inventor has found that HIV inhibition increases with increasing TAR elements until the TAR RNA contains 25 TARs at which point further increases in the number of TAR elements do not appear to increase inhibition. The determining test was done in a transient expression assay and in the case of a stable integration additional TARs would provide further increases in inhibition.

In the construct of the present invention, the promoter is operably linked to the multiple TAR DNA segment so that the promoter controls the amount of TAR RNA produced. While the following examples use the HIV-LTR promoter, multiple TAR elements can be transcribed from various other promoters. For example, a promoter which can be activated by the tat protein might be used. Other promoters (CMV, SV40 or tRNA promoter) can be used, however, these promoters produce a corstitutive expression of the gene product. The advantages using promoters such as HIV-LTR is that they are inducible by the virus. This is more specific than any other promoter. Further, tissue-specific promoters could be useful in these constructs.

The vector used in the construct of the present invention must ensure high efficiency gene transfer to the in vivo target cell, for example, retroviral vectors. Suitable vectors for use in the present invention include vectors which contain a WO 91/10453 PCT/US91/00175 replication origin and a selection marker for propagation in prokaryotes. Vectors may contain more than one promoter. Further, vectors of the present constructs can contain sequences which allow the sitespecific integration of the construct into the chromosome without disturbing the cell function. The vectors can also contain "helper virus" sequences which allow transmission of the construct into the target cells and promote propagation of the vector through further infection.

Using constructs of the present invention, the inventor has shown that the degree of down-regulation of HIV-1 gene expression is dependent on the number of TAR elements in the constructs. This indicates that the use of several excess target nucleotide sequences can be used to down-regulate undesirable gene activity.

It is possible to combine TAR elements in tandem with other elements which inhibit viral expression or which act through inducing protective proteins acting against destructive effects of the viral proteins. For example, the constructs of figures 6 and 7, made by the method of the present invention, can be used to down-regulate undesirable gene activity.

Examples of appropriate elements for incorporation into the construct are transdominant regulatory proteins, antisense sequences, coding sequences of antiviral agents such as interferons or immunosystem stimulating agents. The construct could contain different activation or inhibition response elements as well.

The inhibitory activity of the constructs of the present invention, as described above,can be enhanced by including in the construct a transdominant WO 91/10453 PCT/US91/00175 11 negative mutant of a viral protein, for example a mutant GAG, or the ribozyme-directed against a HIV mRNA, such as, for example, GAGNAM. The constructs can also include a rev-response element (RRE). The RRE element of the construct functions to transport RNA made from the construct out of the nucleus, into the cytoplasm.

The combination of TARs with a ribozyme against a viral mRNA in a single construct provides two different types of inhibition. While TARs inhibit HIV- 1 directed gene expression by sequestering tat, the ribozyme inhibits protein translation by hybridizing to the target RNA and cleaving it. Combining these two inhibition mechanisms increases the possibility of total inhibition. The ribozyme used in the following examples, GAGNAM is directed against GAG mRNA, a particularly good target since it is conserved in the American HIV-isolates.

Constructs containing TARs and trans-dominant mutants of HIV proteins, such as GAG, inhibit both HIV gene expression arn viral assembly. The combination of TARs and mutant GAG provides pure functional inhibition which the virus cannot overcome by mutations.

The constructs of the invention can be made by appropriate means known in the art. The practitioner can prepare multiple target response sequences using purified response sequences which are then ligated in a manner to allow tandem addition of the sequences to provide multiple target response sequences. It should be noted that, while constructs containing multiple activation response sequences have been exemplified, constructs can also contain multiple inhibitory WO 91/10453 PCT/US91/00175 12 response sequences. The use of multiple inhibitory sequences can be expected to allow the practitioner to stimulate activity of a desired promoter. Such constructs containing multiple inhibitory response sequences in tandem can be used, for example, to increase production of a desired product by stimulating the promoter responsible for expression of the desired protein.

The constructs of the present invention can be used in gene therapy by known methods. The method described by David Baltimore (Nature 335:395-396 (1988)) known as "intracellular immunization" can be used. For example, the constructs of the present invention can be introduced into bone marrow cells, including all hematopoietic stem cells. The blood cells can be either of mixed population or of a homogenous population such as lymphocytes. Using the constructs exemplified, the cells of the HIV-infected individual would be used. After introduction of the gene, the cells would be injected back into the patient. To make space for the growth of the implanted cells, the marrow could be partially cleared by irradiation or with a medication before the modified cells are injected.

Blood cells from patients can also be introduced with the vectors of the invention. The cells with the construct would be re-introduced into the patient. In the treatment of HIV infections, the construct must be introduced into CD4+ cells. Since the turn-over of these cells is relatively fast, reintroduction of the protected cells is necessary so long as the viral infection is present. The repeated WO 91/10453 PCT/US91/00175 introduction of such cells will be needed.

The multiple TAR constructs r or TAR+ribozyme constructs of the present invention are believed to produce only inhibitory RNA, not protein products which could be important in gene therapy strategies.

The use of the multiple TAR element construct of thc present invention is very advantageous. For example, the construct provides for specific inhibition of HIV-1 directed gene expression. The expression of the protective gene product is biologically controlled which is distinctively advantageous since constitutive expression of a TAR-containing transcript on normal cell processes in vivo may be deleterious. The usefulness of the construct is not limited by variability between different HIV isolates. Further, the use of the construct can be expanded by the downstream insertion of sequences such as either Sribozymes or trans-dominant mutants of HIV proteins.

EXAMPLES

The followingnon-limiting examples are given to further describe the present invention. While the present invention ;s exemplified using the HIV system and the TAR element, one skilled in the art will appreciate that inhibition of other viruses can be expected to be effected using the method of the present invention.

Plasmid Construction Plasmids containing different numbers of unidirectional TAR elements under the control of HIV- LTR were constructed. (See Figure 2).

"Multimerized" TAR sequences were cloned downstream of WO 91/10453 PCT/US91/00175 14 the authentic TAR sequence of the 5' HIV-1-LTR deletion mutant CD7, lacking the negative regulatory element (NRE) and having higher level of expression as compared to the wild type HIV-I-LTR [Siekevitz et al, Science 238:1575-1578 (1987)]. Plasmid pCD7 (kindly provided by Stepen Josephs) containing a part of the HIV-1 LTR (-278 +63) was digested with restrictions endonucleases and ligated with the multiple, tandem TAR elements. LTR-lTAR, LTR-4TAR and LTR-5TAR contained one, four and five copies of TAR elements, respectively.

For the construction of plasmids LTR-5TAR and LTR-4TAR, two oligonucleotides containing the sequence for the entire TAR element to +63) of HIV-I flanked by half of the palindromic sequence for Dral and SmaI restriction endonuclease recognition sites were synthesized. The oligonucleotides were purified, phosphorylated and annealed. The annealed DNA (TAR) was ligated in the presence of Dral and SmaI allowing only tandem (directional oriented) ligation of the TAR elements. Plasmid CD7-CAT [Siekevitz et al, Science 238:1575-1578 (1987)] containing a part of the HIV-I-LTR (-278 to +63) was digested with restriction endonucleases HindIII and NcoI, ends were filled and ligated with the multiple, tandem TAR elements. Of several E. coli strain tested only one, Bj 5183: F', recBC, endol, gal, met, str, thi, bio, hsd. (kindly gift from F. Lacroute), was able to maintain these plasmids without rearrangements.

LTR-1-TAR was constructed by digestion the CD7-CAT with HindIII and Ncol, ends were filled and relegated.

WO 91/10453 PCT/US91/00175 Two classes of control plasmids were generated: STAR having a deletion of the upstream promoter sequences (TAR sequences are present but not transcribed), and LTR-OTAR having no TAR sequences but contains the upstream promoter sequences (Fig. 2).

LTR-0-TAR was made by digestion of CD7-CAT plasmid [Siekevitz et al, Science 238:1575-1578 (1987)] with PvuII and NcoI, blunt ended and relegated.

plasmid was constructed by deleting the 5' part of the HIV-LTR from the plasmid LTR-5TAR by digesting with XbaI and PvuII, the ends were filled and ligated. The LTR-tat was constructed by digesting pSVL-tat [Rappaport et al, The New Biologist 1:101-110 (1989a)] with SalI and BamHI; the 350 3p tat containing fragment was isolated, and a blunt end ligation was performed with vector CD7-CAT between HindIII and NcoI sites.

All constructs were confirmed by restriction mapping and sequencing.

The cloning strategy allows the formation of direct, but not inverted repeats of the TAR element, since inverted repeats are cleaved by the SmaI and Dral enzymes during ligation. The correct orientation and secondary structure of each element is presumably important for the desired effect, since TAR functions in transactivation only in a position dependant manner [Peterlin et al, Proc. Natl. Acad. Sci. USA 83:9734-9738 (1986)].

Downregulation of transactivation is dependent on the transcription of the TAR elements To determine the effect of multiple TAR elements on HIV-LTR directed gene expression, TAR WO 91/10453 PCT/US91/00175 16 expression plasmids were cotransfected with LTR-CAT [Siekevitz et al, Science 238:1575-1578 (1987)] and LTR-tat or pSVL-tat [Rappaport et al, The New Biologist 1:101-110 (1989a)] in COS cells.

COS-1 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (GIBCO). 2 x 105 cells were plated in 3ml media in 6 well tissue culture plates one day prior transfection. 25 Ag of total plasmid as used for 3 wells. The amount of the different plasmids are indicated on the figures. Plasmid pBR322 was used as carrier DNA. Transfections were carried out with calcium phosphate procedure [Chen, and Okayama, H.

Hol. Cell. Biol. 7:27-45 (1987)]. 48h after transfection cells were collected (SIGMA cell remover reagent) and crude cellular extracts were made in PBS.

The plasmid RSV-Luciferase was included as an internal control to detect the transfection efficiency.

The amount of CAT protein and relative levels of luciferase activity were determined from extracts of transfected cells. CAT protein was assayed with PRIME 3 PRIME ELISA kit according to the manufacture instruction; LUCIFERASE activity was measured according to P.E. Stanley and S.G. Williams [Stanley, P. E. and Williams, S.G. Anal. Biochem 29:381 (1969)] and activities were expressed in arbitrary units (ARU).

As shown in Figure 3A, multiple TAR elements transcribed from HIV-LTR inhibit HIV-LTR directed gene expression in the presence of tat and the downregulation observed is proportional to the number of TAR elements in the construct. LTR-4TAR and inhibited transactivation an average of WO 91/10453 PC1'/US91/90175 17 and 80%, respectively. LTR-1TAR also has a measurable effect resulting in up to 40% downregulation. This reduction represents a cumulative effect of the inhibition of both CAT and tat expression, since both gene products are under the control of the HIV-I-LTR in this experiment. Multiple TAR elements can suppress transactivation when tat is expressed constitutively from the SV40 late promoter (Fig. 3B), albeit to a reduced level. Resu.s presented in Figures 2A and B illustrate that CAT expression is reduced 82% when tat is expressed from HIV-1 LTR compared to a 50% reduction observed with the constitutively expressed tat.

Transcription of the multimerized TAR sequence is required for efficient downregulation and accumulation of steady state competitor RNA occurs only in the presence of tat (See Fig. 3C). Sequences upstream of the TAR element cannot account for the observed effect. The LTR-OTAR plasmid containing no TAR sequences or 5TAR plasmid having a deletion of the upstream promoter sequences, produce no significant effect on HIV-I LTR directed gene expression (See Fig.

3A, 3B, 3C).

Cotransfection was performed with an another human retroviral LTR to determine the specificity of the effect of multimerized TAR RNA. HTLV-I-LTR-CAT (kindly provided by M. Nerenberg) was used as a reporter gene and HTLV-I-LTR-TAR plasmid was included as transactivator of the HTLV-I LTR [Sodroski et al, Science 225:381-385 (1984); Felber et al, Science 229:675- 679 (1985)]. LTR-tat was also supplied for the transcription of the multimerized TAR elements.

Results, presented in Figure 3D, indicate that the WO 91/10453 PCT/US91/00175 18 expression of multiple TAR RNA elements does not affect HTLV-I promoter activity, suggesting that downregulation of gene expression is specific to the

HIV-LTR.

That the multiple TAR RNA elements can spedifically inhibit transactivation of the HIV gene expression is also evidenced in figure 3 wherein the RSV promoter was coupled with LUCIFERASE reporter gene.

The construct was used to verify that the specifically of the multiple TAR RNA elements do not effect the use of heterologous promoters. No significant difference of RSV promoter activity could be detected with TAR RNA or DNA elements. This indicates the relative promoter activities of the HIV-LTR versus RSV promoter as the proportion of CAT and LUCIFRASE expression.

Inhibition of the transactivation parallels the amount of TAR transcripts LTR-CAT and LTR-tat were cotransfected with increasing amount LTR-5TAR plasmid to determine the effect of different amount of TAR transcripts on the transactivation. RNA was isolated using CINNA/BIOTECX RNAzol reagent according to the company protocol. For Northern analysis, RNA was electrophoresed through a 1% formaldehyde/agarose gel. RNA was transferred onto nitrocellulose paper and hybridizing with Nick translated 32P labeled probe as previously described [Maniatis et al, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory (1982)].

Figure 4A illustrates that increasing the amount of LTR-5TAR plasmid results in a proportional decrease in CAT expression (up to Inhibition cannot be due to the competition for limiting 19 Stranscription factors which associate with the HIV-I-LTR since the amount of transfected LTR upstream sequences was kept constant in this experiment. Increasing amounts of transfected LTR-5TAR plasmid results in a similar increase of LTR-5TAR transcripts (See Fig. 4B). From these experiments, it is concluded that downregulation of HIV-1 LTR directed gene expression is dependent upon the relative amount of expression plasmid DNA introduced into cells, in addition to the number of transcribed TAR elements contained in the expression plasmid.

Northern blots show tat and CAT expression appear to be reduced in parallel by multimerized TAR RNA (Figure 4C), which is expected since they are both driven by HIV-

LTR.

The data of figure 4(a-c) supports the teaching that the down-regulation of gene expression is dependent on the number of transcribed target activator nucleotide sequences. The evidence shows that HIV-1 transactivation by tat can be down-regulated using 7TAR RNA elements in tandem up to 97%, and that the down-regulation is a function of the amount of TAR RNA transcription. The Sdata would suggest that a construct containing more than 7TAR elements would provide an even more effective downregulating effect.

25 Constructs Containing More Than 5 TAR Elements The multiple TAR construct was expanded and plasmids containing up to 50 TARs were constructed and tested, (see Figure pSPT18-polyTAR constructs containing between 15 and S 30 45 TARs were constructed by cutting a pSPT18 vector w T e w te (Pharmacia) with XbaI. The ends were then

S

WO 91/10453 PCT/US91/00175 blunt-ended with the Klenow enzyme and dephosphorylated. The insert was prepared by cutting with PvuII and Scal and isolating the STAR containing fragment (455Bp).

The above vector and insert were then ligated together and used to transform E. coli. Plasmid DNA was prepared from single colonies and clones were selected containing big inserts using methods well known in the art. The orientation of the insert was checked with restriction enzyme digestion using SspI and HindIII Sspl.

Next LTR-5TAR-CAT was cloned. The LTR-CAT vector was cut with XbaI HindIII and larger fragments were 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. coll. Single colonies were checked.

LTR-46TAR was prepared by cutting LTR-CAT with HindIII BamHI and isolating the larger fragment containing the LTR-1TAR pBR322. The prepared as described above, was cut with HindIII BamHI and the larger fragment containing the 45TAR was isolated. The isolated fragments were ligated together and E. coli transformed with the ligated product.

and LTR-50TAR were also prepared.

Vector LTR-5TAR-CAT was cut with SalI BamHI and the fragments containing the LTR-5TAR pBR322 were isolated. pSPT-20TAR or pSPT-45TAR were cut with Sail BamHI and fragments containing the 20TAR or the TAR were isolated. The vector and insert were then 'h WO 91/10453 PCT/US91/00175 21 ligated together and E. coli transformed with ligated product.

The results depicted in Figure 5 show that inhibition of the HIV-1-LTR directed gene expression increases with the number of TARs in the construct until 25 TARs are used. Increasing the number of TARs above 25 does not increase the inhibition. Thus it is believe that the tat protein is saturated at this point. It is also possible that the test is not sensitive enough to detect further increases in inhibition.

The LTR-50TAR can be transferred to a retroviral vector, such as DC-vector (Hantzopaulos et al, Pro. Natl. Acad. Sci. USA 86:3519 (1989)]. The LTR- 50TAR is cut with Xbal, filled in with the Klenow ezyme and inserted into the SnaBI site of the DC vector for high efficiency gene transfer. Other vectors which ensure high efficiency gene transfer would be appropriate for use in the present invention.

Construction of Ribozyme-Poly-TAR For the construction of pRRE-ribozyme, the vector pRRE [Daefler et al, Proc. Natl. Acad. Sci. USA 87:4571-4575 (1990)] containing the RRE (rev-response element under the control of a T7 promoter) was cut with BamHI, dephosphorylated and purified. As the insert, a 65Bp long ribozyme PCR fragment [Chang et al, Clinical Biotechnology 2:23-31 (1990)] flanked by BamHI sites was cut by BamHI and purified. This ribozyme is directed against HIV-1 GAG nRNA and was published in Nature 247: 1222 (1990) by N. Sarver et al.

The vector and insert were ligated and an aliquot of the ligation mix was transformed in E. coli.

WO 91/10453 PCT/US91/00175 22 Plasmids were prepared from individual transformants and were tested by restriction enzyme digestion. 8 clones were found containing the insert. These clones were tested in vitro for biological activity and 3 of the 8 clones were found to have the ability to cut a synthetic substrate (substrate gift of J. Rossi).

For construction of LRT-polyTAR-RRE-ribozyme (see Fig. the vector LTR-46TAR is cut with SalI, the ends filled in with the Klenow enzyme and then cut again with HindIII. The DNA is then purified. For the preparation of the insert, pRRE-ribozyme is cut with HindIII and SmaI. The 314Bp fragment is isolated by gel electrophoresis. The vector and insert are then ligated and used to transform E. coli. Individual colonies will be checked.

The LTR-polyTAR-RRE-ribozyme is transferred to a retroviral vector, such as DC-vector [Hantzopaulos et Sal, Proc. Natl. Acad. Sci. USA 86:3519 (1989)], by cutting with XbaI. The big XabI fragment is filled in by Klenow enzyme and inserted in the SnaBI site of the DCvector. Putting the construct in a retroviral vector is necessary for high efficiency gene transfer and the DC vector is preferred. Nevertheless, any vector which ensures high efficiency gene transfer would be appropriate.

Construction of AGAG-Poly-TAR pRRE-AGAG was constructed by cutting the pRRE vector with BamHI, dephosphorylating the ends and purifying the vector. The AGAG pr-tein encoded in the mutant viral DNA HT4(VI-AE-dhfr) [Lorno et al, Cell 59: 113-120 (1989)], can dominantly interfere with the replication of HIV-1. Plasmid DNA HT4(VI-AE-dhfr) was WO 91/10453 PCT/US91/00175 23 cut with BglII and a 1429 Bp fragment containing AGAG was isolated from 1% agarose gel. The vector and insert were ligated and an aliquot of the ligation mix was used to transform E. coli, Plasmids were prepared from individual transformants and were tested by restriction enzyme digestion, EcoRI SphI.

For construction of LRT-polyTAR-RRE-AGAG (see Figi the vector LTR-46TAR is cut with SalI, the ends filled in with the Klenow enzyme. The DNA is then purified. For the preparation of the insert, pRRE AGAG is cut with EcoRV and Smal. The 1.6 kB fragment is isolated by gel electrophoresis. The vector and insert are then ligated and used to transform E. coli.

Individual colonies will be checked. The construct is inserted in a DC-vector as described above.

A plasmid designated LTR-7TAR was deposited in E. coli at the American Type Culture Collection in Bethesda, Maryland on January 17, 1990 under the accession number 68203. Further, the LTR-50TAR plasmid was deposited in E. coli at the American Type Culture Collection in Bethesda, Maryland on October 12, 1990 under the accession number 68446. The plasmids were deposited under the terms of the Budapest Treaty.

All publication mentioned hereinabove are hereby incorporated by reference.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.

WO 91/10453 24 PCr/US91 /001 ANNEX M3 111ternatonal Application No: PCT!

MICROORGANISMS

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Claims (13)

1. A DNA construct for treating viral infections in a mammal comprising a vector and a promoter operably linked to at least two tat activation response elements so that they are transcribed in tandem.

2. The DNA construct according to claim 1 and 50 tat activation response elements.

3. The DNA construct according to claim 2 activation response elements.

4. The DNA construct according to claim 1 promoter is regulatable by a viral product. The DNA construct according to claim 4 promoter is HIV-1 LTR.

6. The DNA construct according to claim 1 vector is pCD7.

7. The DNA construct according to claim 1 comprises a DNA segment encoding a ribozyme viral RNA.

8. The DNA construct according to claim 1 comprises a DNA segment encoding a transdom mutant viral protein.

9. The DNA construct according to claim 8 protein is GAG. having between having 25 tat wherein said wherein said wherein said which further specific for a which further inant negative wherein said 9* C. A DNA construct consisting essentially of a vector and a promoter operably linked to one target response element.

11. A method of treating viral infection comprising the steps of: obtaining cells from a viral infected patient; 30 (ii) introducing the construct according to claim 1 or claim 8 into said cells; and (iii) introducing said cells resulting from step (ii) back to said patient under conditions such that said treatment is effected.

12. The method according to claim 11 wherein said virus 26 is a human immunodeficiency virus (HIV).

13. The method according to claim 11 wherein said cells are bone marrow cells or blood cells.

14. A method of inhibiting HIV-1 replication comprising introducing into viral infected cells said construct according to claim 1 or claim 10 under conditions such that inhibition is effected. The method according to claim 14 wherein said virus is HIV.

16. A method of inhibiting viral replication comprising introducing into a cell infected with said virus the construct according to claim 1, wherein- aproduct of said virus regulates transcription of said elements so that said inhibition is effected. a I go o o* 1 *wl