WO1999057265A1

WO1999057265A1 - Chimeric decoy rnas having synergistic anti-hiv activity

Info

Publication number: WO1999057265A1
Application number: PCT/US1998/004219
Authority: WO
Inventors: Julianna Lisziewicz
Original assignee: Julianna Lisziewicz
Priority date: 1998-05-04
Filing date: 1998-05-04
Publication date: 1999-11-11
Also published as: AU9196798A

Abstract

A construct comprising a unit having a first group comprising at least one unit of the Tat activation response element (TAR), and a second group comprising at least one unit of a Rev binding element, is useful to suppress production of the HIV virus within cells. The preferred inhibitory construct is the combination of Tat-binding and Rev-binding RNA, preferably expressed together from an HIV-inducible promoter. This Tat-binding and Rev-binding chimeric RNA is the first drug which has a strong synergistic antiviral activity among the domains of a single molecule.

Description

CHIMERIC DECOY RNAS HAV ING SYNERG ISTΪC ANTI-HIV ACTIVITY

Field of the invention

The present invention relates generally to the field of treatment of viral diseases in human beings and animals. More specifically, it relates to materials and methods for intracellular immunization against Human Immunodeficiency Virus (HIV) infection. The inventors have found that a construct comprising a unit having a first group comprising at least one unit of the Tat activation response element (TAR), and a second group comprising at least one unit of a Rev binding element, is useful to suppress production of the HIV virus within cells. The preferred inhibitory construct is the combination of Tat-binding and Rev-binding RNA, preferably expressed together from an HIV-inducible promoter. This Tat-binding and Rev-binding chimeric RNA is the first drug which has a strong synergistic antiviral activity among the domains of a single molecule.

Background of the Invention

The tat protein of HIV-1 transactivates viral gene expression and is essential for viral replication. The tat activation response element (TAR) has been localized within the region of the first 44 nucleotides downstream of the transcription initiation site. This region, present in all HIV-1 transcripts, forms an unusually stable stem loop structure, and several lines of evidence suggest that the transcriptional effect of the tat protein is mediated through its interaction with the TAR region of viral RNA. It has been demonstrated that the Tat protein binds to the TAR RNA sequences.

The Rev protein of HIV-1 is another important factor in viral replication. It 2 helps activate HIV-1 gene expression, and part of its mechanism for action involves binding with a portion of the HIV RNA. In the case of Rev, the corresponding RNA protein is termed the Rev response element (RRE) (Malim et al.; HIV-1 structural gene expression requires binding of the Rev transactivator to its RNA target sequence. Cell 60:675-683 (1990). The Rev protein acts after the transcription phase to facilitate both the transport of incompletely spliced viral mRNA from the infected cell's nucleous to the cell's cytoplasm and the production of viral structural proteins (Malim et al.; the HIV-1 Rev transactivator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA 1989, Nature 338:254- 257; Hope et al.; Trans-dominant inhibition of HIV-1 Rev occurs through formation of inactive protein complexes 1992 J. Virol. 66:1849-1855). After a large amount of RNA has been made and small regulatory molecules have been accumulated, several Rev proteins bind to the RRE RNA element and the Rev proteins together with other cellular proteins can transport unspliced and singly spliced HIV-1 RNA from the nucleus to the cytoplasm (Meyer et al.; The HIV-1 Rev transactivator shuttles between the nucleus and the cytoplasm, 1994 Genes Dev. 8:1538-1547).

This RNA is ready to be packaged into a protective envelope, or capsid, and the resulting infectious viral particles, or virions, can then be released from the cell.

Several studies have focused on characterizing the minimum effective portion of the RRE region for HIV-1 RNA. Different candidate versions of the minimal RRE have been reported, several of which are potentially useful as elements of the present invention. For example, studies of minimal RRE for HIV-1 rev have revealed that an 88-base truncated motif including the SL2 region (designated bul) is sufficient to mediate transactivation in vivo and that two copies of this motif function as effectively as the 234-base wild-type RRE (Huang, X., T.J.

Hope, B.L Bond, D.McDonald, K. Grahl, T.G. Parslow 1991 , J. Virol. 65:2131- 2134). Also, a 45-nt transcript encompassing stem-loops (SL) I .A. and IB., and a 13-nt molecule containing part of SLIIB (Lee, et al.; Inhibition of HIV-1 in Human T- 3 cells by a potent Rev response element decoy consisting of the 13-nucleotide minimal Rev binding domain. J. Virol. 68:12 8254-64) has been reported, along with data that indicates that the direct responsiveness to Rev is limited. Other studies using in vitro selection experiments have found RRE mutations in the SL2 region which resulted in tighter Rev binding (Apatamer 1 and 2); it was further demonstrated that tighter binding correlates with their responsiveness to Rev in vivo (Symensma, T.L, L Giver, M. Zapp, G.B. Takle, A.D. Ellington [1996] J. Virol. 70:179-187).

It has been demonstrated that genetic constructs having a vector and a promoter operably linked to at least two DNA segments encoding HIV-1 tat activation response (TAR) elements so that the elements are transcribed in tandem can be used to sequester the tat protein in cells, and thereby inhibit replication of the HIV-1 virus. See USSN 596,299 "Vector with Multiple Target Response Elements Affecting Gene Expression" filed October 15, 1990, by J. Lisziewicz, which is incorporated herein by reference as if set forth in full.

Genetic constructs using multiple copies of DNA that encode TAR RNA (Polymeric-TAR genes) have been developed and explored for their ability to block Tat protein activity. Polymeric TAR was combined with an antisense-faf gene (J. Lisziewicz et al.; An autoregulated dual-function antitat gene for HIV-1 gene therapy. J. Virol. 69:1 206-12 (1995); J. Lisziewicz et al; Antitat gene therapy: a candidate for late-stage AIDS patients. Gene Ther 2:3 218-22 (1995)) and with a ribozyme gene (Lisziewicz et al.; Inhibition of HIV-1 replication by regulated expression of a polymeric Tat activation response RNA decoy as a strategy for gene therapy in AIDS Proc. National Acad. Sci. U.S.A. 90:17 8000-4 (1993)) to augment the inhibition of HIV-1 replication. Both combination genes were shown to be effective inhibitors of HIV-1 replication, mainly because the Tat protein function was inhibited by the product of the polymeric-TAR gene. However, these molecules were not found to significantly contribute to the effect of polymeric-TAR, since they are produced in a low amount and in a Tat-regulated manner. In the case of a polymeric-TAR and ribozyme combination, the antiviral effect of the ribozyme was so little that the highly sensitive PCR test for the presence of RNA could not show any cleaved HIV-1 RNA product. This construct also contains one copy of the wild- type RRE in order to transport the ribozyme from the nucleus to the cytoplasm, not to inhibit HIV or Rev function. (Lisziewicz et al.; Inhibition of HIV-1 replication by regulated expression of a polymeric Tat activation response RNA decoy as a strategy for gene therapy in AIDS Proc. Nat'l Acad. Sci. U.S.A. 90:17 8000-4 1993). In the case of the combination of polymeric-TAR with antisense-faf, the effect of the antisense molecule was also extremely modest compared to the antiviral activity of the polymeric-TAR (J. Lisziewicz et al.; An autoregulated dual-function antitat gene for HIV-1 gene therapy. J. Virol. 69:1 206-12 (1995); J. Lisziewicz et al; Antitat gene therapy: a candidate for late-stage AIDS patients. Gene Ther 2:3 218-22 (1995)).

Brief Summary of the Invention

It is an object of the present invention to provide a means for inhibiting viral production within cells. It is also an object of the invention to provide a means for preventing and treating lentivirus infections. It is yet another object of this invention to treat or prevent viral infections with a minimum of side effects. Yet a further object of this invention is to prevent or treat viral infections within living cells while yet preserving the viability and function of the cells.

The subject invention concerns materials and methods to inhibit viral replication and to treat viral infections in humans and animals. The materials and methods of the subject invention are described in terms of lentiviruses, but one of ordinary skill in the art will recognize they are applicable to a variety of viruses. In one embodiment, the subject invention pertains to new RNA molecules comprised of selected RNA elements, preferably including one or more TAR element transcribed in tandem with one or more Rev binding elements transcribed in tandem, more preferably including 2-7 TAR elements and 2-4 Rev binding elements, and most preferably including 5 TAR elements and 2 Rev binding elements. Rev binding elements are known in several forms, and preferred forms include the wild-type RRE, Aptamer 1 (Apt1), Aptamer 2(Apt2), bul and 2 or more bul (2bul, 3bul, etc.).

Another embodiment of the present invention is a DNA construct having a vector and a promoter operably linked to a unit having at least two to seven, more preferably five DNA segments encoding HIV-1 Tat activation response (TAR) elements so that the TAR elements are transcribed in tandem, and at least one, more preferably at least two, DNA segments encoding HIV-1 Rev binding elements so that the Rev binding elements are transcribed in tandem. The DNA construct preferably includes a promoter capable of being regulated by HIV-1 Tat protein, and is preferably a primate lentivirus long terminal repeat (LTR) promoter such as the LTR promoter for Human Immunodeficiency Virus -1 "(HIV-1) LTR".

In another embodiment of the present invention, the constructs have two to five, preferably three, repeating units. In yet another embodiment, the present invention relates to articles including the RNA or DNA constructs and a delivery system.

For DNA constructs, the preferred delivery systems include viral gene delivery vectors, including adenovirus, herpesvirus, adeno-associated virus, SV-40 virus, retrovirus and lentivirus vectors as well as non-viral gene delivery systems, including liposomes, virosomes, and polyethylenimine conjugates and other conjugates. Among the non-viral gene delivery systems, virosomes are preferred. Most preferred are retrovirus or lentivirus vectors capable of integrating the construct into the genetic material in the nucleous of the target cell. Where retrovirus or lentivirus vectors are used, it is preferable to modify the cells in a transient fashion by loading them with dNTP. (See USSN 08/989,301 titled Materials and Methods for Gene Transfer, filed December 11 , 1997), in order to be able to transduce, that is, add the foreign genetic material to the quiescent (non- dividing) cells.

For RNA constructs, the preferred delivery systems include viral gene delivery systems and non-viral gene delivery systems. Preferred are the non-viral gene delivery systems, including liposomes, virosomes, and polyethyleneimine and other conjugates.

The genetic constructs of the present invention are preferably delivered to cells using high efficiency transfection techniques such as antibody-mediated delivery of genes to cells. In the present case, a gene delivery complex compatible with a specific type of targeted cell is formed from a carrier, a delivery particle, and the DNA construct. Suitable materials and techniques are described in USSN

60/058,933, "Method of Delivering Genes into Antigen Presenting Cells", filed September 15, 1997, by J. Lisziewicz, which is incorporated herein by reference as if set forth in full.

The subject invention also concerns a method for inhibiting viral replication which comprises administering a combination of multiple-TAR and multiple-REV binding elements, which the inventors have demonstrated to be advantageously included in a single molecule. These multiple elements can be administered in the form of a single molecule which demonstrates an enhanced ability to inhibit viruses dependent upon the activities of Tat and Rev proteins. The genetic construct of the subject invention preferably also includes an inducible promoter to direct the expression of the TAR and Rev binding elements. Suitable promoters include for example, an HIV-1 -LTR promoter and modifications thereof. These constructs would work if they are expressed from constitutive promoters such as cytomegalovirus, SV40 promoter and Moloney murine leukemia virus LTR. However, inducible production of the product from the construct is advantageous as it limits the activity of the construct to a time and place where it is needed, thereby minimizing potential side effects.

An advantage of the present invention is that the DNA constructs which combine TAR and Rev binding elements as described herein yield greatly enhanced activity over similar constructs using the elements separately. Another advantage of the present invention is that it allows lower concentrations of the constructs to be used. Yet another advantage is that the constructs are active only in infected cells, and remain quiescent in uninfected cells. As a result, the potential side effects are minimized. Further, the combination of multiple TAR and RRE elements in the same antiviral vector targets two important viral proteins at the same time. This provides further constraints for any escape mutants, as such a mutant has to confer simultaneous mutations in both Rev and Tat proteins. Furthermore, this fusion molecule suppresses both virus replication and virus expression from infected cells.

Brief Description of the Drawings

Fig. 1 is a schematic representation of the polymeric-TAR and Rev binding

DNA constructs, each of which has a promoter (LTR) and variable amounts of TAR and Rev binding elements.

Fig. 2 depicts the known structure of the wild-type HIV-1 RRE and its variants aptamer 1 (Apt1), aptamer 2 (Apt2) and bul. Fig. 3 is a comparison of anti-Rev activity of several different constructs, each having an LTR promoter and 5 TAR elements, with several Rev binding variants, including the wild-type RRE (wtRRE), Apt1 , Apt2 and bul. One variant, bul, is shown as both 1 and 2 elements (bul and 2bul). Fig. 4 is a comparison of anti-TAT activity of various DNA constructs, each of which has a promoter and 5 TAR elements, and variable types of Rev binding elements, shown as the Apt1 and 2bul variants. Fig. 5 is a comparison of the anti-HIV activity of different DNA constructs having variable amounts of TAR and variable types of Rev binding elements. LTR- OTAR is a control which is effectively the promoter only, and which does not have antiviral activity; LTR-4TAR is in prior art; the Rev binding elements are shown as 2bul and Apt1 variants. Fig. 6 compares the anti-HIV activity of three different DNA constructs at varying concentrations. LTR-4TAR is in the prior art. Variable amounts of TAR are shown with one Rev binding element identified as the Apt1 variant.

Fig. 7 compares the anti-HIV activity of three different DNA constructs at varying concentrations. LTR-4TAR is the prior art. Variable amounts of TAR are shown with two copies of the bul variant of the Rev binding element.

Fig. 8 depicts the mechanism of action of the constructs of the present invention.

Fig. 9 a schematic representation of the multi-unit polymeric-TAR and Rev binding constructs. Two examples are shown, for a non-viral gene delivery system (Plasmid constructs) and for a viral gene delivery system (Retrovirus vector construct).

Detailed Description of the Invention

The subject invention provides materials and methods to inhibit viral replication in humans and animals. Specifically provided herein are unique genetic constructs and gene therapy methods to inhibit or prevent infection by lentiviruses, including HIV. 9

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. Fig. 1 is a schematic representation of various polymeric-TAR and Rev binding constructs studied by the inventor. All constructs were made using the procedure described in Example 1 , below. All constructs contain a CD7-LTR element as a promoter, which is derived from the wild type HIV-1 -LTR, by deletion of the negative regulatory element (NRE). A plasmid construct, LTR-5TAR-CAT, was used as the backbone to extend the LTR-5TAR gene sequence with the previously described Rev binding elements. Wild type RRE, bul (Huang, et al.; Minimal Rev-response element for type 1 HIV. J. Virol. 65:2131-4 (1991) Apt1 and Apt2 (Symensma, et al,: RNA aptamers selected to bind HIV-1 Rev in vitro are Rev responsive in vivo. J. Virol., 70:1 179-87 (1996)) were amplified with primers 5 ' C T A T A G G T C G A C A C A T T G T A A C A G A T C A G a n d 5 ' -

CCAAATGGGATCCAGAGATTTATTACTCC, which embedded Sail and BamHI sites. These products were inserted between the Sail and BamHI sites of vector pLTR-5TAR-CAT to replace the CAT gene. Tandem bul, termed 2bul, were obtained by ligating a second bul PCR product 3' of LTR-5TAR-bul. Plasmids pLTR-5TAR-Apt1 and pLTR-5TAR-2bul were treated with Bglll and relegated to obtain pLTR-1TAR-Apt1 and pLTR-1TAR-2bul constructs.

Fig. 2 depicts the known structure of the HIV-1 RRE and its variants Apt1 , Apt2 and bul. Apt 1 and 2 has been selected because they had been reported to bind more strongly to the Rev protein in vitro than the wild type RRE. (Symensma, et al,: RNA aptamers selected to bind HIV-1 Rev in vitro are Rev responsive in vivo.

J. Virol., 70:1 179-87 (1996)) Bul has been also previously described (Huang, et al.; Minimal Rev-response element for type 1 HIV. J. Virol. 65:2131-4 (1991)) as a minimal Rev-binding domain in the wild type RRE. 10

Fig. 3. reports Anti-Rev activity of constructs having both TAR and Rev binding elements as percent Rev inhibition. To perform the analysis, a cell line was altered to produce an easily measured protein (CAT) in response to the presence of the Rev protein. A human kidney cell line ( 293T cells) was used for the transient transfection experiment. Transfection efficiency as high as nearly 90% was achieved using the following Ca₃(PO₄)₂-based protocol with the 293T cells. One microgram of DNA was used for each well of a 24-well plate. Plasmid DNA was first mixed with equal volume of 0.5 M CaCI₂, vortexed and then mixed with buffer containing 50 mM Hepes (pH=7.1), 280 mM NaCI, and 1 mM NaH₂PO₄ at a 1 to 1 ratio followed by continuous vortexing. The mixture was then plated on top of the cells and subsequently incubated in an incubator at 37(C with 5% CO₂ for overnight. Cells were then washed with PBS and covered with fresh DMEM medium containing 10% FCS. Forty-eight hours later, cell numbers were counted and cells were lysised following the protocol as described in the CAT ELISA system provided by Boehringer Mannheim. An ELISA was performed to quantify CAT protein present in the cell lysis. The values as shown are the averages of three experiments. 50 ng pDM128 and 4 ng pCMV-Rev plasmid DNA was used to induce Rev-dependent CAT expression (Huang et al; J. Virol 65:2131-4 (1991)), and with 0.4 ng LTR-Tat to induce the expression of TAR-RRE genes. LTR-OTAR and carrier plasmid Bluescript was added so that the total amount of HIV-1 LTR was 200 ng in each transfection. LTR-OTAR was used as control. Forty-eight hours after transfection, the same number of cells were lysed and CAT protein was measured by ELISA according to the manufacturer's protocol (Boehringer Mannheim, Indianapolis, IN). The values as shown are the averages of three separate experiments.

This figure demonstrates that different Rev-binding elements (Apt1 , Apt2 bul, 2bul, and wild type RRE) combined with the polymeric-TAR molecule are capable 11

of interrupting the function of the Rev protein. Based on these data, 2bul was found to inhibit the function of the Rev protein as effectively as wild type RRE. We have chosen to further study the anti-HIV-1 activity of two hybrid molecules, LTR-5TAR- 2bul and LTR-5TAR-Apt1. The anti-Rev data here, which is also confirmed in the anti-HIV studies reported in Figs. 6 and 7, shows that Apt1 expressed alone from HIV-LTR can inhibit a maximum of about 50% of Rev activity in contrast to 2bul alone which inhibited HIV-1 through inhibition of the activity of the Rev protein up to 80%. These results indicate that the most effective construct is the 5TAR-2bul combination.

Fig. 4 compares the anti-Tat activity of 5TAR, 5TAR-Apt1 , and 5TAR-2bul constructs. LTR-CAT was cotransfected with LTR-Tat as described above at Fig. 3, and each of the antiviral genes as described earlier (Lisziewicz, J., et al.: Tat regulated production of multmerized TAR RNA inhibits HIV-1 gene expression. New Biologist 3: 82-90, 1991). LTR-OTAR was used as a control. Forty-eight hours after transfection, the same number of cells were lysed and CAT protein was measured by ELISA according to the manufacturer's protocol (Boehringer Mannheim, Indianapolis, IN). Values are the averages of three separate experiments. This figure demonstrates that the most effective Rev binding elements (Apt1 and 2bul) in the combination molecule does not degrade the anti-Tat activity of the polymeric-TAR molecule.

Fig. 5 demonstrates the anti-HIV-1 activity of the different TAR and Rev binding constructs. To demonstrate the inhibition of HIV viral production, different antiviral vectors were cotransfected with pLW-int6, which encoded an integrase defective variant of HIV-1. The transient transfection was performed as described above at Fig. 3. At 72 hours post-transfection, p24 was measured from the cell culture supernatant using an ELISA system provided by Coulter. 12

LTR-OTAR is the control which does not have an antiviral activity. The polymeric-TAR construct, LTR-4TAR, is a prior art construct known to sequester the Tat protein, and it is used in this assay to demonstrate the state of art of the polymeric-TAR technology. New constructs expressing Rev-binding motifs from HIV-1 LTR, 2bul and Apt1 , have been shown in Fig. 3 to sequester Rev and therefore, as demonstrated in this figure, can inhibit HIV-1 production. This figure also clearly demonstrates the superior anti-HIV activity of the combination constructs LTR-5TAR-2bul and LTR-5TAR-Apt1.

Fig. 6 demonstrates the anti-HIV-1 activity of the Apt1 Rev binding element alone and in combination with the polymeric TAR element. This experiment was done as described in Fig. 5 using LTR-OTAR as a control. Different amounts of LTR-5TAR-Apt1 plasmids were used in cotransfection to assess anti-HIV activity. Inhibition of HIV-1 replication is dependent on the concentration of the antiviral molecules which is also depicted on the figure by the trendlines of the different antiviral molecules. These trendlines describe that antiviral activity islogarithmicallyy increasing with the concentration of the antiviral RNA product, (or, drug). Mathematical analysis of the trendlines resulted in the following logarithmically equation between the concentration of the antiviral molecules (x) and % of HIV-1 inhibition (y).

LTR-4TAR y=41.9 Ln(x) + 9.7 R2=0.94

LTR-1TAR-Apt1 y=20.0 Ln(x) + 13.8 R2=0.71

LTR-5TAR-Apt1

y= 9.1 Ln(x) + 78.8 R2=0.92

The mathematical analysis demonstrates that both polymeric-TAR containing constructs have a very effective antiviral activity at high concentration in contrast to the Apt1 construct, which even in high concentration does not inhibit HIV-1 more 13 than 50%. However, at low concentration, none of the single constructs, polymeric- TAR (LTR-4TAR) and Rev binding element (LTR-1TAR-Apt1) inhibited HIV-1 significantly, but the combination construct (LTR-5TAR-Apt1 ) is highly effective.

Fig. 7. demonstrates the anti-HIV-1 activity of the 2bul Rev binding element alone and in combination with the polymeric TAR element. T;his experiment was done as described in Fig. 5 using LTR-OTAR as a control. Different amounts of LTR-5TAR-2bul plasmids were used in cotransfection to assess anti-HIV activity. Inhibition of HIV-1 replication is dependent on the concentration of the antiviral molecules which is also depicted on the figure by the trendlines of the different antiviral molecules. These trendlines describe that antiviral activity increases logarithmically with the concentration of the antiviral RNA (or, drug). Mathematical analysis of the trendlines resulted in the following logarithmically equation between the concentration of the antiviral molecules (x) and % of HIV-1 inhibition (y).

LTR-4TAR y = 41.9 Ln(x) + 9.7 R2 = 0.94

LTR-1TAR-2bul y = 36.4 Ln(x) + 8.9 R2 = 0.92

LTR-5TAR-2bul

y = 9.2 Ln(x) + 78.9 R2 = 0.95

The mathematical analysis demonstrates that both polymeric-TAR containing constructs have a very effective antiviral activity at high concentration. The 2bul construct alone was less effective at high concentration than the other two, however it could reach inhibition up to 80%, which suggest that 2bul is a more effective inhibitor that Apt1 , which even in high concentration did not inhibit HIV-1 more than 50%(See Fig. 6). At low concentration, (similarly to the other construct, see figure 6) none of the single constructs, polymeric-TAR (LTR-4TAR) and Rev binding element (LTR-1TAR-2bul) inhibited HIV-1 significantly, in contrast to the combination construct (LTR-5TAR-2bul), which was highly effective. 14

The data for both the LTR-5TAR-Apt1 and the LTR-5TAR-2bul constructs appeared, by inspection, to show strong synergy. For quantitative estimation of the effect, we used the interaction index (I) of Berenbaum ,M.C; Synergy, Additivism and Antagonism in Immunosuppression. Clin. Exp. Immunol. 28:1-18, (1997)) which is based on the most commonly used definition of interaction (due to Loewe

S. and Muischnek, H. Effect of Combinations: Mathematical Basis of Problem. Arch. Exp. Pathol. Pharmakil. 114:313-326, (1926)).! can be computed according to the equation

I = (CX,1 in combination/CX,1) + (CX,2 in combination/CX,2) where CX,1 in combination and CX,2 in combination are the concentrations of elements 1 and 2, respectively, in the combined construct required to reduce HIV-1 production by X%. CX,1 and CX,2 are the concentrations of non-combination constructs required to achieve the same X% decrease. For an additive effect, 1 = 1 ; for synergy, I <1 ; for antagonism, I >1. I is computationally equivalent to the "combination index" introduced for mutually exclusive interaction in the "median effect analysis" (Chou, T.C., and Talalay, P. Generalized Equations for the Analysis of Inhibitions of Michaelis-Menten and Higher Order Kinetic Systems with two or More Mutually Exclusive and Nonexclusive Inhibitors. Eur. J. Biochem. 115:207- 216 (1981)). The individual dose response curves for separate and combined constructs were analyzed by fitting a third degree polynomial spline (with smoothing function I = 0.3) to the log-log transformed data using the program package JMP (SAS Institute, Cary, NC). This procedure yielded reasonable fits to all of the data, and led to conservative estimates of the degree of synergy (i.e., to conservative estimates of deviation of the interaction index from unity). Spline fits (Cf. Suhnel, J. Evaluation of Synergism and Antagonism for the Combined Action of Antivral

Agents. Antiviral Res. 13:23-40) (1990)) were used because the dose-response curves were not well modeled by logistics, hence, we could not use the "median- effect" (Chou, T.C., and Talalay, P. Generalized Equations for the Analysis of 15

Inhibitions of Michaelis-Menten and Higher Order Kinetic Systems with two or More Mutually Exclusive and Nonexclusive Inhibitors. Eur. J. Biochem. 115:207-216 (1981)). ) or "Combo" (Bunow and J.N. Weinstein. COMBO: A New Approach to the Analysis of Drug Combinations in Vitro. Annals of the New York Academy of

Science 616:490-494, (1990)) forms of analysis, and the experimental design was not appropriate for three dimensional modeling response surface modeling (Bunow et al. COMBO, N.Y. Acedemy of Science 616:490-494, (1990); Greco, W.R., Bravo, G. And Parsons, J.C. "The Search for Synergy: A Critical Review from a Response Surface Perspective. Pharmacol. Reviews 47: 331-385, (1995); Pritchard, M.N. and

Shipman C, Jr. "A Three Dimensional Model to Analyze drug-drug Interactions." Antiviral Res. 14: 181-206 (1990)). Parenthetically, much greater degrees of apparent synergy would be found using the Webb "partial products" definition (Webb, J.L. Effect of More Than One Inhibotor In Enzymes and Metabolic Inhibitors, Vol. 1 , pp. 66-79 and 487-512, Academic Press, N.Y. (1963)) of interaction, but we did not consider the partial products formalism appropriate for this type of study. The many complex issues involved in assessments of synergy and antagonism have been cogently reviewed by Greco, et al., (The Search for Synergy; A Critical Review from a Response Surface Perspective. Pharmacol. Reviews 47: 331-385, (1995)).

By inspection of the data depicted in Figs. 6 and 7, there appears to be a pronounced synergistic interaction between the TAR and Rev binding elements in both types of combined constructs. Very conservative quantitative estimates of this effect were obtained in terms of the Berenbaum "interaction index" (I). For the LTR- 5TAR-Apt1 constructs, we found I = 0.37 at 50% inhibition; for the LTR-5TAR-2bul constructs, I = 0.24 at 50% inhibition. That is, these highly conservative estimates indicate that the combined constructs would be approximately 3- and 4-fold more potent, respectively, than would be predicted on the basis of additive effects. 16

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Fig. 8. demonstrates the mechanism of action for the constructs of the present invention. In an uninfected cell (no HIV-1) the TAR-RRE (that is, polymeric- TAR and one or more Rev binding elements) gene is not expressed because the Tat protein is not present to activate gene expression. Therefore, the antiviral RNA

(drug) is not produced. The figure, however depicts an HIV-1 infected cell (see integrated provirus, HIV-1). In this cell, the Tat protein is produced by the provirus and activates the expression of HIV-1 LTR. As soon as the TAR-RRE gene is activated, the polymeric-TAR-RRE RNA (the drug) is produced in the nucleus and the polymeric-TAR part of the molecule sequesters the Tat protein and the polymeric-RRE part of the molecule sequesters the Rev protein. Sequestering Tat results in inhibition of the expression of all HIV-1 genes (including the rev gene). Sequestering the Rev protein results in inhibition of the transport of single spliced and unspliced HIV-1 RNA from the nucleus to the cytoplasm. Therefore, both gene expression and provirus production is blocked in cells containing the chimeric antiviral gene.

Fig. 9 is a schematic representation of the multi-unit constructs. Non viral gene delivery systems may use the plasmid construct. This specific construct contains 3 copies of the TAR-RRE chimeric molecule, however depending on the size limitations, two and even one copy of the chimeric molecule can be used. If there is no size limitation on the gene delivery system, the use of higher numbers of units of the chimeric molecule is preferred, because, theoretically, maximal inhibition of HIV production will result. However, practically we expect complete inhibition of HIV with 3 units of the chimeric TAR-RRE because the strong synergistic antiviral activity of these molecules. The other example is the retrovirus vector containing the 3x(5TAR-RRE) antiviral gene. Retrovirus vectors (including lentivirus vectors) are preferred gene delivery vehicles for these constructs because they can efficiently integrate the antiviral gene into the chromosome of the target 18

cell. This can be an important feature if the gene is introduced into the hematopoietic stem cells in order to repopulate the immune system with HIV- resitant cells.

Claims

19What is claimed:

1. An RNA molecule comprising a unit having a first group comprising at least one DNA segment encoding Tat activation response (TAR) element and a second group comprising at least one DNA segment encoding Rev binding element, the groups having synergistic antiviral activity.

2. An RNA molecule as in Claim I, wherein the first group comprises 2-7 TAR elements transcribed in tandem, and the second group comprises 2-4 Rev binding elements transcribed in tandem.

3. An RNA molecule as in Claim 2, wherein the first group comprises 5TAR elements and the second group comprises 2 Rev binding elements.

4. An RNA molecule as in any one of Claims 2 to 3, having at least 2 units.

5. An RNA molecule as in any one of Claims 2 to 3, having at least 3 units.

6. An RNA molecule comprising a unit having a first group comprising at least one TAR element and a second group comprising at least two Rev binding elements so that the rev binding elements are transcribed in tandem.

7. An RNA molecule as in Claim 6, wherein the first group comprises 2-7 TAR elements transcribed in tandem, and the second group comprises 2-4 Rev binding elements. 20

8. An RNA molecule as in Claim 7, wherein the first group comprises 5 TAR elements and the second group comprises 2 Rev binding elements.

9. An RNA molecule as in any one of Claims 7 to 8, having at least 2 units.

10. An RNA molecule as in any one of Claims 7 to 8, having at least 3 units.

11. A DNA construct comprising a promoter operably linked to a unit having a first group comprising at least one DNA segment encoding Tat activation response

(TAR) element, and a second group comprising at least one DNA segment encoding Rev binding element.

12. A DNA construct as in Claim 11 , wherein the first group comprises two to seven TAR elements so that the TAR elements are transcribed in tandem, and the second group comprises two to four DNA segments encoding Rev binding element so that the Rev binding elements are transcribed in tandem.

13. A DNA construct as in Claim 12, wherein the first group comprises 5 TAR elements and the second group comprises 2 Rev binding elements.

14. A DNA construct as in Claim 12 or 13 having at least 2 units.

15. A DNA construct as in Claims 12 or 13, having at least 3 units.

16. The DNA construct according to Claim 11 , wherein the promoter is a primate lentivirus long terminal repeat (LTR) promoter. 21

17. The DNA construct according to Claim 16, wherein the primate lentivirus LTR is the Human Immunodeficiency Virus- 1 (HIV-1) LTR.

18. The DNA construct according to Claim 11 , wherein the promoter is capable of being regulated by HIV-1 Tat protein.

19. The DNA construct according to Claim 16, wherein the Rev binding elements are selected from the group consisting of wild-type RRE, Aptamer 1 , Aptamer 2, bul and 2 or more bul, and mixtures thereof.

20. The DNA construct according to Claim 11 , further comprising a vector selected from the group consisting of viral gene delivery vectors, including adenovirus, herpesvirus, adeno-associated virus, SV-40 virus, retrovirus, and lentivirus vectors.

21. The DNA construct according to Claim 20, wherein the vector is a retrovirus or lentivirus vector capable of transducing quiescent target cells.

22. An article comprising the construct according to any one of Claims 1 , 6, or

11 ,, further comprising a non-viral gene delivery system, including liposomes, virosomes and polyethyleneimine and other conjugates.

23. A method of inhibiting lentivirus replication, the steps comprising introducing a construct according to any one of Claims 1 , 6, or 11 into a cell population including hematopoietic stem cells, bone marrow cells, cord blood cells, peripheral blood cells, lymphocytes, macrophages, dendritic cells, and mixtures thereof. 22

24. A method of treating or preventing lentivirus infections, the steps comprising introducing a construct according to any one of Claims 1 , 6, or 11 into a cell population including hematopoietic stem cells, bone marrow cells, cord blood cells, peripheral blood cells, lymphocytes, macrophages, dendritic cells, and mixtures thereof.