WO2004041194A2 - Inhibition of hiv-1 replication - Google Patents

Abstract

Compositions for the inhibition of HIV-1 comprising the use of polyamide nucleic acids (PNAs) that bind to either the transactivation response element or primer binding sequence of the HIV virus are taught. The PNAs may be bound to a membrane transducing peptide, such as transportan. The methods for treating patients infected with HIV or for protecting non-infected patients against HIV infection using the anti-HIV compositions are also taught.

Description

SPECIFICATION

INHIBITION OF HIV-I REPLICATION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of United States Application Serial Number 60/423,015, filed November 1, 2002.

GOVERNMENT INTEREST

This research was supported by the grants NIAID (AI42520) and NCI (CA72821). The government may have certain rights in the invention.

FIELD OF THE INVENTION

This invention is in the field of viral inhibition. Particularly, the field of the present invention is HIV-1 inhibition via several mechanisms of inhibiting transcription and replication of the HIV-1 virus. The invention is also in the field of treating patients with viral infections, specifically HIV infections.

BACKGROUND

Various publications or patents are referred to in parentheses throughout this application to describe the state of the art to which the invention pertains. Each of these publications or patents is incorporated by reference herein. Complete citations of scientific publications are set forth in the text or at the end of the specification.

[54704-8057/LA033020.002] -1- Acquired immune deficiency syndrome (AIDS) has reached worldwide epidemic proportions in spite of enormous efforts for the prevention of AIDS. Currently, clinical treatment of AIDS patients with highly active antiretroviral therapy (HAART) involves a combination of drugs targeting the two key human immunodeficiency virus type 1 (HIV-1) enzymes, namely the reverse transcriptase and protease. HAART has been successful in reducing the viral load (Aalen et al., 1999; Centers for Disease Control and Prevention, 1998). Major impediments to successful HIV-1 therapy include the rapid emergence of drug- resistant viral strains, the persistence of integrated provirus in host cells, and the abundance of latently infected cells. Replication of HIV-1 occurs through a multistep reverse transcription process, in which the single-stranded viral RNA genome is converted into a double-stranded proviral DNA intermediate prior to integration into the host chromosome. The process of reverse transcription is carried out by the virally encoded reverse transcriptase, which utilizes the cellular tRNA₃ ^Lys bound to the primer-binding site (PBS) near the 5' nontranslated region (U5) of the viral RNA genome as primer. During the assembly of the HIV-1 virions, several cellular tRNAs including tRNA-^Lys are packaged into the virion particles, but HIV-1 utilizes only tRNA₃ ^Lys for the initiation of reverse transcription (Jiang et al, 1993). The tRNA₃ ^Lys primes to the PBS, an 18-nucleotide sequence that is located downstream of the U5 region of the 5' long terminal repeats (LTR) and spans from nucleotides 183-201 of the viral RNA genome. The 3 '-terminal 18 nucleotides of the primer fRNA₃ ^ys are complementary to the PBS region (Ratner et al, 1985). HIV-1 mutants that use reverse-transcription primers other than the natural tRNA₃ ^Lys exhibit reduced replication efficiencies and revert to the wild-type tRNA₃ ^ys sequence upon prolonged culturing (Das et al, 1995). Apart from the interaction of tRNA.^Lys with the PBS, the A-loop region located upstream of the PBS has been implicated in

[54704-8057/LA033020.002] -2- the selection and stabilization of fRNA-^Lys on the viral genome (Li et al, 1997). This suggests the importance of the primer-binding sequence as well as the A-loop sequence during viral replication.

In recent years, it has become apparent that identification of novel viral targets and antiviral agents are needed to empower anti-HIV-1 therapeutic strategies. The unique U5 region (1-333 nucleotides) of the HIV-1 genome comprises a number of regulatory regions essential for viral replication, which may be potential targets for drug intervention. These critical domains are as follows: (i) PBS (nucleotides 183-201); (ii) the A-loop region (nucleotides 168-173), located upstream of the PBS and essential for the selection and interaction of tRNA₃ ^Lys primer; (iii) the LTR sequences at the 5 ' and 3 ' ends, essential for viral transcription and integration; and (iv) the tAms-activation response element (TAR), essential for viral gene expression via transcriptional activation and which extends between nucleotides + 1 and +59 to form a unique, stable stem-loop RNA structure

In attempting to arrest retroviral replication, one promising method has been the use of peptide nucleic acid (also known as polyamide nucleic acid or PNA) oligomers that bind selectively to complementary DNA or RNA sequences and inhibit translation and replication. PNA are nucleic acid analogues containing polynucleobase linked with peptide backbone instead of the sugar-phosphate backbone. A PNA targeted to the PBS region of the viral genome blocks reverse transcription of U5 PBS HIV-1 RNA primed with the DNA primer or synthetic tRNA₃ ^Ly\ Unlike oligonucleotides, PNA are not confounded by nonspecific interactions. Moreover, PNA exhibit an added advantage in that they can invade the duplex DNA/RNA by invasion or displacement of one of the DNA strands and bind to their target site. These molecules are also chemically stable and resistant to cleavage by nucleases and proteases. It has been shown that PNA targeted to the coding region of the Ha-ras mRNA

[54704-8057/LA033020.002] -3- effectively blocked the formation of the translation initiation complex and polypeptide chain elongation. Likewise, PNA targeted to the telomerase RNA blocked its activity, resulting in progressive telomere shortening and causing immortal human breast epithelial cells to undergo apoptosis. Removal of antitelomerase PNA from the cell culture restored the telomere length.

Because HIV-1 gene expression is regulated through a complex interplay of specific cis-acting DNA elements within its long terminal repeat (LTR) with host cell proteins/factors as well as with its own accessory proteins, another approach to controlling HIV is focusing on one of these cis-acting elements called trans-activation response region (TAR) in the 5'-LTR of viral genome. TAR is essential for transcriptional activation by the transactivator protein Tat (Isel and Karn, 1999; Jeang et al.,1999; Veschambre et al, 1995). A three nucleotide U- rich bulge located between +23 and +25 of TAR has been identified as the site of Tat binding (Long and Crothers, 1999). Tat (meaning transactivator of transcription) is a small HIV protein essential for both viral replication and progression of the disease. The primary role of Tat is in regulating productive and processive transcription from the HIV-1 LTR. Natural or induced mutations that destabilize TAR by disrupting base pairing in the stem region abolish Tat-stimulated transcription resulting in premature transcription termination at random locations downstream of the viral RNA start site.

In vitro, Tat binds to bulge region of TAR RNA but does not recognize sequences in the loop of the TAR hairpin that are essential for transactivation (Roy et al., 1990). The interaction of Tat with TAR region requires cellular factors such as cyclin T/CDK9 that bind to the terminal loop region of TAR (Wei et al., 1998). Tat is involved in recruiting cellular kinases that phosphorylate the C-terminal domain of RNA pol II, resulting in a more processive RNA Pol II complex (Bieniasz et al., 1999; Chen et al., 1999; Fujinaga et al.,

[54704-8057/LA033020.002] -4- 1998; Isel and Karn, 1999; Ivanov et al., 1999; O'Keeffe et al., 2000; Ramanathan et al., 1999; Romano et al., 1999; Napolitano et al., 1999; Wei et al., 1998). These reports underscore the importance of the bulge and loop region of TAR in viral gene regulation.

Transcription activation by Tat occurs through TAR and requires the proper folding of the TAR RNA hairpin structure (Cullen, 1993; Jones and Peterlin, 1994). It has been demonstrated that both an intact loop sequence and an intact Tat-binding site are critical structural motifs of the TAR element, and there is no complementation in cis between TAR element carrying mutations in loop or in the Tat-binding site (Churcher et al., 1995). A potential barrier in this interaction would result in down regulation of transcription. Given the functional importance of Tat-TAR interaction, both the Tat and TAR element represent attractive targets for drug design. A number of reports have suggested various chemicals, genetic inhibitors, Tat peptide analogs, TAR RNA decoys, TAR circle, TAR ribozyme, extra cellular anti-Tat monoclonal antibody and single-chain anti-Tat antibodies, among others, to sequester Tat's function, thereby reducing transcription and viral load. These agents affect the interaction between Tat and TAR, thereby preventing transcriptional activation of HIV-1 genome either by steric hindrance, sheer displacement mechanism or by deprivation of the functional molecules. Recently, it has also been shown that shielding the bulge-loop region of TAR with PNA and other oligo analogues in an anti- sense fashion inhibits HIV-1 reverse transcription (Boulme et al., 1998). Transfection of an anti-TAR PNA complementary to the stem-loop and bulge regions of HIV-1 TAR can effectively inhibit the Tat-mediated transactivation of HIV-1 LTR in CEM cells (Mayhood et al., 2000). It has also been shown that a 12-mer PNA and its analogues inhibit Tat-dependent transcription in HeLa cell nuclear extract (Arzumanov et al., 2001). Thus, the Tat protein of HIV-1 is a potent transactivator of viral gene expression and is essential for viral replication.

[54704-8057/LA033020.002] -5- While there has been significant progress made in the field of HIV-1 inhibition of replication, new and optimized inventions are always sought. Any improvement in the field of treating retroviruses would be very helpful because of the difficult nature of treating such highly mutable viruses. Further, any improvement in treating the HIV retrovirus would be a significant improvement in the art because of the large number of people worldwide who are infected, the massive resource expenditure used to treat HIV, and the deadly results of the infection.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, a PNA of sixteen (16) nucleotides in length targets the PBS (PNA_PBS) and A-Loop sequences of the HIV-1 genome, destabilizing packaged tRNA₃ ^Lys and inhibiting replication. Preferably, the PNA_PBS has a sequence of

CGCCACTGCTAGAGAT (SEQ ID NO:l). However, any 16-mer PNA_PBS that inhibits replication is contemplated.

Another aspect of the present invention is a composition of the 16-mer PNA_PBS and a pharmaceutically acceptable carrier for administration to a subject with a retrovirus. Preferably, the retrovirus is HIV and the subject is human. In this aspect, the PNA_PBS invades the duplex region of the tRNA₃ ^ys and viral RNA region and destabilizes the priming process.

Another aspect of the present invention contemplates a method of treating a subject infected with a retrovirus or virion comprising administering a therapeutically effective amount of 16-mer PNA_PBS to a patient. The dose administered and frequency of administration would be preferentially determined by a treating physician based on the individual requirements of the subject. Preferably, the PNA_PBS is administered in a pharmaceutically acceptable carrier.

[54704-8057/LA033020.002] -6- Also contemplated is a kit comprising an antiviral agent and instructions for use. Preferably, the kit contains 16-mer PNA_PBg or PNA conjugated to a membrane transducing peptide.

In a separate aspect of the present invention, a PNA of 13 to 16 nucleotides is used to block the TAR region of HIV-1, thus blocking reverse transcription. Preferably, the PNA_TAR is 16 nucleotides in length. More preferably, the PNA^ is conjugated to a membrane transducing peptide. Most preferably, the MTD is transportan.

Methods of the present invention contemplate the PNA^-transportan affecting the target by permeating the lipid bilayer of both cells containing the virus and the virus itself. Preferably, the virus is HIV-1. Another embodiment of the invention contemplates that the

PNA_TAR-transportan is able to inactivate HIV-1 viruses circulating in plasma prior to entry and infection of a new cell.

The invention is also embodied in compositions of PNA-.^ with or without the MTD and methods of treatment using the composition. Preferably, the composition would comprise PNA-^-transportan and a pharmaceutically acceptable carrier. The method of treating a viral infection teaches administering the composition in a pharmaceutically effective amount to treat the virus. Preferably, the virus is HIV-1.

The present invention can be further understood through the following definitions, figures, and four sets of experiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring particularly to the drawings for the purpose of illustration only and not limitation, there is illustrated:

[54704-8057/LA033020.002] -7- FIG. 1 shows the sequences and configurations of PNA_SCB and the U5 PBS HIV-1 RNA.

FIG. 2 is a gel -shift analysis testing the binding specificity of PNA_PBS to its target sequence on the HIV-1 genomic RNA. FIG. 3 shows the effect of PNA_PBS (Fig. 3 A) or PNA_SCB (Fig. 3B) on the formation of the (tRNA-^{L s}-U5 PBS RNA) complex.

FIG. 4 is a polyacrylamide-urea gel displaying the effect of PNA_PBS on a tRNA-^1'5'8 - primer extension assay.

FIG. 5 is a polyacrylamide-urea gel showing PNA_PBS-mediated inhibition of reverse transcription in disrupted HIV-1 virions (Fig. 5A) and purified vRNA-tRNA (Fig. 5B).

FIG. 6 is a bar graph showing that PNA_PBS inhibits HIV-1 replication in T cells as measured by the percentage of luciferase activity inhibited.

FIG. 7 is an agarose gel showing quantitation of integrated HIV-1 cDNA.

FIG. 8 is the general structure and sequence of PNA with various anti-TAR PNAs of varying lengths also shown.

FIG. 9 is a gel showing the specificity of interaction of P A-.^ to its target sequence on the HIV-1 genome.

FIG. 10 depicts both a gel showing the effect of anti-TAR PNA on reverse transcription of HIV-1 TAR RNA and a diagram of PNA with position 43 of the PNA marked on both.

FIG. 11 depicts both a gel and a diagram showing the inhibition of reverse transcription of TAR RNA as a function of individual anti-TAR PNA concentration.

FIG. 12 is a bar graph showing the effect of anti-TAR PNA on Tat-mediated transactivation in CEM cells.

[54704-8057/LA033020.002] -8- FIG. 13 is a series of four bar graphs showing the effect of anti-TAR PNA on HIV-1 production in CEM cells.

FIG. 14 illustrates the sequence of PNA-transportan conjugates.

FIG. 15 illustrates the binding specificity of PNA_TAR-transportan conjugate to its target sequence.

FIG. 16 is a gel and a diagram showing the effect of PNA-^-transportan conjugate on reverse transcription on TAR RNA primed with 17-mer DNA primer.

FIG. 17 is two photos showing the cellular uptake of PNA-transportan conjugate.

FIG. 18 is a series of two bar graphs showing Tat-mediated transactivation of HIV-1 LTR.

FIG. 19 is a series of two bar graphs illustrating the effect of PNA-^-transportan conjugate on Tat-mediated transactivation of HIV-1 LTR.

FIG. 20 is an RNase protection assay.

FIG. 21 is a chart showmg the effect of PNA-^-transportan conjugate on HIV-1 production in chronically HIV-1 infected H9 cells.

FIG. 22 is photograph of a gel showing PNA_TAR-transportan inhibiting transcription of HIV-1 mRNA in chronically HIV-1 infected H9 cells.

FIG. 23 is a graph showing [³H]thymidine incorporation into cellular DNA in chronically infected HIV-1 H9 cells. FIG. 24 is a series of fluorescence intensity charts showing uptake of PNA_TAR- transportan by CEM and Jurkat cells by measuring relative cell count.

FIG. 25 is a series of three-dimensional charts showing the uptake of Flu-tagged anti- TAR PNA transportan conjugate by Jurkat cells at different temperatures.

[54704-8057/LA033020.002] -9- FIG. 26 is two plots showing the uptake of ¹²⁵I-labeled anti-TAR-PNA transportan conjugate by Jurkat cells.

FIG. 27 is a bar chart illustrating the antiviral efficacy of anti-TAR PNA transportan conjugate. FIG. 28 is an illustration of internalization of PNA^-transportan in HIV-1 virions.

FIG. 29 is a gel showing abortive endogenous reverse transcription in HTV-1 virions pretreated with PNA_rAR-transportan conjugate.

FIG. 30 is a bar chart showing the infectivity of HIV-1 virions treated with PNA-transportan conjugate in the RMPI medium in the presence or absence of 10% FCS.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

Various terms relating to the biological molecules of the present invention are used throughout the specification and claims.

"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated," as the term is employed herein.

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.

"Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and

RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising

[54704-8057/LA033020.002] _10- DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques that are well known in the art. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racernization, selenoylation,

[54704-8057/LA033020.002] - 11 - sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

"Conjugate" generally means paired, and specifically means any two components, such as PNA and MTD, which are joined together.

A "target" is any object of the products or methods of the present invention. Specifically, a target is cell, tissue, organ, system, or subject containing the virus or threatened by the virus or the virus itself. The target is any physical location upon which the products and methods of the invention can effectively work.

[54704-8057/LA033020.002] -12- "Inhibit" or "inhibition" means to curb, stop, or restrain, such as in stopping the progression of a virus.

A "coding sequence" or "coding region" refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed. "Affect" or "affecting" means to act upon or influence so as to effect a response.

"Therapeutically effective amount" is an amount of a compound that, when administered, brings about a desired therapeutic effect, such as treating the target disease. The precise therapeutically effective amount is an amount of the composition, such as the conjugate of the present invention, which will yield the most effective results in terms of efficacy of treatment in a given subject will depend upon the activity, pharmacokinetics, pharmacodynamics, and bioavailability of a particular ERR antagonist, physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), the nature of pharmaceutically acceptable carrier in a formulation, and a route of administration, among other potential factors. Those skilled in the clinical and pharmacological arts will be able to determine these factors through routine experimentation consisting of monitoring the subject and adjusting the dosage. Remington: The Science and Practice of Pharmacy (Gennaro ed. 20^th edition, Williams & Wilkins PA, USA) (2000).

The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the compound or conjugate of the present invention from one tissue, organ, or portion of the body, to another tissue, organ, or portion of the body. Each component must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of

[54704-8057/LA033020.002] -13- the formulation. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenecity, or other problems or complications, commensurate with a reasonable benefit risk ratio. The conjugate may also be encapsulated with liposomes. A "route of administration'' for a novel compound or composition can be by any pathway known in the art, including without limitation, oral, enteral, nasal, topical, rectal, vaginal, aerosol, transmucosal, transdermal, ophthalmic, pulmonary, and/or parenteral administration. A parenteral administration refers to an administration route that typically relates to injection. Parenteral administration includes, but is not limited to, intravenous, intramuscular, intraarterial, intraathecal, intracapsular, infraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, via intrasternal injection, and/or via infusion.

"Treatment" of or "treating" a disease may mean preventing the disease by causing clinical symptoms not to develop, inhibiting the disease by stopping or reducing the symptoms, the development of the disease, and/or slowing the rate of development of the disease, relieving the disease by causing a complete or partial regression of the disease, reducing the risk of developing the disease, or a combination thereof.

The terms "promoter", "promoter region" or "promoter sequence" refer generally to transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. The typical 5' promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels

[54704-8057/LA033020.002] -14- detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the bmding of RNA polymerase.

The term "contacted" when applied to a cell, tissue or organ means the process by which the compounds or conjugates of the present invention are delivered to the target cell, tissue or organ, or placed in direct proximity of the cell, tissue, or organ.

"Retaining the binding property" as applied to the variants discussed herein means that the variants bind with the at least 90% of the binding strength of the PNA with a sequence of SEQ ID NO: 1. Binding strength is defined as the binding energy required to break the bond between the object binding and the object being bound.

EMBODIMENTS

One aspect of the invention is a novel 16-mer PNA, which targets the PBS (PNA_PBS) and A-Loop sequences of the HIV-1 genome. The specific PNA_PBS has a sequence of CGCCACTGCTAGAGAT (SEQ ID NO: 1). This sequence is shown to be important because a scrambled PNA does not bind. Only the PNA_PBS sequence or variants that retain the binding properties of the PNA_PBS described above and are substantially the same are contemplated. The PNA_?BS binds the PBS region of HIV, destabilizes packaged tRNA ₃ ^Lys and inhibits replication.

Another aspect of the invention, novel PNAs of 13 to 16 nucleotides is used to block the TAR region of HIV-1, thus blocking reverse transcription. Preferably, the PNA.^ is 16 nucleotides in length. More preferably, the PN -_AJ. is conjugated to a membrane transducing peptide. Most preferably, the MTD is transportan.

[54704-8057/LA033020.002] -15- Methods of this aspect of the present invention contemplate the PNA_TAF.-transportan permeating the lipid bilayer of both cells containing the virus and the virus itself. Preferably, the virus is HIV-1. Another preferred embodiment of the virus contemplates that the PNA-transportan is able to inactivate HIV-1 viruses circulating in plasma prior to entry and infection of a new cell.

Both the PNA^-transportan and the PNA_PBS sequence, which may be connected to transportan, can be used, either together or separately, in pharmaceutical compositions. The composition comprises one or both of the PNAs and a biologically acceptable carrier for administration to a subject with a retrovirus. Preferably, the retrovirus is HIV and the subject is human. In this aspect, the PNA_PBS invades the duplex region of the tRNA₃ ^ys and viral RNA region and destabilizes the priming process in vivo. PNA-^-transportan blocks the TAR region of the virus and prevents replication in that manner. When the priming process is disrupted, the HIV cannot continue to replicate and the progression of the virus is halted. The composition is administered in an amount that effectively inhibits HIV-1 activity. A method for the treatment of a disease associated with a HIV-1, which involves administering to a subject a pharmaceutical composition that includes an effective amount of such a compound that inhibits HIV-1 activity, is also within the scope of the present invention. The dose administered and frequency of administration would be preferentially determined by a treating physician based on the individual requirements of the subject. The present invention encompasses pharmaceutical compositions prepared for storage or administration that comprise a therapeutically effective amount of one or more compounds of the present invention in a pharmaceutically acceptable carrier. The therapeutically effective amount of a compound of the present invention will be in the range of about l.mu.g kg to about 50 mg kg. The particular dosage will depend on the route of

[54704-8057/LA033020.002] - 1 - administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration, as well as the characteristics of the specific compound: for example, potency, bioavailability, metabolic characteristics, etc. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the mode of administration can be tailored to achieve optimal efficiency and will be contingent on myriad factors recognized by those skilled in the medical arts, including weight, diet, and concurrent medication. The therapeutically effective amount of the compounds of the present invention can range broadly depending upon the desired effects and the therapeutic indication. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences (A. P. Gennaro, ed.; Mack, 1985). For example, sterile saline or phosphate-buffered saline at physiological pH may be used. Preservatives, stabilizers, dyes, and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid may be added as preservatives (Id. at 1449). Antioxidants and suspending agents may also be used (Id.).

The pharmaceutical compositions of the present invention may be formulated and used as tablets, capsules, or elixirs for oral administration; suppositories for rectal or vaginal administration; sterile solutions and suspensions for parenteral administration; creams, lotions, or gels for topical administration; aerosols or insufflations for intratracheobronchial administration; and the like. Preparations of such formulations are well known to those skilled in the pharmaceutical arts. The dosage and method of administration can be tailored to achieve optimal efficacy and will depend on factors that those skilled in the medical arts will recognize.

[54704-8057/LA033020.002] -17- When administration is to be parenteral, such as intravenous on a daily basis, injectable pharmaceuticals may be prepared in conventional forms, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid prior to injection; or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, or the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (e.g. liposomes) may be utilized.

Also within the scope of the present invention is a kit comprising an antiviral agent and instructions for use, for the delivery of the therapeutic agents described herein. Preferably, the kit contains a 16-mer PNA_PBS and/or PNA-^ conjugated to a membrane transducing peptide. The kit may include the therapeutic composition of the invention in an aqueous form (i.e., a solublized PNA suspended in a stable buffer such as EDTA). The instructions will furnish steps to make the compounds used for formulating nucleic acid molecules. Additionally, the instructions will include methods for testing compositions of the invention that entail establishing if the nucleic acid molecules are damaged upon administration. The kit may also include notification of FDA approved use and instructions

EXPERIMENT SET #1— 16-MER PNA_PBS INHIBITS HIV-1 REPLICATION

I. OVERVIEW

A PNA targeted to PBS and A-Loop sequence (PNA_PBS) exhibits high specificity for its target sequence and prevents tRNA₃ ^Lys priming on the viral genome. Also demonstrated is that PNA_PBS is able to invade the duplex region of the tRNA₃ ^Ly -viral RNA complex and destabilize the priming process, thereby inhibiting the initiation of reverse transcription. The

[54704-8057/LA033020.002] - 18- endogenously packaged tRNA₃ ^Lys bound to the PBS region of the viral RNA genome in the HIV-1 virion is efficiently competed out by PNA_PBS, resulting in near complete inhibition of initiation of endogenous reverse transcription. Examination of the effect of PNA_PBS on HIV-1 production in CEM cells infected with pseudotyped HIV-1 virions carrying luciferase reporter revealed dramatic reduction of HIV-1 replication by nearly 99%. Analysis of the mechanism of PNA_PBS-mediated inhibition indicated that PNA_PBS interferes at the step of reverse transcription.

In summary, a 16-mer anti-PBS PNA (also called PNA_PBS), targeting five nucleotides of the PBS sequences and 11 nucleotides upstream of PBS in the 5' nontranslated region of the viral genome, can specifically sequester its target sequence and prevent tRNA₃ ^ys priming on the viral genome. Further, PNA_PBS is able to compete out the packaged fRNA-^{L s}, thereby destabilizing it and inhibiting the endogenous reverse-transcription process in the virions. PNA_PBS also dramatically inhibits HIV-1 replication in CEM cells infected with pseudotyped HIV-1 virions by predominantly interfering with the reverse-transcription process. Scrambled PNA has no influence either on the reverse transcription process or on the replication of the HIV-1 -infected cells, thus indicating the specificity of PNA_PBS. These findings show that PNA_PBS blocks the process of HIV-1 replication.

This experiment demonstrates that the potential of PNA complementary to the 5 nucleotides of the PBS and 11 nucleotides of the A-loop region (PNA_PBS) to inhibit virus replication in cell culture. PNA_PBS stoichiometrically binds to the PBS/A-loop region of the U5 PBS RNA (Fig. 2) and efficiently displaces the bound tRNA^ primer (Fig. 3A). By contrast, scrambled PNA (Fig. 3B) as well as an oligonucleotide having identical sequence as PNA_PBS (results not shown) were unable to disrupt the preformed (tRNA₃ ^Lys -U5 PBS RNA)

[54704-8057/LA033020.0021 -19- complex. This suggests that unlike an oligonucleotide, the uncharged PNA_PBS is able to invade the duplex region of tRN A₃ ^Lys-primed viral R A genome and disrupt this interaction.

Finding that the binding of HIV-1 RT to the tRNA₃ ^LyB alone is not affected by PNA_PBS, further suggests that the affinity of PNA_PBS is highly specific toward its target sequence on the viral genome (Fig. 3 A, lanes 9 and 10). Sequestering the PBS region of the viral genome by the complementary PNA_PBS has a strong destabilization effect on the formation of enzyme tRNA₃ ^L -viral RNA ternary complex. This interaction may, in turn, prevent initiation of HIV- 1 reverse transcription and transition of the initiated primer to elongation.

Next, the effect of PNA_PBS on the natural tRNA^-primed initiation of reverse transcription catalyzed by HIV-1 RT was investigated. In the absence of PNA_PBS or with scrambled PNA, the tRNA₃ ^{L s}-derived reverse transcription yielded a full-length (-)-strand strong stop DNA product of predicted size (257 nucleotides). Incubation of PNA_PBS with U5 PBS RNA preprinted with t NA₃ ^Lys resulted in significant reduction in the initiation of reverse transcription and its subsequent elongation (Fig. 4A; lanes 2-7). This shows that the ability of PNA_PBS to destabilize the tRNA-^Lys primer by invading the duplex region of the viral RNA. The inhibitory effect of PNA_PBS was more pronounced when incubated with unprimed U5 PBS RNA (Fig. 4B; lanes 2-7), showing that PNA_PBS once bound to its target can effectively interfere with the priming process. It was therefore interesting to determine whether PNA_PBS could inhibit the process of reverse transcription in the disrupted virions containing the endogenous tRNA₃ ^Lys-primed viral RNA genome and other viral components/ proteins.

The results indicate that incubation of the disrupted virions with PNA,,-,-. distinctly inhibited the reverse-transcription process as a function of PNA_PBS concentration (Fig. 5A; lanes 1-8), thus indicating the ability of PNA_PBS to sequester its target in the disrupted virions.

154704-B057.LA033020.002] -20- Purified vRNA-tRNA complex isolated from HIV-1 virions also yielded the characteristic 257- nucleotide product (Fig. 5B, lane C), while the disrupted virions yielded products longer than 257 nucleotides (Fig. 5A). This indicates that the longer products with the disrupted virions arose as a result of strand transfer. This experiment proves that the interaction of PNA_PBS with the PBS sequences on the viral genome blocks the base pairing interaction between PBS and the 3' terminal 5 nucleotides of tR A₃ ^ys, resulting in destabilization of the tRNA₃ ⁵ primer on the viral genome. Integrity of both the PBS region and the A-loop structure is essential for viral infectivity. Interestingly, all the naturally occurring HIV-1 strains isolated to date from clinical samples harbor the PBS complementary to the tRNA₃ ^ys. The results of the present investigation indicate that PNA_PBS can inhibit HIV-1 replication in cell culture (Fig. 6). The observation, that the amounts of proviral DNA integrated in the host DNA decrease at increasing PNA_PBS concentration (Fig. 7), shows that the mechanism of PNA_PBS-mediated interference may be regulated at the level of reverse transcription.

II. EXPERIMENTS

1.1 Binding specificity of PNA-^ to U5 PBS HIV-1 RNA

Labeled U5 PBS HIV-1 RNA was used to determine the binding specificity of PNA_PBS targeted to the PBS region of HIV-1 genome. The sequence of U5 PBS RNA encompassing the TAR (R), 5' UTR, and PBS regions, as well as PNA_PBS and scrambled PNA (PNA_SCB) are shown in Figure 1. The sequences of PNA_PBS and PNA_SCB are as shown in the box, and contain 16 and 17 bases, respectively, linked with polyamide backbone. The 3' terminal 18 nucleotides of tR-NA₃ ^Lys, which primes the PBS region are as indicated. The sequences within the PBS and 5' UTR interacting with the PNA_PBS are underlined. The design of the sequence of PNA_PBS was such that five of its N-terminai bases are complementary to the 3' terminal

[54704-8057/LA033020.002] -21- PBS bases spannin nucleotides 183-187, while the remaining C-terminal bases are complementary to the sequences upstream of the PBS (172-182 nucleotides).

To ascertain the ability of PNA_PBS to interact with PBS sequences on the viral RNA, gel-retardation assays were performed with ³²P-labeled 200-base-long U5 PBS RNA transcript (5 x 10³ Cerenkov cpm) at varying concentrations of PNA_PBS or scrambled PNA (Figure 2). In this binding affinity and specificity assay, lanes 1 through 10 represent molar ratios of PNA_PBS to U5 PBS RNA of 0.0, 0.1. 0.3, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, and 5.0, respectively. Titration of PNA_PBS with U5 PBS RNA at molar ratios of PNA_PBS to U5 PBS RNA less than 1 resulted in a stoichiometric band shift of the labeled U5 PBS RNA (Figure 2, lanes 2-10). As seen Figure 2, at molar ratios of 0.1, 0.3, 0.5, 0.6, 0.7, 0.8, and 0.9 of PNA_PBS to labeled U5 PBS RNA, the extent of band shift corresponded to 12, 27, 52, 58, 67, 72, and 87%, respectively. The percentage corresponded to the amount of U5 PBS RNA retarded due to PNA binding. A complete shift in the mobility was achieved at equimolar or molar excess of PNA_PBS to U5 PBS RNA (Figure 2, lanes 9 and 10). A similar titration was carried out in the presence of scrambled PNA. Lanes 11 through 15 represent gel shift with PNA_SCB at molar ratios of PNA_SCB to U5 PBS RNA of 0.1 , 0.5, 1.0, 2.5, and 5.0, respectively. Scrambled PNA at fivefold molar excess of U5 PBS RNA exhibited no shift in the mobility of the labeled U5 PBS RNA (Fig. 2, lanes 11-15). The labeled U5 PBS RNA bands were quantified on phosphorlmager using Image-Quant software (Molecular Dynamics). The experimental results indicated that the gradient of band shift observed as a function of PNA_PBS concentration is due to sequence-specific interaction between PNA_PBS and U5 PBS RNA.

[54704-8057/LA033020.002] -22- 1.2 Disruption of binding interaction between tRNA3^Lys and U5 PBS RNA by PNAPRS

To determine the effect of PNA_PBS on the formation of the (tRNA^-viral U5 PBS

RNA) complex as well as on the (HIV-1 RT- tR A₃ ^Lys) complex, gel-retardation analysis was executed (Figure 3). Incubation of U5 PBS RNA preprimed with the labeled tRNA₃ ^Lys in the presence or absence of PNA_PBS (Fig. 3A) or PNA_SCB (Fig. 3B) is shown with conditions as stated for the individual lanes. The complex formed was resolved on a native gel and analyzed on a phosphorlmager.

Lanes 1-4 show incubation of the preformed complex in the presence of PNA_PBS or PNA_SCB at molar ratios of PNA to U5 PBS RNA of 0.0, 1.0, 2.5, and 5.0 respectively. The incubation resulted in the formation of a distinct complex between the tRNA^^⁵ and viral RNA as discerned by a shift in their mobility (lane 1). Incubation of this preformed (tRNA₃ ^Ly-viral RNA) complex with increasing concentrations of PNA_PBS resulted in the disruption of this complex in a concentration-dependent manner as noted by a significant decrease in the intensity of the slower migrating complex (lanes 2-4). These results show that the chargeless PNA_PBS is able to invade the tRNA₃ ^ys-primed duplex region of the viral RNA resulting in the fRNA-¹'⁵ displacement. (Lane 5 was merely ³²P-labeled tRNA₃ ^{L s} alone.)

In lanes 6-8, U5 PBS RNA was preincubated with PNA_PBS or PNA_SCB at molar ratios of PNA to U5 PBS RNA of 1.0, 2.5, and 5.0, respectively, followed by further incubation with tRNA₃ ^Lys. This complex formation was completely blocked when PNA_PBS was incubated with the U5 PBS RNA prior to its priming with the tRNA₃ ^Lys, suggesting that PNA_PBS once bound to its target sequence prevents t A^⁵ priming binding. Significantly, an oligonucleotide having identical sequence as PNA_PBS was unable to disrupt the preformed complex of (tRNA₃ ^ys-U5 PBS RNA), although it was able to block tRNA₃ ^Lys priming when preincubated

[54704-8057/LA033020.002] -23- with the U5 PBS RNA prior to priming with tRNA₃ ^{L s}. This indicates that the exact sequence of the 16-mer PNA as disclosed in the present invention is required for binding to the PBS region.

Incubation of the tRNA₃ ^Lys with HIV-1 RT, in the absence of PNA_PBS (lane 9) or in its presence (lane 10) resulted in complete supershift in the mobility of the HIV-1 RT-tRNA₃ ^ys- binary complex, thus suggesting that PNA_PBS has no influence on the binding of tRNA_j ^Ly5 with the viral enzyme. A similar experiment was carried out in the presence of scrambled PNA (Fig. 3B). As shown in the figure, scrambled PNA neither disrupted the preformed complex of

PBS RNA (lanes 2-4) nor prevented the formation of this complex when preincubated with the U5 PBS RNA prior to its priming with tRNA₃ ^Lys (lanes 6-8). Similar to PNA_PBg, scrambled PNA did not affect tRNA₃ ^Lys binding to HIV-1 RT (lanes 9 and 10). These experiments clearly indicate the potential of PNA_PBS in efficiently disrupting the (viral RNA- tRNA₃ ^Lys) complex.

1.3 Blockage of reverse transcription as a function of PNA_m concentration Since initiation of HIV-1 reverse transcription requires the priming of HIV-1 genome with the natural tRNA₃ ^ys primer, the influence of PNA_PBS on the initiation process of reverse transcription was examined. For this purpose, U5 PBS RNA annealed to natural tRNA₃ ^ys was incubated at varying molar ratios of PNA_PBS or scrambled PNA to TP. Reverse transcription was initiated by HIV-1 RT in the presence of labeled dNTP-Mg²⁺. The reactions were resolved on a denaturing polyacrylamide-urea gel and analyzed on a phosphorlmager.

The results of this experiment are shown in Figure 4. Reverse transcription of tRNA₃ ^Lys-primed reaction, the control without PNA, resulted in the synthesis of (-)-strand strong stop DNA (ssDNA) of expected length (257 nucleotide) (Fig. 4A, lane 1). Lanes 2-7 show the reverse transcription products in the presence of PNA_PBS at 2.5, 5.0, 7.0, 8.0, 9.0, and

[54704-8057/LA033020.0021 -24- 10.0 molar rations of PNA_PBS to TP, respectively. In those lanes, incubation of the template primer with PNA_PBS significantly decreased the synthesis of ssDNA as a function of PNA_PBS concentrations. The position of molecular markers is indicated to the right of the gels.

PNA_PBS invaded the duplex region of the template primer and displaced tRNA-¹^ from the viral genome, thereby inhibiting initiation as well as elongation of reverse transcription at all concentrations. Lanes 8-10 represent the reactions carried out in the presence of PNA_SCB at

2.5, 7.0. and 10.0 molar ratio of PNA_SCB to TP, respectively. Scrambled PNA had no influence on the reverse transcription under identical conditions.

In another set of experiments, the PBS sequence of U5 PBS RNA was blocked by preincubating with PNA_PBS and then allowed to interact with tRNA₃ ^ys prior to initiation of reverse transcription. The results shown in Figure 4B clearly demonstrate the efficacy of PNA_PBS in preventing the ssDNA synthesis on tRNA3 Lys primer (lanes 2-7). At as low as 2.5 molar ratio of PNA_PBS to TP, near complete absence of ssDNA product was noted (lane 2). The nonspecific scrambled PNA exhibited no effect on reverse transcription under identical conditions (Fig. 4B, lanes 8-10). These results demonstrate that PNA_PBS, once bound to its target sequence, cannot be competed out by tRNA₃ ^Lys.

1.4 PNA_pg. mediated inhibition of the endogenous reverse transcription in the disrupted HTV-l virions

Since PNA_PBS significantly inhibited the in vitro initiation and subsequent extension of tPvNA.^Lys-primed reverse transcription, it was of interest to see whether such inhibition could also be seen with the isolated HIV-1 virion particles. The efficacy of PNA_PBS in invading the tPvNA₃ ^ys-prirned duplex region in the disrupted HIV-1 virions was investigated, as was blocking the process of reverse transcription. Aliquots of disrupted virions were incubated in the presence of varying concentrations of PNA_PBS or scrambled PNA for a stated time.

[54704-8057/LA033020.002] -25- Reverse transcription was then catalyzed by the endogenous HIV-1 RT present in the disrupted virions, by supplementing the reaction mixture with a cocktail of dNTP-Mg ⁺ including labeled dCTP. The result of this analysis is presented in Figure 5 A. A similar set of reactions was carried out in the case of the isolated HIV-1 RNA-tRNA incubation mix, except that extraneous HIV-1 RT was included in the assay (Fig. 5B). The reaction products were resolved on a denaturing polyacrylamide-urea gel and analyzed by phosphorimaging.

As shown in Figure 5A, in the absence of PNA_PBS (lane C), substantial amounts of reverse-transcription products, smaller as well as larger than the expected size of (-) ssDNA (257 nucleotide), were obtained. The presence of larger products may be attributed to the efficient strand transfer reaction, whereas incomplete (-)ssDNA products may have accumulated due to their inability to participate in the strand transfer reaction. Lanes 1-8 represent reactions carried out at PNA_?BS concentrations of 25 nM, 50 nM, 100 nM, 250, nM, 500nM, 750 nM, 1 μM and 5 μM, respectively. The position of molecular markers is indicated in the lane marked M. Incubation of the disrupted virions with PNA_PBS significantly decreased the reverse- transcription products. This decrease corresponded to both the initiation and the subsequent elongation products and correlated with the concentration of PNA_PBS (lanes 1-8). At 25 nM PNA_PBS concentration, approximately 30% inhibition of reverse transcription was obtained (lane 2), which gradually increased to 95% inhibition at 750 nM PNA_PBS concentration (lane 6). In contrast, incubation of the disrupted virions with scrambled PNA (5 μM) did not influence the endogenous reverse-transcription process (lane marked S), thus indicating the specificity of PNA_PBS for its target sequence. Positions of the molecular markers are shown in the lane marked M.

[54704-8057/LA033020.002] -26- In another set, the efficacy of PNA_PBS in blocking the process of reverse transcription on purified vRNA isolated from HIV-1 virions was examined. The tRNA primer remains bound to the viral genomic RNA upon isolation and can be visualized in a tRNA-primer extension assay upon addition of RT enzyme and dNTPs (Fig. 5B). Unlike in the case of disrupted virion particles, tRNA extension on the purified vRNA yielded a prominent 257-nt- long tRNA-cDNA product (lane C). The identity of this 257-nt-long tRNA-cDNA product was established by NaOH-mediated degradation of the tRNA part, leaving a 181-nt cDNA product. Incubation with increasing concentrations of PNA_PBS in the reaction mixture resulted in a corresponding decrease of the reverse transcription product (lanes 1-8), thus indicating the efficacy of PNA_PBS in displacing the bound tRNA from its complementary PBS sequence on the HIV-1 viral RNA and disrupting reverse transcription. Further, the specificity of interaction of PNA_PBS in targeting its corresponding sequence was corroborated by the observation that scrambled PNA had no effect on the reverse-transcription process (lane S).

1.5 Inhibition of HIV-1 replication in CEM cells bv anti-PBS PNA Since anti-PBS PNA inhibited the reverse-transcription process in the disrupted virions, the next experiment investigated their efficacy in HIV-1 -infected cell cultures. T cell lymphocytes, infected with the pseudotyped HIV-1 virus carrying the firefly luciferase reporter, were transfected with varying concentrations of the individual anti-PBS PNA or scrambled PNA. Cell extracts were then normalized for the total protein and analyzed for quantitative levels of luciferase expression to evaluate the effect of varying concentrations of PNA_PBS on HIV-1 production in these cells. Luciferase activity obtained in the infected T cells in the absence of PNA was arbitrarily set at 100% and activity obtained in the presence of PNA was calculated relative to this value. These results presented in Figure 6 represent the percentage luciferase activity at indicated concentrations of PNA_PBS in CEM (Fig. 6A) and

[54704-8057/IA033020.0021 -27- Jurkat cells (Fig. 6B) at 48 hours posttransfection. The concentrations at which luciferase activity was obtained are shown in A and B. The amount of PNA_SCB transfected corresponds to 10 μg. In Figure 6, the values shown are an average of three sets of experiments and the bar represents standard deviation. Substantial inhibition in virus production as indicated by a corresponding decrease in the luciferase activity was seen in both the T cell types. The extent of inhibition was correlated directly with the concentrations of PNA_PBS. At 0.5 μg PNA_PBS concentration, a modest inhibition of approximately 12-14% was obtained, which increased to 54-58% inhibition at 1 μg PNA_PBS. A near abolishment of virus production (approximately 1-2% luciferase activity) was obtained at 10 μg of PNA_PBS. By contrast, scrambled PNA at similar concentration exhibited no inhibition of virus production, thus indicating the specificity of PNA_res-mediated inhibition. These results clearly demonstrate the antiviral efficacy of PNA_PBS.

1.6 Mechanism of PNA_p-.--mediated inhibition of virus production A substantial reduction in the expression of the luciferase reporter in both the CEM and Jurkat cells suggested inhibition of transcription of the HIV-1 mRNA. The next experiment was conducted to test the possibility that the decreased transcription may be due to lower levels of integrated HIV-1 cDNA into host cell genome. A decrease in cDNA synthesis would be a direct consequence of PNA_PBS interference in the reverse-transcription process as observed in tRNA primer extension analysis in vitro (Fig. 4), as well as in disrupted HIV-1 virions (Fig. 5A). Therefore, the amounts of integrated HIV-1 cDNA in the host cell genome were analyzed. For this, molar excess of oligomeric PBS primer was primed with 1 μg of total DNA isolated from the PNA_PBS transfected, infected T cell lymphocytes. The annealed PBS primer was extended using radiolabeled nucleotides and

[54704-8057/LA033020.002] -28- HIV-1 RT and products were resolved on an agarose gel and analyzed by phosphorimaging. The results of the quantitation of integrated HIV-1 cDNA are presented in Figure 7.

The HIV-1 cDNA population integrated into the host genomic DNA was estimated from total DNA isolated from HIV-1 infected T cell lymphocytes, transfected with varying amounts of PNA_Pas or PNA_SCB. One microgram of total DNA was hybridized with the 17-mer PBS primer, complementary to the primer binding sequence (nucleotides 183-201 of the HIV-1 genomic RNA) and extended with HIV-1 RT in the presence of dNTPs and Mg²⁺. The products were resolved on an agarose gel, which was dried and analyzed on a phosphorlmager. Extension of the annealed PBS primer on the integrated viral DNA in the host DNA resulted in a prominent product corresponding to approximately 2-2.5 kb in size (lanes 2-9). A complete absence of product in the uninfected cells (lane 1) established the identity of this 2- to 2.5-kb product as a result of PBS primer extension after annealing specifically to its complementary sequence on the integrated viral DNA. The intensity of this product decreased quantitatively with increasing PNA_ΪBS concentrations (lanes 3-8) in both CEM and Jurkat cells, thus indicating that the population of viral DNA present in total DNA decreased with increasing PBS concentration. Lanes 2-8 represent DNA products obtained at 0, 0.5, 1.0, 2.5, 5.0, 7.5, and 10 μg of PNA_PBS concentration. Interestingly, scrambled PNA did not affect the amount of product synthesis (lane 9). Lane 9 exhibits similar intensity of the product in PNA_SCB-transfected CEM or Jurkat cells. The size of the products is approximately 2-2.5 kb. The position of the lambda HmdIII-digested markers is indicated on the right. The intensity of the product was quantified using ImageQuant software (Molecular Dynamics). The value obtained in the absence of PNA (Lane 2) was arbitrarily set at 100% and the values obtained in the presence of PNA were calculated relative to 100%. These values are indicated at the

[54704-8057/LA033020.002] -29- bottom of each lane. These results indicate that the mechanism of PNA_PBS inhibition is directly related to interference at the step of reverse transcription.

HI. MATERIALS AND METHODS

DNA modifying enzymes were purchased from Roche Molecular Biochemicals. HPLC-purified human placental tRNA₃ ^Ly$ was obtained from BIO S&T (Canada). Total DNA isolation kit (Qia p DNA kit) was a product of Qiagen Inc. Cell culture media, fetal bovine serum (FBS), and transfection reagents were either from Life Technologies or Roche Molecular Biochemicals. Luciferase assay kit was from Promega. Tritiated dNTPs, γ-32P-ATP, and α-32PdNTPs were the products of Perkin-Elmer Life Sciences. Pore-size filters measuring 0.45 μm were from Schleicher & Schuell. ELISA p24 antigen kit was from Abbott Laboratories. DNA oligomers were synthesized at the Molecular Resource Facility at UMDNJ. Polyamide nucleic acid oligomers were synthesized at Applied Biosystems Inc. All other reagents were of the highest available purity grade and purchased from Fisher. Plasmid and clones

The wild-type homodimeric (p66/p66) HIV-1 RT was purified using the expression vector, pKK-RT66, constructed in the laboratory (Lee et al, 1997). An HIV-RNA expression clone pHIV-PBS was a generous gift from Dr. M. A. Wainberg (Arts et al, 1994). This clone contains a 947-bp fragment (+473 to +1420) of pHλHXB2 HIV-1 proviral clone and was used to generate RNA transcript corresponding to the U5 PBS region. Another HIV-1 RNA expression clone, pU5-PBS constructed in this laboratory (Kaushik et al, 2001), was used to transcribe shorter HIV-1 RNA (200 nucleotide) for gel-shift analysis. This clone contains a 183-bp fragment corresponding to nucleotides 473-656 of pHλHXB2 HIV-1 proviral clone comprising PBS, U5, and part of the R region. An additional 17 nucleotides flanking the 5^r

[54704-8057/LA033020.002] -30- terminus are derived from the plasmid vector. The plasmid, pVSV-G, encoding the vesicular stomatitis virus protein G under the control of the CMV immediate-early promoter was purchased from BD Biosciences Clontech. The plasmid, pHlV-_l __CSF__Iu(,env(-), was a kind gift from Dr. Planelles (Planelles et al, 1995). This replication-defective HIN-1 clone lacks the envelope gene and has a firefly luciferase reporter cloned in place of nef in the HIN-1JR- CSFenv(-) cassette.

Isolation of p66/51 HIV-1 RT

The recombinant clone pKK-RT66 encoding the wild-type p66 HIV-1 RT was expressed in JM109 and purified as described before (Lee et al, 1997). To generate the heterodimeric HIV-1 RT (p66/p51), the homodimeric enzyme (p66/ρ66) was subjected to proteolytic cleavage by HIV-1 protease as follows: The p66/p66 species of HIV-1 RT was incubated with HIV-1 protease at 1:1 molar ratio in a buffer containing 0.1 M potassium phosphate, pH 7.5, 1.0 M ΝaCl, 1 mM DTT, and 1 mM EDTA. Following 16 h incubation at

4°C, the extent of proteolytic cleavage was monitored by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue staining. The heterodimeric enzyme was isolated on a phosphocellulose column (Lee et al, 1997) and the final enzyme preparation was stored at -70°C in a buffer containing 50 mM Tris-HCl pH 7.5,

100 mM ΝaCl, 1 mM DTT, and 50% glycerol. Protein concentrations were determined by using the Bio-Rad colorimetric kit as well as by spectrophotometric measurements using E'₂₈₀ = 2.62 x 10^s M^'1 cm^"1 for p66/51 heterodimer.

Preparation of 32P-labeled US PBS RΝA template

The plasmid ρU5-PBS was used to transcribe labeled RΝA for assessing the binding affinity of PΝA_PBS to its target sequence on the HIV-1 genomic RNA, as described previously (Kaushik et al, 2001). Briefly, the plasmid was linearized with the restriction enzyme Xhol

[54704-8057/LA033020.Q02] -31- and internally labeled with α-³²P-UTP (3000 Ci/mmol; Amersham Life Sciences) using T7 RNA polymerase (Roche Molecular Biochemicals). The plasmid was digested with DNase I free of RNase and the labeled transcript was extracted with phenol-chloroform and alcohol precipitated. The RNA product was dissolved in DEPC-treated water containing 10 M DTT, further purified by G-50 spin column, and stored at -70°C.

Gel retardation assay

The ³²P-labeled U5 PBS RNA (5 x 10³ Cerenkov cpm) was incubated with PNA_PBS or scrambled PNA at varying molar ratios for 3 h at 37°C in a binding buffer containing 50 mM Tris-HCl, pH 7.8, 60 mM KC1, 5 mM MgCl₂, 10 mM DTT, 10% glycerol, 0.01% bovine serum albumin, 0.01% NP-40, and 500 ng of poly[r(I-C)], in a final volume of 15μ. Three microliters of RNA gel-loading dye (0.27% bromphenol blue and 20% glycerol) were added to the samples and subjected to non-denaturing gel analysis on a 8% polyacrylamide gel in Tris-borate-EDTA (TBE) buffer, pH 8.2. The gels were routinely prerun at 100 V for 30 min at 4°C in TBE buffer. The (U5 PBS RNA-PNA) complexes were resolved at a constant voltage of 150 V at 4°C. The gel was dried and analyzed on a phosphorlmager (Molecular

Dynamics). The percentage of labeled U5 PBS RNA retarded as a result of PNA binding was quantified by Image-Quant.

Labeling of tRNA.¹"

The HPLC purified human placental tRNA₃ ^ys obtained from BIO S&T was 3' end- labeled with [³²P]pCp by T4 RNA ligase (Ausubel et al, 1998). The labeled product was extracted three times with phenol chloroform, precipitated with alcohol, lyophilized, and suspended in TE buffer. This was further purified on a NAP-10 column to remove the unincorporated radiolabeled nucleotides.

[54704-8057/LA033020.002] -32- Preparation of R-U5-PBS HIV-1 RNA template

The plasmid, pHJN-PBS, was used to transcribe the 495-base-long U5-PBS RΝA template as described earlier (Arts et al, 1994). Briefly, the plasmid pHIV-PBS was linearized with the restriction enzyme Accl and transcribed using T7 RΝA polymerase and other reaction components from Roche Biochemicals. After in vitro transcription reaction, the DΝA template was removed by DΝase I digestion, followed by phenol-chloroform extraction and alcohol precipitation. The RΝA products were dissolved in DEPC-treated water containing 10 mM DTT, further purified by G-50 spin column, and stored at -70°C. .

Effect of PΝA^ on the formation of ftRΝA3 Lvs-U5 PBS RNA) complex U5 PBS RNA was preprimed with labeled tRNA₃ ^Ly! (7.5 x 10³ Cerenkov cpm) and incubated at 37°C for 2 h with PNA_PBS or scrambled PNA at varying molar ratios of PNA_PBS to U5 PBS RNA. The incubation buffer contained 50 mM Tris-HCl, pH 7.8, 10 mM DTT, 0.01% BSA, 60 mM KC1, and 5 mM MgCl₂. In another set, U5 PBS RNA was preincubated with PNA_PBS or scrambled PNA at 37°C for 1 hour and then supplemented with the labeled tRNA₃ ^Lys and further incubated for 2 hours. In a separate set of experiments, 1 μM HIV-1 RT was incubated with labeled tRNA₃ ^{L s} (7.5 x 10³ Cerenkov cpm) in the absence or presence of PNA_PBS or scrambled PNA. Three microliters of RNA gel loading dye were added to 15 μL of the reaction mixture. The (U5-PBS RNA-tRNA₃ ^ys) complex formed was resolved by electrophoresis on a 6% non-denaturing polyacrylamide gel at 150 V in TBE buffer. The gels were dried and analyzed on a phosphorlmager.

Reverse transcription of U5-PBS RNA template primed with natural tRNA,^tø

U5 PBS RNA was annealed with the natural tRNA₃ ^Lys at 2:1 molar ratio. Reverse- transcription reactions were carried out by incubating 5 nM of U5 PBS RNA/tRNA₃ ^{L s} template primer with 50 nM HIV-1 RT, in a reaction mixture containing 50 mM Tris-HCl, pH 7.8, 10 mM DTT, 100 μg/mL BSA, 5 mM M gCl_2, and 50 μM each of dATP, dTTP,

[54704-8057/LA033020.002] -33- dGTP, and 2 μM of α-³²P-dCTP (0.5 μCi/pmol). Reactions were initiated by the addition of enzyme and terminated by the addition of 20 mM EDTA. The samples were extracted with phenol-chloroform, precipitated with alcohol, and lyophilized. Ten microliters of 1 X Sanger's gel loading solution was added to the samples (Sanger et al, 1977) and the products were resolved on an 8% polyacrylamide-urea gel.

The effect of PNA_PBS on in vitro natural tRNA₃ ^Lys-primed reverse transcription was assessed as follows. In one set, varying molar ratios of PNA_PBS or scrambled PNA were incubated with U5 PBS-RNA template preprimed with the tRNA₃ ^Ly5 at 37°C for 2 h. In another set, PNA_PBS or scrambled PNA was first incubated with U5 PBS-RNA template at 37°C for 2 h, followed by priming with tRNA₃ ^Ly\ Reverse transcription on both these sets was assessed as described above.

Cell culture and transfection

CEM and Jurkat T cell lymphocytes were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 4 mM _L-glutamine, 100 U/ml of penicillin, and 100 μg/ml of streptomycin at 37°C and 5% C0₂. The chronically HIV-1 -infected H9 cells (H9_L were maintained under identical conditions except that they were supplemented with 20% fetal calf serum. Human 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum and 1% penicillin- streptomycin. For transfection in CEM, the cells were grown to mid-log phase, washed with phosphate- buffered saline without Ca⁺² or Mg^+Z, resuspended in RPMI-1640 medium (5.0 x 10^δ cells in 250 μl), and electroporated at 250 V/900 microfarad capacitance using the Bio- Rad Gene pulsar II. Jurkat and 293T cells were transfected in accordance with the manufacturer's protocol using the X-tremeGENE Q2 Transfection reagent (Roche Molecular

[54704-8057/LA033020.002] -34- Biochemicals) or the calcium-phosphate transfection system (Life Technologies), respectively.

Isolation of HIV-1 virions

H9_LAI cells were used as the source of the HIV-1 virus. These chronically HIV-1- infected cells were extensively washed to remove previously produced virions, suspended in 25 ml of RPMI 1640 complete medium at a cell density of 10⁴ cells/ml, and grown for 3 days in a 37°C incubator. The culture medium was centrifuged at 1200 rp for 7 min to remove cells. An aliquot of the cell supernatant was used for p24 antigen estimation. The virus- containing supernatant was filtered through a 0.45-μm pore-size filter (Schleicher & Schuell), and the virions were pelleted by centrifugation at 28,000 φm for 45 min in a Beckman SW28 rotor. The HIV-1 virion pellet was disrupted in 300 μl buffer containing 50 mM Tris- HC1, pH 7.8, 100 mM KC1, 0.5 mM EDTA, and 0.2% NP-40 and used as a source of tRNA₃ ^Lys- primed HIV-1 viral RNA genome, endogenous RT, and other packaged viral components.

Isolation of HIV-1 vRNA HIV-1 virions isolated from H9_LAI cells as described above were used as a source for extracting the virion RNA. The virions were resuspended in 500 μl of buffer containing 50 mM Tris-HCl (pH 7.8), 100 mM NaCl, 2.5 mM EDTA, 1.0% SDS, and 300 μg of proteinase K and incubated at 37°C for 30 min, followed by 3X extraction with phenol-chloroform- isoamyl alcohol (25:24:1). RNA was precipitated in 0.3 M Na-acetate (pH 5.2) and 70% ethanol at -80°C, centrifuged at 16,000 g for 30 min, washed with 70% ethanol, and air-dried. Contaminating DNA was removed by digestion with DNase I (RNasefree; Roche Biochemicals) as per the supplier's instructions. RNA was reextracted with phenol- chloroformisoamyl alcohol (25:24:1), precipitated with 0.3 M Na acetate and 70% ethanol, and pelleted at 16,000 g for 30 min. Following 70% ethanol washing, the air-dried RNA

[54704-8057/LA033020.002] -35- pellet was resuspended in lOOμl of 25 mM Tris-HCl, pH 7.8/0.5 mM EDTA, and stored at -

20°C.

Endogenous reverse transcription in the disrupted HIV-1 virions and tRNA-primer extensions on associated viral RNA in the presence of PNA The effect of PNA_PBS on endogenous reverse transcription in the disrupted HIN-1 virions as well as on viral RΝA-associated tRΝA was assessed as follows: Disrupted HIN-1 virions (lOμl) were incubated at 4°C for 3 h and subsequently at 37°C for 30 min, in the presence or absence of varying concentrations of PΝA_PBS or scrambled PNA in a 15 μl volume. The components of the incubation mix were 50 mM Tris-HCl, pH 7.8, 10 mM DTT, 66 mM KCl, 0.3 mM EDTA, 0.14% NP-40, and 1 U/μl RNasin. Reverse transcription was initiated by the addition of 5 μl of 4 X RT buffer (40 mM MgCl₂, 40 mM dithiothreitol, 200 mM KCl, 120 μg of actinomycin D/ml, 100 μg BSA/mL, 40 μM each of dATP, dGTP, dTTP, 4 μM dCTP), and 5 μCi of -³²P-dCTP (3000 Ci/mmol). The labeled cDNA product generated at 37°C for 5 min was further chased for 30 min by the addition of 1 μl deoxynucleoside triphosphate (dNTP) mix (10 mM each dNTP) and the reaction was quenched with 50 mM EDTA. Following phenol-chloroform extraction and alcohol precipitation, the pellet was resuspended in formamide loading buffer, heated at 90°C for 5 min, resolved on a denaturing 6% polyacrylamide-urea gel, and analyzed by phosphorimaging.

In another set, similar reactions were carried out with the isolated HIV-1 RNA- tRNA₃ ^Lys , except that 5 μl of purified HIV-1 RNA-tRNA was used per reaction and 150 ng of recombinant HIV-1 RT was included in the assay.

T cell infections

Pseudotyped HIV-1 virion stocks were produced by cotransfection of ρHIV-_m._{CSF. lttt}env(-) (Planelles et al, 1995) and pVSV-G (BD Biosciences Clontech) in 293T cells. Culture medium was harvested at 24, 48, and 72 h posttransfection; an aliquot was removed

[54704-8057/LA033020.002] -36- for p24 antigen quantification using the ELISA p24 antigen kit (Abbott Laboratories), and the virus stock was frozen at -80°C. To determine the effect of PNA_PBS on HIV-1 production in T cells, CEM and Jurkat cells (5 X 10⁶ cells) were infected at 37°C for 1 h with the pseudotyped HIV-1 virions in the presence of 10 μg/ml of polybrene to achieve multiplicities of infection (m.o.i.) of 10. The cells were extensively washed with PBS to remove the viruses, resuspended in 5.0 ml of complete RPMI medium, and incubated for another 1 h at 37°C. The infected T cells were then transfected in the presence or absence of varying amounts of the individual PNA_PBS or scrambled PNA and grown in 10 ml of complete RPMI media. Forty- eight hours posttransfection, the cells were harvested and analyzed for firefly luciferase reporter and HIV-1 cDNA levels as described below.

Luciferase assay

Luciferase assays were performed as per the Promega' s luciferase assay protocol.

Forty-eight hours posttransfection, the cells were washed with PBS and lysed in 100 μl of the reporter lysis buffer (Promega). The cell extracts were normalized for total protein (Bio-Rad) and assayed for firefly luciferase activity in a 96-well fluorotrac plate using a Packard Top Count Luminescence Counter. The results of at least three separate transfections were analyzed for each experiment.

Total DNA isolation and estimation of integrated HrV- 1 cDNA

To estimate the HIV-1 cDNA integrated into the host cell chromosome, total DNA was isolated from the transfected T cells using the Qiamp DNA kit (Qiagen Inc.). A 17-mer PBS oligomer with a sequence complementary to the PBS region of the HIV-1 genomic RNA was used as a probe for analyzing the amounts of HIV-1 cDNA integrated into the host genomic DNA by oligomer extension analysis. Hybridization of the probe to the HIV-1 cDNA was achieved by incubating 1 μg of total DNA with 50 pmol of 17-mer PBS oligomer

[54704-8057/LA033020.002] -37- in IX annealing buffer (50 mM Tris-HCl, pH 7.8 and 50 mM KCl) at 75°C for 7 min and allowing it to cool gradually to room temperature.

Extension reactions were carried out in a buffer containing 50 mM Tris-HCl, pH 7.8, 10 mM DTT, 60 mM KCl, 10 mM MgCl., 100 μg BSA/mL, 10 μM each of dATP, dGTP, dTTP, 1 μM dCTP, 5 μCi of -³²P-dCTP (3000 Ci/mmol) and 200 ng of heterodimeric HIV-1 RT. The labeled cDNA product generated at 37°C for 10 min was chased with cold dNTP mix (500 μM each) for an additional 45 min at 37°C and quenched with 50 mM EDTA. The cDNA product was phenol-chloroform extracted, alcohol precipitated, and the pellet resuspended in 15 μl of 1 X loading buffer (10% glycerol and 0.01% each xylene cyanol and bromphenol blue). The products were resolved on 0.8% agarose gel; the gel was dried and subjected to phosphorimager analysis.

EXPERIMENT SET #2— PNA_TAR INHIBITS HIV-1 REPLICATION

I. OVERVIEW

In summary, four anti-TAR PNAs of different length have been designed such that they either complement the entire loop and bulge region (P A-.^,_; and P A-^._u) or are short a few sequences in the loop (PNA^^) or in both the loop and bulge (PNA_j^..). Tests then examined their functional efficacy in vitro as well as in HIV-1 infected cell cultures. All four anti-TAR PNAs showed strong affinity for TAR RNA, while their ability to block in vitro reverse transcription was influenced by their length. In marked contrast to PNA^..,., and P A_j^_.,₃, the two longer PNA.^ constructs were able to efficiently sequester the targeted site on TAR RNA, thereby substantially inhibiting Tat-mediated transactivation of the HIV-1 LTR. Further, a substantial inhibition of virus production was noted with all the four anti-

[54704-8057/LA033020.002] -38- TAR PNA, with PNA..^.. exhibiting a dramatic reduction of HIV-1 production by nearly 99%. These results demonstrate that PNA_TAR.16 is an anti-HIV agent.

In order to identify the appropriate length of anti-TAR PNA for efficient blocking of Tat-mediated transactivation, four anti-TAR PNAs of varying length ranging from 12- to 16- mer (PNA^-,..., PNA_T^_.-_J, PNA_TAI..₁₅ and PN ^_.,.) were designed and coupled with the luciferase reporter gene constructs. See Figure 8. It was discovered that a 16-mer PNA complementing both the loop and bulge regions of TAR efficiently inhibits Tat-mediated transactivation of HIV-1 LTR. Further, transfection of this 16-mer PNA in HIV-1 -infected CEM cells effectively blocks HIV-1 production, thus suggesting that anti-TAR PNA is an attractive candidate for antiviral therapy.

Activation of transcriptional elongation occurs following the recruitment of Tat to the transcription machinery via a specific interaction with an RNA regulatory element called TAR, a 59-residue RNA leader sequence in the long terminal repeat (LTR) (Karn, 1999). The main advantage of targeting the TAR element is that it is conserved and folds into a stable stem-loop structure. Any mutational changes in TAR that destabilize this structure also abolish Tat-TAR interaction.

The sequences of four anti-TAR PNAs of varying lengths and the region of TAR RNA that they target are shown in Figure 8. All four anti-TAR PNAs used in this study were able to bind to TAR, although subtle differences in their ability to gel-shift TAR RNA were noted at lower molar ratio of PNA_TAR to TAR RNA (Fig. 9, panels A, B, C, and D). PNA^-, is CCAGGCTCAGAT (SEQ ID NO: 2). P A-^ is CCAGGCTCAGATC (SEQ ID NO: 3). P A^. is TCCCAGGCTCAGATC (SEQ ID NO: 4). PNA-.^ is TCCCAGGCTCAGATCT (SEQ ID NO: 5). This difference in their binding ability is correlated with their lengths (Fig. 9, panels A, B, C, and D; lanes 2 and 3). The binding

[54704-8057/LA033020.002] -39- specificity of the anti-TAR PNAs is supported by our observation that scrambled PNA did not influence the mobility of TAR RNA (Fig. 9, panel 5).

The findings with the four anti-TAR PNAs used in this study, exhibiting pronounced blockage of reverse transcription on TAR RNA are consistent with this observation (Fig. 10). It may be pointed out, however, that the extent of blockage varied with the temperature of incubation of the anti-TAR PNA as well as the PNA_TAR itself (Figs. 10 and 11). While PNA_TA-,_.1S and PNA-.^_... exhibited near complete blockage of reverse transcription, some extension of the product beyond the targeted site was noted in the case of PNA-,^₃ and PNA_TAR.12 (Fig. 10). The observation that inhibition of reverse transcription on TAR RNA was markedly reduced when P A-.^_.^ was incubated at ambient temperature with the pre- annealed template primer suggests that the TAR stem-loop structure may be more stable at ambient temperatures and that smaller P _j^ are relatively less efficient in sequestering this region.

This contention is supported by the findings in CEM cells co-transfected with the pHIV-1 LTRLuc and pCMV-Tat reporter gene constructs in the presence and absence of the four different anti-TAR PNA and nonspecific scrambled PNA (Fig. 12). A substantial increase in the luciferase activity upon co-transfection with pCMV-Tat indicated a significant stimulation of the basal level of transcription of the HIV-1 LTR. All of the four anti-TAR PNAs were able to sequester the targeted site on the reporter gene construct in cell culture. However, it may be noted that decrease in the length of the anti-TAR PNA by shifting the target by a few nucleotides upstream or downstream resulted in a significant decrease in Tat- mediated transactivation. These results are not surprising since the TAR domain is minimally required for Tat response either directly or via its interaction with other transcription factors including the Tat binding pyrimidine bulge, the TAR RNA upper stem, and the loop

[54704-8057/LA033020.002] -40- sequences (Harrich et al., 1994.). Furthermore, the data clearly demonstrate that FNA_TAR.16 can inhibit HIV-1 replication in cell culture (Fig. 13). This is not svu rising since disruption of the Tat-TAR interaction by this PNA is expected to inhibit virus production.

II. EXPERIMENTS

2.1. Binding specificity of PN -^ to TAR RNA

Labeled TAR RNA corresponding to nucleotides +1 to +82 of the HIV-1 LTR was transcribed in vitro using T7 RNA polymerase and used for determining the binding specificity of the individual PNA^. The sequence of the HIV-1 TAR, PN -^, mismatched PNA_j,^ and scrambled PNA are shown in Fig. 8. Four anti-TAR PNAs of different length that either complement the entire loop and bulge region of TAR (PNA-.^_.^ and PNA-.^.,,) or are short of a few sequences either in the loop (P A_j.^-) or in both the loop and bulge (PNA_TA-,_,₂) were used in this study. Gel mobility shift assays determined the relative ability of the individual anti-TAR PNAs to bind with TAR RNA (Fig. 9). Anti-TAR PNAs or scrambled PNA at indicated concentrations, were incubated with 6.4 nM of the labeled TAR RNA transcript for lhr at 37°C in binding buffer and subjected to native polyacrylamide gel electrophoresis. The RNA-PNA complexes were resolved at a constant voltage of 120 V at 4 °C and visualized by phosphorimaging. The extent of gel shift was determined by quantifying the probe RNA band on the phosphorimager using Image-Quant software (Molecular Dynamics). Panels A, B, C, D and E represent gel-shift of labeled TAR RNA in the presence of PN ^_j._.^, PNA-^_.j., PN -^_j, PNA-.^_..- and PNA_SCB, respectively. Lanes 1-8 indicate gel- shift carried out at the following concentrations of the individual anti-TAR PNA or scrambled PNA: 0, 2.5, 5.1, 9, 16, 32, 48 and 64 nM, respectively. The percent of labeled TAR RNA retarded due to PNA binding is as indicated.

[54704-8057/LA033020.002] -41- As seen in Figure 9, a distinct shift in the mobility of TAR RNA was observed due to the formation of a specific [PNA_TAR-TAR RNA] complex (panels A, B, C and D; lanes 2-8). This mobility shift was concentration-dependent, as is evident from an incomplete shift seen at lower concentrations of PNA^. to TAR RNA (lanes 2 and 3) and a complete shift seen at higher concentrations (lanes 4-8). Although all the anti-TAR PNAs displayed strong affinity for TAR, the extent of gel retardation at lower concentrations varied with the length of the individual PNA; higher binding was noted with PNA-,^_... and PNA..^_.,-, as compared to PNA_τAR-₁₂ and PNA-,^-,., (lanes 2 and 3). The slower moving complex was not seen with scrambled PNA (panel E), thus illustrating the specificity of this interaction. 2.2. Inhibition of reverse transcription of TAR RNA in the presence of anti-TAR PNA

Since PNA-RNA or PNA-DNA duplexes exhibit higher Tm values than the corresponding RNA-DNA or DNA-DNA duplexes (Lee et al., 1998), it was interesting to examine if the individual anti-TAR PNA was able to block reverse transcription of HJN-1 TAR. Ability to block reverse transcription would have multiple effects on viral replication besides influencing Tat-mediated transactivation. For this propose, TAR RΝA primed with the labeled 17-mer DΝA primer was incubated in the absence or presence of the individual anti-TAR P A or scrambled P A at 37 °C followed by initiation of reverse transcription by HIV-1 RT. The results are presented in Figure 10. The individual anti-TAR PΝA or scrambled PΝA (1 μM) were pre-incubated at 37 °C with the TAR RΝA template primed with the 5'-³²P labeled 17-mer DΝA primer. Reverse transcription reactions were initiated by the addition of enzyme and dΝTP mix and aliquots were withdrawn at 5, 10, 15 and 20 min of incubation at 25 °C and quenched with the Sanger's stop dye. Control set represents the reactions carried out in the absence of anti-TAR PΝAs or scrambled PΝA. The position of the

[54704-8057/LA033020.002] -42- 17-mer primer is indicated. The position marked as 43 corresponds to the beginning of the loop region on TAR RNA targeted by the anti-TAR PNA.

In Figure 10, PHA-,^_..- and PNA-^_.^ caused a prominent pause in reverse transcription at the 42-mer position prior to the loop site targeted by these two PNAs. Likewise, the other two anti-TAR PNAs, PNA-,^_.,. and PN -.^.. also exhibited a prominent pause at nucleotide position 44, prior to the targeted site. In addition, another minor pause at nucleotide 43 was also seen, but appeared to be a natural pause on this template as noted from the control set carried out in the absence of PNA. These results show that the individual anti- TAR PNA bind to their target site on TAR and block reverse transcription. This blockage occurs because anti-TAR PNA inhibit the strand displacement activity of HIV-1 RT. Interestingly, while complete blockage at nucleotide position 42 was seen in case of PNA_TAR._]6 and PNA_TAR.15, further extension of some of the accumulated products beyond position 44 were observed with PNA_TAR.13 and PNA-^- upon prolonged reaction. Reverse transcription of HIV-1 TAR in the presence of scrambled PNA was similar to the control, indicating the specificity of the interaction of the individual anti-TAR PNA with its target sequence.

It is possible that incubation of PNA^- and RNA template at 37 °C may have facilitated their interaction by destabilizing the secondary structure of TAR. In order to evaluate whether PN -^ is able to invade the stem-loop of TAR and block reverse transcription at ambient temperature, individual PNA^ with the pre-primed HIV- 1 TAR at 25 °C were incubated at various concentrations. The pattern of reverse transcription products seen in Figure 11 indicates a distinct difference in the ability of the various PNA.^ to block reverse transcription of TAR. The TAR RNA template primed with the 5α-³²P labeled 17-mer DNA primer was incubated in the absence or presence of increasing concentrations of PNA-^ or scrambled PNA at 25 °C for 2 h. Reverse transcription was initiated by the addition of the

[54704-8057/LA033020.002] -43- four dNTP mix and HIV-1 RT. The reaction products were analyzed on a denaturing 8% polyacrylamide-urea gel and subjected to phosphorimager analysis. Lane 1 in each set represents the control reaction carried out in the absence of PNA. Lanes 2-9 represent extension of the 17-mer primer in the presence of the indicated PNA at 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 μM concentration, respectively. The position of the 17-mer primer is indicated on the left. The position marked as 43 corresponds to the beginning of the loop region on TAR RNA targeted by the anti-TAR PNA.

As shown in Figure 11, PNA_TAR.16,

were able to invade and block reverse transcription in a concentration dependent manner at ambient temperature and the pattern was similar to that observed at 37 °C. The highest inhibition was observed with

PNA^_j._.... In contrast, the ability of PNA-,^_... to block reverse transcription was greatly diminished at an ambient temperature. The higher efficiency of P -^_..- in sequestering

TAR and blocking reverse transcription suggests that targeting the entire stem-loop and bulge region-spanning nucleotides 19-34 in the HIV-1 LTR is essential for maximal efficiency. 2.3. Anti-TAR PNA blocks tat-mediated transactivation of the HIV- 1 LTR in cell culture

Results of the above in vitro experiment demonstrated that the four different anti-TAR

PNAs of varying lengths and sequences differed in their ability to block reverse transcription on HIV-1 TAR. Since HIV-1 Tat enhances transcription elongation via interacting with TAR, it was of interest to probe which of the four anti-TAR PNAs could block the function of Tat in cell culture. To this end, a reporter plasmid construct expressing the firefly luciferase under the control of the HIV-1 LTR was used. CEM cells were transfected, with the reporter plasmids, in the absence or presence of the pCMV-Tat along with varying amounts of the individual anti-TAR PNA or nonspecific scrambled PNA. The expression of luciferase was

[54704-8057/LA033020.0021 -44- then estimated by the Dual Luciferase Assay kit. Expression of Renilla luciferase driven by the CMV promoter did not significantly change in the absence or presence of the Tat expression clone or in the presence or absence of anti-TAR PNA, and was therefore used as a control to normalize for transfection efficiency (data not shown). On the other hand, expression of the firefly luciferase driven by the HIV-1 LTR directly correlated with the concentrations, as well as the lengths, of the individual P A^, thus pointing to the promoter specificity of anti-TAR PNA. These results are presented in Fig. 12, as the percent inhibition of Tat-mediated transactivation of the HIV-1 LTR at the indicated concentrations of the individual P A-^ or scrambled PNA calculated from the respective ratios of the firefly and Renilla luciferase activities.

Specifically, CEM cells (5.0*10⁶ cells in 250 μl) were transfected with the reporter plasmids pHIV-1 LTR-Luc (2 μg) and pCMV-R.Luc (0.6 μg) in the absence or presence of Tat expression vector, pCMV-Tat (1.0 μg). The individual PNA_TAi or scrambled PNA (PNA_SCB) were cotransfected at 1.0, 2.5 and 5.0 μg concentrations. The transfected cells were harvested 18 h post-transfection and analyzed for the individual luciferase activities.

Expression of Renilla luciferase driven by the CMV promoter did not significantly change either in the absence or presence of the Tat expression clone or in the presence or absence of anti-TAR PNA, and was, therefore, used as a control to normalize for transfection efficiency.

Expression of the firefly luciferase driven by the HIV-1 LTR correlated with the concentrations as well as the lengths of the individual PNA_TAR. These results are presented as the percent inhibition of Tat-mediated transactivation of the HIV-1 LTR at the indicated concentrations of the individual PNA-.^ or scrambled PNA calculated from their respective ratios of the firefly and Renilla luciferase activities. The results are expressed as mean values along with standard deviations of three independent experiments.

[54704-8057/LA033020.002] -45- As seen from Figure 12, the individual anti-TAR PNA significantly inhibited the Tat- mediated transactivation of the HIV-1 LTR. The extent of inhibition varied with the individual PN ^. as well as its concentration. Of the four anti-TAR PNAs, PN .^_.,, was most effective in inhibiting Tat-mediated transactivation. Co-transfection of as low as 1 μg PNA-.^._!- resulted in 75% inhibition of Tat-mediated transactivation, which subsequently increased to 88% and 97% inhibition at 2.5 and 5.0 μg, respectively. The percent inhibition with PNA_TAR.15 at these concentrations ranged from 40 to 87%. The extent of inhibition in case of PNA_TAR.]3 and PNA^-._.,-, was significantly lower with a maximum of 40-50% inhibition seen at the highest concentrations tested. Furthermore, it was noted that transfection of anti- TAR PNA at the indicated concentrations had no adverse effect on cell viability (data not shown), thus indicating that these molecules are probably not toxic to the cells.

2.4. Inhibition of HIV- 1 production in CEM cells by anti-TAR PNA

Since a concentration-dependent gradient of inhibition of Tat-mediated transactivation of the HIV-1 LTR was noted with the anti-TAR PNA of varying lengths, their efficacy in HIV-1 -infected cell cultures was investigated. The pseudotyped HIV-1 virion-infected lymphocyte CEM CD4+ cells were transfected with varying concentrations of the individual anti-TAR PNA (PNA-.^), mismatched anti-TAR PNA (PNA-.^-,^), or nonspecific scrambled PNA (PNA_SCB). The effect of anti-TAR PNA on HIV-1 production in CEM cells was monitored by analyzing the expression of the firefly luciferase reporter cloned in place of nef in the HIV-_1JR.CSFenv(-) cassette (Planelles et al., 1995). The firefly luciferase activity was normalized to the total protein in the cell extract. Luciferase expression obtained in the absence of PNA in the mock-transfected HIN-1 infected CEM controls was taken to be hundred percent and that obtained in the presence of PΝA was calculated relative to this value. These results, presented in Fig. 13, are as the percent luciferase activity obtained

[54704-8057/LA033020.002] -46- relative to the control at indicated concentrations of the anti-TAR PNA, mismatched TAR PNA or scrambled PNA.

In Figure 13, the effect of anti-TAR PNA on HIV-1 production was monitored by analyzing the expression of the firefly luciferase in the cell extracts at 48h post-transfection. Luciferase activity was monitored in a 96-well fluorotrac plate using a Packard Top Count Luminescence Counter. The firefly luciferase activity was normalized to the total protein in the cell extract. Luciferase expression obtained in the absence of PNA in the mock- transfected HIV-1 -infected CEM controls was taken to be hundred percent and the extent of luciferase expression in the presence of the PNA was calculated relative to this value. These results are presented as the percent luciferase activity obtained relative to the control at the indicated concentrations of individual PNAs. Panels A, B, C, D represent transfections carried out at 1.0, 2.5, 5.0 and 10.0 μg of the individual PNAs. The values shown are the average of three sets of experiments. The bars represent the standard deviation.

A substantial inhibition of virus production was seen with all four anti-TAR PNAs. The extent of inhibition was concentration-dependent and varied depending on the length of the individual PNA-,^. Of the four anti-TAR PNAs, PNA^-._..,; complementing the entire loop and bulge region of the TAR as well as a single nucleotide in the stem, was most effective in suppressing virus production exhibiting a dramatic abolishment at the highest concentration (10 μg). In contrast, P A^_.,. having the same sequence as PNA_TAR.I6 except for a single nucleotide complementing the stem, was significantly less effective than

in suppressing virus production, exhibiting an inhibition ranging from 50 to 85%.

Further decrease in the length of the anti-TAR PNA such that they are short of few sequences in the loop (PNA^,.) or in both the loop and bulge (PNA-^_.,-) resulted in a further decrease in their efficacy to inhibit HIV-1 production. Thus, PNA_TAR.13 and PN ^_.,., exhibited

[54704-8057/LA033020.002] -47- inhibition ranging from 10-70%). Scrambled PNA exhibited no inhibition of virus production at similar concentrations, indicating the specificity of anti- TAR PNA. A mismatched 16-mer anti-TAR PNA (PNA_TAR-_M having three nucleotide mismatches in the bulge region of the TAR, exhibited substantially reduced antiviral efficacy (6-40% inhibition of virus production). These results clearly demonstrate the importance of targeting the bulge region of the TAR.

III. MATERIALS AND METHODS

PNA oligomers

The PNA oligomers targeted to TAR regions of HIV-1 genome as well as scrambled PNA were synthesized at Applied Biosystems Inc. (Fig. 8). The general structure of PNA, where bases are linked with peptide bonds, is shown. The sequence of the individual anti- TAR PNA of varying lengths, the mismatched anti-TAR PNA (PNA-,^_.^) as well as the 17- mer scrambled (PNA_SCB) used in the experiments, are listed. Secondary structure of the HIV- 1 TAR RNA genome corresponding to the RNA stem-loop and bulge, where the complementary anti-TAR PNA binds, as shown in the box.

Plasmid constructs

The plasmid pEM-7 encoding the HIV-1 TAR under the control of the T7 promoter was used for transcribing the wild type TAR RNA (Gunnery et al., 1992) for gel shift analysis and primer extension studies. The plasmids, pHIV-1 LTRLuc, pCMV-Tat (pcDNA3-Tat), pCMV-R.Luc and pcDNA3.1 were used in the transfection experiments to investigate the effect of PNA^ on HIV-1 LTR. The plasmid pHIV-1 LTR-Luc (a kind gift from Dr. M. B. Mathews) contains the firefly luciferase gene cloned downstream of the HIV-1 LTR. The . plasmid pCMV-Tat, encodes for the Tat protein under the control of the CMV promoter (Fujinaga et al, 1999). The plasmid ρCMV-R.Luc (Promega Corp.) encodes for the Renilla

[54704-8057/LA033020.002] -48- Luciferase downstream of the CMV promoter andpcDNA3.1 (Invitrogen Coφ.) encodes for the CMV promoter.

Transcription of HIV- 1 TAR RNA template

HIV-1 TAR RNA template was transcribed after initially linearizing the plasmid pEM-7 with Hzrcdffl as described previously (Mayhood et al., 2000). For preparing the unlabeled transcript, in vitro transcription reaction was carried out using T7 RNA polymerase in accordance with the Manufacturer's protocol (Roche Molecular Biochemicals). The internally labeled transcript was similarly prepared except that the rNTP mixture contained 1 mM each of ATP, GTP, CTP and 20μM α-³²P UTP (specific activity: 1 μCi/10 pmol; Perkin- Elmer Life Sciences Inc.). Following the transcription reaction, 25 U of DNase I (RNase free) was added and further incubated for 30 min to digest the DNA. The labeled transcript was purified by 10% polyacrylamide-urea gel electrophoresis. The radioactive band was excised from the gel, extracted in 0.5 M ammonium acetate, and desalted on a NAP-10 column (Pharmacia Inc). It was then lyophilized and dissolved in 10 mM Tris- ΗC1, pΗ 7.8, 60 mM KCl and 10 mM DTT and stored at -70 °C. The specific radioactivity of the resulting purified transcript was determined by A260 absorbance and Cerenkov counting.

Gel retardation assay

The affinity and specificity of the various anti-TAR PNAs for the TAR RNA was evaluated by gel mobility shift analysis. Varying concentrations of anti-TAR PNAs or scrambled PNA were incubated with 6.4 nM ³²P-labeled TAR RNA transcript (5000 Cerenkov cpm) for 1 h at 37 °C in a binding buffer containing 50 mM Tris-ΗCl, pΗ 7.8, 60 mM KCl, 5.0 mM MgCl₂, 10 mM DTT, 10% glycerol, 0.01% NP-40 and 500 ng of rQnC). Three microliters of RNA gel loading dye (0.27% bromophenol blue and 30% glycerol) were added to the samples and subjected to electrophoresis on a native 6% polyacrylamide gel in

[54704-8057/LA033020.002] -49- Tris-Borate buffer. The gels were pre-run at 120 V for 30 min at 4 °C in Tris-Borate buffer, pH 8.2. The RNA-PNA complexes were resolved at a constant voltage of 120 V at 4 °C for 3 h and subj ected to phosphorlmager analysis (Molecular Dynamics).

Reverse transcription of TAR RNA primed with 17-mer DNA primer Reverse transcription catalyzed by HIN-1 RT on TAR RΝA in the presence or absence of the individual anti-TAR PΝA or scrambled PΝA was monitored by gel extension analysis. To this end, the 17-mer DΝA primer was 5 '-labeled using α-³ P-ATP and T4 polynucleotide kinase according to the standard protocol and annealed in a 2:1 molar ratio of RΝA template to primer. The individual anti-TAR PΝAs at the indicated concentrations were pre-incubated with 10 nM of the annealed template-primer either at 37 °C or at 25 °C for the indicated times in a reaction buffer containing 50 mM Tris-HCl, pH 7.8, 10 mM DTT, 100 μg/ml BSA, 60 mM KCl and 5 mM MgCl₂ and used in the extension reaction. Reverse transcription was initiated by the addition of 50 nM of HIV-1 RT and 100 μM each of the 4 d TP mix. The reactions were performed at 25 °C and terminated by the addition of equal volume of Sanger's gel loading solution. The products were resolved on an 8% polyacrylamide-urea gel and visualized on a phosphorlmager.

Tissue culture and transfection

Lymphocyte CEM (12D7) cells were maintained in complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin at 37 °C in 5% C02 containing humidified air. For transfections, the cells were grown to mid-log phase, washed with phosphate-buffered saline (PBS) without Ca⁺² or Mg⁺², resuspended in unsupplemented RPMI-1640 medium (5.0xlO^δ cells in 250 μl). The cells were then electroporated at 250 V and 900 microfarad capacitance with optimum amounts of the plasmids pHIN-1 LTR-Luc and pCMV-Tat, using a Bio-Rad Gene pulsar II.

[54704-8057/LA033020.002] -50- In order to monitor the efficiency of transfection, the cells were co-transfected with the reporter plasmid, pCMV-R.Luc. The effect of anti-TAR PNA on Tat-mediated transactivation of the HIV-1 LTR was monitored by co-transfecting the individual anti-TAR PNAs at the indicated concentrations. In order to determine the specificity of Tat-TAR interaction, a 17-mer control PNA containing scrambled sequence was co-transfected in an independent experiment. The transfected cells were plated in 10 ml of serum free RPMI-1640 media, allowed to recover from the effects of electroporation at 37 °C for 2 h and then grown in 10 ml of complete RPMI-1640 medium. Eighteen hours post-transfection, the cells were harvested and analyzed for luciferase activity. To monitor the effect of anti-TAR PNA on cell viability, an aliquot of the transfected cell culture was withdrawn prior to harvesting and examined using the calcein AM component from the Live-Dead viability kit (Molecular probes) as per the Manufacturer's protocol.

Production of pseudotyped HIV

Pseudotyped HIV-1 virions were produced in 293T cells by co-transfection of pHIV- UR-CSF-lucenv(-) (Planelles et al. 1995) with the pVSV-G retroviral vector, encoding the vesicular stomatitis virus protein G under the control of the CMV immediate-early promoter (BD Biosciences Clontech.) using the calcium phosphate transfection system (Life Technologies). Virus stocks were harvested at 24, 48 and 72 h post-transfection, an aliquot was removed for p24 antigen quantitation using the ELISA p24 antigen kit (Abbott Laboratories) and the remaining stock was frozen at -80 °C.

Infections

The effect of anti-TAR PNA on HIV-1 production was monitored in CEM cells infected with the pseudotyped HIV-1 virions expressing the firefly luciferase reporter. Briefly, pseudotyped HIV-1 virions in the presence of 10 μg of polybrene/ml were added to

[54704-8057/LA033020.002] -51- 5xl0^δ CEM cells in a final volume of 1.0 ml to achieve multiplicities of infection of 10. The cell cultures were incubated at 37 °C for 1 h, cells were gently spun out, washed with PBS and resuspended in 1.0 ml of complete RPMI medium. The infected cells were further incubated for 1 h in a 37 °C incubator and then transfected with varying amounts of the individual anti-TAR PNA, scrambled PNA or the mismatched TAR PNA as described above. A mock transfection of infected cells was similarly carried out. The cells were grown in 10 ml of complete RPMI medium. Forty-eight hours post-transfection, the cells were harvested and expression of the pseudovirus was analyzed by estimating the firefly luciferase activity.

Luciferase assays Luciferase assays were performed by using the Promega Dual Luciferase assay kit.

The transfected cells were harvested, washed once with PBS without Ca⁺² or Mg⁺² and resuspended in 50 μl of the reporter lysis buffer (Promega). Cell lysis was carried out by incubating the samples at room temperature for 15 min on a rocking shaker. The lysate was centrifuged at 15,000 rpm for 10 min and the cell extracts were assayed for firefly and Renilla luciferase activity in a 96-well fluorotrac plate using a Packard Top Count Luminescence Counter. The results of at least three separate transfections were analyzed for each experiment.

EXPERIMENT SET #3— PNA^ CONJUGATED WITH MEMBRANE PERMEATING PEPTIDE VECTOR RETAINS AFFINITY FOR TAR

I. OVERVIEW

In summary, this experiment set proves that anti-TAR PNA (PNA-.^), upon conjugation with a membrane-permeating peptide vector (transportan) retained its affinity for TAR in vitro similar to the unconjugated analog. The conjugate was efficiently internalized

[54704-8057/LA033020.002] -52- into the cells when added to the culture medium. Examination of the functional efficacy of the PNA_TAB-transρortan conjugate in cell culture using luciferase reporter gene constructs resulted in a significant inhibition of Tat-mediated transactivation of HIV-1 LTR. Furthermore, PNA_TAR-transportan conjugate substantially inhibited HIV-1 production in chronically HIN-1 -infected H9 cells. The mechanism of this inhibition is regulated at the level of transcription. These results demonstrate the efficacy of PΝA_τAR~transportan as an anti- HIV agent.

Effective antisense inhibition in cells requires efficient cell entry. Though PΝAs have been shown to inhibit gene expression and have several advantages over conventional DΝA or RΝA antisense oligomers, their use as an alternative therapy has been limited chiefly by their inability to cross the cell membrane efficiently. The present studies used the transportan peptide vector for biodelivery of PΝA_TAR. Transportan is a 27-amino-acid chimeric peptide consisting of 13 amino acids from the amino terminus of the neuropeptide galanin and 14 amino acids from the amino terminus of the wasp venom toxic peptide mastoparan. Transportan penetrates every cell type in a rapid and efficient way and is localized mostly in membranous structure; upon prolonged incubation, it concentrates in the nucleus. In moderate concentration (10 μM), it does not affect the growth of cells in culture. These unique properties of the chimeric transportan peptide make it potentially useful as a vector for biodelivery of different proteins or nonprotein drugs into cells. To ascertain the ability of the PNA-transportan conjugate to enter the cells, the cells were tagged it with TAMRA, a fluorescent probe, and its uptake in the cell was monitored. A substantial accumulation of the fluorophore in the cell in 12 hours (Table 1) indicated efficient uptake, though the overall kinetics of its internalization was slower than with transportan alone. In marked contrast to an earlier report demonstrating that cell type did not

[54704-8057/LA033020.002] -53- influence the cell-penetrating ability of transportan (Pooga et al., 1998), the uptake of the PNA-transportan conjugate by CEM and Jurkat cells varied significantly (Table 1). This may be attributed to a difference in the morphology and membrane composition of these two cells, or it is likely that the presence of the PNA moiety influences uptake. Nonetheless, the accumulation of the PNA-transportan conjugate in the cells is encouraging and suggests that the transportan vector may be used effectively for biodelivery of this class of compounds.

PNA_TAR-transportan substantially inhibited Tat-mediated transactivation of the HIV-1 LTR in both CEM and Jurkat cells (Fig. 18). The PNA-peptide conjugate may have dual functions; it may prevent the TAR-Tat interaction by sequestering the TAR sequence of the nascent RNA, or it may prevent transcription directly by interacting with the TAR sequence on the proviral DNA. One may visualize that the large transportan moiety may interfere with the PNA, targeting its site inside the cell. However, this did not appear to be true in this study, since the PNA_TAR-transportan conjugate displayed similar binding affinity for its target sequence on the TAR RNA (Fig. 15) and was also able to block reverse transcription to a similar extent as the unconjugated PNA_TAR(Fig. 1 ). Moreover, a substantial inhibition of Tat- mediated transactivation was also achieved by PNA_TAR-transportan conjugate in cell culture (Fig. 19). The mechanism of this inhibition appeared to be at the level of transcription (Fig. 20).

The results demonstrating that PNA_j^-transportan can enter chronically HIV-1- infected H9 cells and inhibit virus production are very promising (Fig. 21). In summary, the studies have demonstrated that cellular uptake of anti-TAR PNA is significantly enhanced upon conjugation with a transportan peptide, resulting in effective blockage of the Tat-TAR interaction at relatively low inhibitor concentrations and inhibiting transcription. These studies with transportan peptides as the vector for biodelivery of PNA have created a

[54704-8057/LA033020.002] -54- therapeutic niche for the application of PNA against HIV-1. Thus, the approach using PNA- transportan to target the critical regions of the HIV-1 genome in the quest for novel inhibitors of HIV-1 appears quite promising. Optimal chemical modification of the existing transporter peptides by treatment with polyethylene glycol in order to increase the circulating half-life of PNA-transporter conjugates will further advance the efforts to develop potent antiviral therapeutics targeting viral genes.

II. EXPERIMENTS

3.1 Synthesis of PNA and PNA-transportan conjugates

PNA and PNA-transportan conjugates were synthesized at Applied Biosystems. The PNA molecules are linked to the transportan peptide through a disulfide linkage, as shown in Figure 14. Secondary structure of the HIV-1 TAR RNA is also shown in this figure. Arrows indicate the TAR sequences interacting with the PNATAR transportan conjugate. The molecular mass of the PNA^-transportan conjugate was analyzed by matrix-assisted laser desoφtion ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The observed mass of the conjugate (7,382.3 Da) corresponded to the calculated mass (7,377.3 Da) with a variance of -0.07%.

3.2 Affinity of PNA_TAR-transportan conjjugate to TAR

In order to ascertain whether PNA^. conjugated with the transportan peptide vector retained its binding affinity for its target sequence on the TAR RNA, we performed gel mobility shift assays with the ²P-labeled 82-base-long TAR RNA transcript and various concentrations of PNA^-transportan conjugate, scrambled PNA-transportan conjugate, or unconjugated PNA TAR (Fig. 15A to C). Titration of PNA^-transportan conjugate with TAR RNA at molar ratios of the conjugate to TAR RNA of less than 1 resulted in a stoichiometric band shift of the labeled TAR RNA (Fig. 15 A, lanes 2 to 4).

[54704-8057/LA033020.002] -55- In Figure 15, the affinity of the PNA-^-transportan conjugate (A) for its target sequence on the TAR RNA was assessed by gel mobility shift analysis as described in Materials and Methods: Unconjugated PN -.^ (B) was included as a positive control, and scrambled PNA-transportan conjugate (PNA_SCB-transportan) (C) was included as a negative control. The specificity of this interaction was determined by analyzing the relative gel mobility shift upon interaction of PNA_TA-,-transρortan conjugate with a mutant TAR RNA (TAR BS; panel D) as well as with an unrelated RNA such as the HIV-1 RT RNA (panel E). In panel A, lanes 1 through 9 represent molar ratios of PNA_TA-,-transportan conjugate to TAR RNA of 0, 0.1, 0.5, 0.8, 1.0, 2.5, 5, 7.5, and 10, respectively. Panel B and panel C show gel mobility shifts performed at similar ratios as indicated in panel A except that unconjugated PNA,.^ and scrambled PNA-transportan conjugate, respectively, were used. Similar ratios are also shown in panels D and E except that the mobility shift of the PNA_TAE.-transportan conjugate was carried out with the TAR BS and HIV-1 RT RNA probes, respectively. The extent of gel shift was determined by quantifying the probe RNA band on the Phosphorlmager using Image-Quant software (Molecular Dynamics). The percentage of labeled TAR RNA retarded due to PNA binding is indicated.

As shown in the figure, at molar ratios of 0.1, 0.5, and 0.8 of the conjugate to labeled TAR RNA, the extent of band shift corresponded to approximately 8, 45, and 87%, respectively. A complete shift in the mobility was achieved at an equimolar ratio or a molar excess of the conjugate to TAR RNA (panel A, lanes 5 to 9). A similar titration was carried out in the presence of unconjugated PNA^-, (panel B, lanes 2 to 9) and scrambled PNA- transportan conjugate (panel C, lanes 2 to 9). As shown in the figure, the binding of PNA.^ to the TAR RNA was similar to that obtained with the PNA-^-transportan conjugate, suggesting that conjugation of PNA-.^ with the transportan peptide vector had no effect on its

[54704-8057/LA033020.002] -56- binding affinity. Scrambled PNA-transportan conjugate at similar molar ratios did not result in any gel shift, suggesting the specificity of the interaction. Further evidence of this specificity was demonstrated by our observation that interaction of PNA_TAR-transportan conjugate with TAR BS, a 63-base-long mutant TAR RNA carrying a deletion in the stem and bulge region (panel D), and with HIV-1 RT RNA, an unrelated RNA (panel E), also exhibited no shift in mobility.

3.3 Reverse transcription of TAR RNA region is blocked by PNA_TAR-transportan conjugate

Since the PNA^-transportan conjugate displayed affinity for TAR RNA similar to unconjugated PNA_TAR, it was interesting to examine if the conjugate could effectively block reverse transcription of TAR RNA. Ability to block reverse transcription, in turn, would have multiple impacts on viral replication besides influencing Tat-mediated transactivation. For this purpose, the TAR RNA transcript primed with the labeled 17-mer DNA primer was incubated in the absence or presence of 100 nM PNA_rAR-transportan conjugate, PNA_TAR, or scrambled PNA at 37°C for one hour, followed by initiation of reverse transcription by HIV-1 RT. The results are presented in Figure 16. The reaction products were analyzed on a denaturing 8% polyacrylamide-urea gel and subjected to Phosphorlmager analysis. Lanes 1 through 4 in each set represent the extension reactions at 25°C for 5, 10, 15, and 20 min, respectively. The control set represents reactions carried out in the absence of PNA-,^- transportan conjugate, unconjugated PNA_TAR, or scrambled PNA-transportan conjugate. The arrow indicates the position on the gel corresponding to the sequence targeted by^'PNA.^- transportan conjugate as well as unconjugated PNA_TAR on TAR RNA.

PNA-.^ and PNA-^-transportan conjugate showed a similar pattern of reverse transcription, with a prominent pause at nucleotide position 42, prior to the loop site targeted

[54704-8057/LA033020.002] -57- by these two PNAs. These results show that conjugation of PNA^, with transportan did not alter the ability of PNA..^ to block reverse transcription of TAR RNA.

3.4 Cellular uptake of PNA-transportan and its intracellular localization

To evaluate the uptake of the PNA-transportan conjugate in our system, a transportan-conjugated 10-mer PNA tagged with a TAMRA fluorophore probe was used. The entry of the dye-linked conjugate into the cells was then monitored by fluorescence microscopy and analyzed by fluorescence-activated cell sorting (FACS) analysis. The results are shown in Fig. 17 and Table 1. It was observed that the PNA-transportan conjugate is able to enter the cells in a time-dependent manner, confirming that transportan linked with PNA retained its ability to cross the cell membrane (Fig. 17). CEM cells were incubated with 70 nM TAMRA-tagged PNA-transportan conjugate in a six- well plate m RPMI 1640 medium in the absence of serum. At various times, aliquots of the cells were washed, resuspended in PBS, and examined by fluorescence microscopy for monitoring cellular uptake. The panel on the left is the fluorescence image, and that on the right is the phase-contrast image of the same field.

At 12 hours, approximately 90% of the CEM cells were found to display fluorescence, as against 52% of the Jurkat cells (Table 1 — values represent averages of three independent experiments). This suggests that the PNA-transportan conjugate can efficiently cross the membrane of both cell types, although the kinetics of cell entry varied. Furthermore, the PNA-transportan conjugate had no detrimental effect on cell viability up to the 10 μM concentration tested in both CEM and Jurkat cells (results not shown). These results demonstrate that membrane-permeating peptide vectors such as transportan may be effective carriers for intracellular delivery of PNA.

[54704-8057/LA033020002] -58- TABLE 1. Time course of TAMRA-tagged PNA-transportan conjugate uptake in cells Uptake of PNA-transportan (%) at time posttransfection:

Cells lh 2h 4h 6h 12h

CEM 5 12 35 55 90 Jurkat 0 3 16 27 52

3.5 Inhibition of Tat-mediated transactivation of HIV-1 LTR by PNA_TAT,-transportan conjugate

Since the transporter peptide was found to be an efficient vehicle for biodelivery of PNA, we examined the abihty of PNA_TAR-transportan conjugate to block Tat-mediated transactivation of HIV-1 LTR in CEM and Jurkat cells. Using the reporter plasmid, the optimum amounts of Tat required for the expression of the HIV-1 LTR in these cells was first established. For this, the highly sensitive assay system involving expression of Renilla luciferase and firefly luciferase. Expression of the firefly luciferase (pHIV-1 LTR-Luc) and Renilla luciferase (pCMV-R.Luc) was under the control of the HIV-1 LTR and CMV promoter, respectively. The cells were transfected with these two plasmids in the presence of various concentrations of the Tat expression vector (pCMV-Tat).

In the Jurkat cells, the transfected cells were activated with PMA and Ca²⁺ ionophore for expression of the reporter plasmids. When Tat was expressed in trans, the firefly luciferase was transactivated several fold, while the Renilla luciferase expression, used as an internal control to monitor transfection efficiency, remained constant. Expression of the firefly luciferase in CEM as well as Jurkat cells was found to be proportional to the concentration of pCMV-Tat used in the transfection. The amount of pCMV-Tat for optimum stimulation of HIV-1 LTR in CEM cells (100-fold stimulation) and Jurkat cells (150-fold stimulation) was 1 μg and 3 μg, respectively (Fig. 18).

[54704-8057/LA033020.0021 -59- In Figure 18, the indicated amounts of pCMV-Tat were cotransfected along with a plasmid cocktail comprising pHIV-1 LTR-Luc (4 μg) and pCMV-R.Luc (1 μg) in Jurkat and CEM cells (5.0 _ 106 cells) in order to establish the extent of Tat-mediated transactivation of the HIV-1 LTR. The highly sensitive luciferase reporter was used for this analysis. The extent of stimulation of the HIV-1 LTR was monitored as a function of the expression of the firefly luciferase gene, with the Renilla luciferase reporter serving as an internal control for normalizing the transfection efficiency. The results are expressed as the ratio of firefly to Renilla luciferase activity. Panels A and B represent the extent of transactivation in CEM and Jurkat cells, respectively, as a function of pCMV-Tat concentration. The results are presented as an average of three independent experiments. The bars represent the standard deviation.

Using this standardized system, we determined the functional ability of the PN ^- transportan conjugate to inhibit Tat-mediated transactivation of the HIN-1 LTR. The results are shown in Fig. 19. As shown in the figure, CEM and Jurkat cells (5.0 _ 106 cells), cotransfected with the plasmid cocktail comprising pHIV-1 LTR-Luc (4 μg) and pCMV- R.Luc (1 μg) in the presence or absence of the Tat expression clone pCMV-Tat, were incubated with the indicated concentrations of the PΝA-^-transportan, unconjugated PΝA..^, transportan, or scrambled PΝA-transportan (PΝA_SCB-transportan) under the conditions described in Materials and Methods. At 20 h (CEM) or 28 h (Jurkat) posttransfection, cell lysates were assayed for firefly and Renilla luciferase activities. The results are presented as the relative ratio of firefly to Renilla luciferase activity. The amounts of the various plasmids, transportan, and PNA used in the experiment are indicated. The results are presented as an average of three independent experiments. The bars represent the standard deviation.

In Figure 19, the PNA-^-transportan conjugate substantially inhibited Tat-mediated transactivation of the HTV-l LTR, as judged from the expression levels of luciferase in both

[54704-8057/LA033020.002] -60- CEM and Jurkat cells. Approximately 55% inhibition was achieved at 500 nM PNA-peptide conjugate, which gradually leveled off to 70 to 80% inhibition at higher concentrations. Unconjugated P .^, scrambled PNA-transportan, and transportan itself had no effect on the luciferase activity under identical conditions. These results demonstrate that P A-.^- transportan present in the culture medium is able to enter the cells and prevent Tat-mediated transactivation by efficiently sequestering the TAR region of the HIV-1 LTR.

3.6 PNA_y^-transportan inhibits at the level of transcription

A substantial reduction in the expression of the luciferase reporter in both CEM and

Jurkat cells suggested that the PNA_TAR-transportan inhibits the Tat-mediated transactivation of the HIV- 1 LTR. To determine if the inhibition was at the level of transcription, we performed RNase protection assays. CEM and Jurkat cells transfected with an HIV-1 LTR promoter- driven luciferase reporter plasmid in the absence or presence of a Tat expression plasmid were treated with various concentrations of PNA-^-transportan, PNA_TAR, scrambled PNA- transportan, or transportan and harvested at the specified times for total RNA isolation. In addition, the Renilla luciferase reporter driven by a CMV promoter was included as a control for monitoring transfection efficiency. The RNA samples were hybridized with the 32P- labeled pSPluc_ and pSPrluc probes, digested with the RNase A and TI mix, and examined by denaturing gel electrophoresis for protected fragments corresponding to the firefly and Renilla transcripts, respectively. A representation of one such analysis is presented in Fig. 20. In Fig. 20(A), Jurkat cells were transfected with 4 μg of pHIV-1 LTR-Luc and 1 μg of pCMV-R.Luc in the absence or presence of pCMV-Tat (2.5 μg). The cells were grown in the presence of PNA^-transportan (0.5 to 5.0 μM), PN .^ (1.0 and 5.0 μM), transportan (1.0 and 5.0 μM), or scrambled PNA-transportan (1.0 and 5.0 μM). At 28 h posttransfection, total RNA was isolated from the cells and hybridized with RNA probes for the different reporter

[54704-8057/LA033020.002] -61- transcripts (firefly and Renilla luciferase). The pSPrluc probe coding for the Renilla RNA served as a transfection control. The firefly RNA levels were normalized to the Renilla RNA signal. The lanes are as follows: mock-transfected cells (lane 1); luciferase expression in the absence of Tat (lane 2); and luciferase expression in the presence of Tat and the indicated inhibitors (lanes 3 to 12). (B) CEM and Jurkat cells were transfected with 1 μg of pCMV- R.Luc and incubated in the absence or presence of PNA^-transportan, unconjugated PN -^, transportan, or scrambled PNA-transportan (PNA_SCB-transportan) at 5 μM in order to investigate the promoter specificity of PNA_TAR-transportan. Total RNA was isolated from cells and subjected to the RNase protection assay with the pSPrluc probe. The protected fragments were analyzed on a denaturing polyacrylamide-urea gel and visualized on a Phosphorlmager. Renilla RNA was normalized to total RNA in the cell. The lane marked control represents Renilla RNA signal in transfected cells in the absence of the ihibitor.

Our results indicated that the accumulation of firefly RNA was inversely proportional to the concentration of PNA_TAR-transportan in CEM and Jurkat cells (Fig. 20A). This provides direct evidence that the PNA-^-transportan interferes with the transcription process, presumably by binding to the TAR region of the HIV-1 LTR, thereby preventing Tat- mediated transactivation. Neither the unconjugated PNA_TAR, transportan itself, nor scrambled PNA-transportan affected the firefly RNA expression levels, indicating the specificity of the interaction of PNA_TAR-transportan with its target sequence. Furthermore, the effect of PNA-^- transportan on the HIV-1 LTR appeared to be promoter specific, as noted from our observation that PNA_TAR-transportan, PNA_TAR, transportan, and scrambled PNA-transportan did not affect the expression levels of the Renilla RNA driven by the CMV promoter (Fig. 20B)

[54704-8057/LA033020.002] -62- 3.7 Effect of PNA-^-transportan on HIV-1 production in chronically HIV-1-infected H9 cells

Since PNA_TAR-transportan was able to enter the cells and interfere with Tat-mediated transactivation, its antiviral efficacy in H9 cells chronically infected with HIV-1 was examined. These cells were grown in the absence or presence of PNA^-transportan (1.0 to 5.0 μM) (A), unconjugated PNA_TAR (5.0 μM), transportan (5.0 μM), or scrambled PNA- transportan (PNA_SCB-transportan) (5.0 μM) (B) for 3 days, and their culture supernatants were analyzed for levels of p24 antigen by ELISA, since the levels of p24 antigen provided a measure of viral titers. The results are shown in Fig. 21. The levels of p24 antigen decreased by 60%> at 1 μM PNA-^-transportan conjugate. Further increases in the concentration of the PNA_TAR-transportan conjugate resulted in a substantial decrease (70 to 90%) in the p24 levels, suggesting its potential for blocking HIV-1 production (Fig. 21 A). Our observation that unconjugated PNA-.^ at similar concentrations did not decrease viral production indicated the efficacy of PNA_TAR-transρortan. Furthermore, the effect of PNA-^-. transportan appeared to be quite specific, since neither scrambled PNA-transportan nor transportan by itself decreased HIV-1 production under identical conditions (Fig. 2 IB).

3.8 PNA-^-transportan inhibits transcription of HIV-1 mRNA in chronically HIV-1- infected H9 cells

The experiment set also examined the mechanism by which PNA_rAR-transportan inhibited HIV-1 production in chronically HIV-1 -infected H9 cells. In Figure 22, total RNA was isolated from chronically HIV-1 -infected H9 cells treated with the indicated concentrations of PNA_TAR-transportan or 5.0 μM PNA^-,, transportan alone, or scrambled PNA-transportan (PNA_SCB-transportan). The RNA was hybridized with the pGEM23 probe and digested with the RNase A and T, mix, and the protected fragments were separated on a polyacrylamide-urea gel and detected by Phosphorlmager analysis. The undigested probe

[54704-8057/LA033020.002] -63" corresponds to a 195-base fragment, whereas HIV-1 mRNA initiating at +1 of the HIV-1

LTR protected an 83-nucleotide fragment of the riboprobe corresponding to the anti-TAR sequence. A decrease in levels of HIV-1 mRNA in cells treated with PNA_TAR-transportan suggested that the inhibition occurred at the transcription step. The specificity of the effect of PNA_rAR-transportan was seen from the observation that neither transportan itself nor scrambled PNA-transportan altered the HIV-1 mRNA levels. Furthermore, PNA^. alone also did not influence the levels of HIV-1 mRNA synthesis, indicating the efficiency of transportan as a vehicle for delivering PNA_TAR to its target site.

3.9 PNA_TAR-transportan conjugate has no effect on cellular proliferation of chronically HIV-1-infected H9 cells

The incoφoration of [³H]thymidine into cellular DNA was examined in order to determine if 5.0 μM PNA_TAS.-transportan conjugate impacted cellular proliferation of chronically HIV-1 -infected H9 cells. The cells were grown in the presence or absence of P -^-. -transportan and supplemented with 10 μCi [met . >/-³H]thymidine/ml. At the indicated time points, aliquots were withdrawn, and the DNA was precipitated with TCA onto glass fiber filters to determine thymidine (TdR) incoφoration. The results of this analysis are shown in Fig. 23. The total amount of radioactivity was determined by scintillation counting, and the results are represented as counts per minute incorporated per milligram of protein. The results are expressed as average values along with the standard deviation for three independent experiments. As shown in the figure, similar amounts of [³H]thymidine were incoφorated into cellular DNA in both the treated and untreated cells at all time points analyzed, indicating that cellular proliferation was not altered in the presence of PNA_TAR- transportan.

[54704-8057/LA033020.002] -64- III. MATERIALS AND METHODS

PNA oligomers

The sequence of PNA_TAR-transportan conjugate, tetramethyhhodamine (TAMRA)- labeled PNA-transportan conjugate, and scrambled- PNA-transportan conjugate are shown in Fig. 14. PNA oligomers and their conjugated derivatives were obtained from Applied Biosystems. Secondary structure of the HIV-1 TAR RNA is as shown. Arrows indicate the TAR sequences interacting with the PNATAR-transportan conjugate. The inset corresponds to the sequences of PNA_TAK, TAMRA-tagged PNA, and scrambled PNA conjugated with the 27-amino-acid transportan peptide. To avoid the problem of precipitation of PNA at high concentrations, the working stocks of PNA oligomers as well as the PNA-transportan conjugates were maintained at a 100 μM concentration and stored at 4°C as recommended. The stocks were prepared by dissolving the PNA in water and heating to 50°C for 10 min, followed by incubation at 37°C for 30 min prior to quantification. Unused portions of the PNA stocks were aliquoted after 2 weeks, lyophilized, and stored at 4°C. The TAMRA-labeled PNA was protected from light to avoid photobleaching during its preparation and during the experiments.

Plasmid constructs

Plasmids for expression in mammalian cells were as follows: pHIV-1 LTR-Luc (a kind gift from M. B. Mathews), containing the firefly luciferase gene cloned downstream of the HIV-1 LTR; pcDNA3-Tat (pCMVTat), encoding the 72-amino-acid Tat protein under the control of the cytomegalovirus (CMV) promoter(Fujinaga, et al., 1999); pCMV-R.Luc (Promega, Madison, Wis.), for expressing Renilla luciferase under the control of the CMV promoter; and cDNA3.1 (Invitrogen Coφ., Carlsbad, Calif.), encoding the CMV promoter.

[54704-8057/LA033020.002] -65- Plasmids used for preparing transcripts for l shift assays pEM7 and pTAR-BS were used for generating the wild-type TAR RNA and its mutant derivative carrying a deletion at the top of the stem (Gunnery et al., 1992), and pET- 28a-RT was used for transcribing a 427-base-long HIV-1 reverse transcriptase coding fragment (Kaushik, et al., 1996).

Plasmids used for generating probes for RNase protection assay

The plasmids used for generating probes for the RNase protection assay were as follows: plasmids pSP-luc+ and pSP-rluc (Promega), encoding the firefly and Renilla luciferase gene cassettes, respectively, and having an opposing T7 promoter located downstream of the luc+ and rluc insert, whereas pGEM23 (Laspia, et al., 1989) has an opposing SP6 promoter located downstream of the TAR gene insert.

Cell culture and transfection

CEM and Jurkat T-cell lymphocytes were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 4 mM L-glutamine, 100 U of penicillin, and 100 μg of streptomycin per ml at 37°C in 5% CO_z-containing humidified air. The chronically HIV- 1 -infected H9 cells were maintained under identical conditions except that they were supplemented with 20% fetal calf serum. Jurkat T cells (5.0 X 10^s cells in 300 μl of RPMI 1640 supplemented with 20% fetal calf serum) were transfected with requisite amounts of the experimental plasmids pHIV-1 LTR-Luc and pCMV-Tat by electroporation using a Bio-Rad gene pulser II at 280 V and 975 μF capacitance.

In order to monitor the efficiency of transfection, the cells were cotransfected with the reporter plasmid pCMV-R.Luc. The cells were stimulated with phorbol 12-myristate 13- acetate (PMA; 20 ng/ml) and the calcium ionophore A23187 (1 μM) at 24 h posttransfection.

CEM cells were washed with equal volumes of phosphate-buffered saline (PBS) (without Ca²⁺ or Mg²⁺) before transfection. After washing, cells were resuspended in unsupplemented

[54704-8057/LA033020.002] -66- RPMI 1640 medium (5.0 X 10⁶cells in 250 μl) and electroporated with experimental and reporter plasmids at 230 V and 800 μF capacitance. Cells were then grown in 10 ml of complete RPMI 1640 medium.

Cellular uptake of PNA-transportan conjugate The uptake of TAMRA-tagged PNA-transportan conjugate was examined in CEM cells and Jurkat cells. Briefly, the log-phase cells were washed with PBS and resuspended in a six-well plate in serum-free RPMI medium at a cell density of 3 X 10^s cells/ml. The TAMRA-PNA-transportan conjugate (75 nM) was added to the culture medium, and the cells were incubated at 37°C. At various time intervals, aliquots (700 μl) of the cells were removed, washed with PBS, and examined by fluorescence microscopy. Simultaneously, the effect of PNA-transportan conjugate on cell viability was examined using the calcein AM component from the live-dead viability kit (Molecular Probes) as per the manufacturer's protocol.

Electrophoretic mobility shift assay The affinity and specificity of the PNA_rAR-transportan conjugate for the TAR RNA was examined by gel mobility shift analysis using the wild-type TAR RNA probe, a mutant TAR RNA probe, and the HIV-1 reverse transcriptase RNA probe. The ³²P-labeled TAR and TAR BS mutant RNA probes were prepared by in vitro transcription of the linearized plasmid templates as described previously (Mayhood et al, 2000). The 427-base HIV-1 reverse transcriptase (RT) RNA probe was generated by digesting pET-28a-RT with EcøRV and transcribing it using the T7 RNA polymerase as per the manufacturer's protocol (Roche Biochemicals). TAR RNA transcript (5 X 10¹ Cerenkov cpm) was incubated with PNA-.^- transportan conjugate, unconjugated PNA^-., or scrambled PNA-transportan conjugate at various molar ratios. In another set, PNA_j^-transportan conjugate was incubated with the

[54704-8057/LA033020.002] -67- mutant TAR BS or the HIV-1 RT radiolabeled probe at various molar ratios in order to evaluate its binding specificity.

Incubations were carried out for 3 h at 37°C in a binding buffer containing 50 mM Tris-HCl (pH 7.8), 60 mM KCl, 5 mM MgCl₂, 10 mM dithiothreitol (DTT), 10% glycerol, 0.01% bovine serum albumin, 0.01% NP-40, and 500 ng of poly (rlrC), in a final volume of 15 μl. Then, 3 μl of RNA gel loading dye (0.27%) bromophenol blue and 20% glycerol) was added to the samples and subjected to gel retardation analysis on native 6% polyacrylamide gel in Tris-borate-EDTA (TBE) buffer, pH 8.2. The gels were routinely run at 100 V for 30 min at 4°C in TBE buffer. The various complexes were resolved at a constant voltage of 150 V at 4°C. The gels were dried, visualized on a phosphorlmager (Molecular Dynamics), and quantified using Image-Quant software (Molecular Dynamics).

Reverse transcription of TAR RNA transcript

Reverse transcription catalyzed by HIV-1 RT on the TAR RNA transcript in the presence or absence of PNA^-transportan conjugate, unconjugated PNA-.^, or scrambled PNA-transportan conjugate was monitored by extension of 5 '-³²P-labeled 17-mer DNA primer annealed with the TAR RNA template. The PNA^-transportan conjugate or P -.^ at 1 μM concentration was incubated with 10 nM annealed template-primer at 37°C for 1 h in a reaction buffer containing 50 mM Tris-HCl (pH 7.8), 10 mM DTT, 100 μg of bovine serum albumin per ml, 60 mM KCl, and 5 M MgCl. and used in the extension reaction. Reverse transcription was initiated by the addition of 50 nM HIV-1 RT and 100 μM each of the four deoxynucleoside triphosphates. The reaction mixture was incubated at 25°C, and the reaction was terminated at the indicated time by the addition of an equal volume of Sanger's gel loading solution. The products were resolved on an 8% polyacrylamide-urea gel and visualized by Phosphorlmager.

[54704-8057/LA033020.002] -68- Assay for Tat-mediated transactivation of HIV-1 LTR

The amounts of Tat required for the transactivation of the HIV-1 LTR were optimized in Jurkat and CEM cells. In Jurkat cells, the plasmid pHIV-1 LTR-Luc (4 μg) was cotransfected with pCMV-R.Luc (1 μg) and various amounts of the Tat expression clone (pCMV-Tat) and electroporated under standard conditions as described above. The cells were plated in 10 ml of complete RPMI 1640 medium and stimulated with PMA and the Ca²⁺ ionophore A23187 at 24 h posttransfection. Following 8 h of stimulation, the cells were harvested and assayed for luciferase activity.

In another set of experiments, CEM cells were transfected with identical amounts of the plasmid cocktail comprising pHIV-1 LTR-Luc and pCMV-R.Luc and various amounts of plasmid pCMN-Tat under standard reaction conditions and assayed for luciferase activity at

16 h posttransfection.

Tat-mediated transactivation of HIV-1 LTR in cell culture in the presence of PΝA,,_AR- transportan conjugate The effect of the PNA^-transportan conjugate on Tat-mediated transactivation of

HIV-1 LTR was assessed in both CEM and Jurkat cells as described below. CEM and Jurkat cells (5 X 10⁶ cells) were electroporated with a plasmid cocktail comprising pHIV-1 LTRLuc (4 μg) and pCMV-R.Luc (1 μg) in the presence or absence of optimal amounts of plasmid pCMV-Tat as determined from the above experiment. The Jurkat and CEM cells were allowed to recover from the effects of electroporation in complete RPMI medium containing 10% fetal bovine serum for 12 h and 3 h, respectively.

To facilitate the uptake of PNA-transportan conjugate, the cells were washed with PBS and resuspended in RPMI medium containing various concentrations of the PNA.,^- transportan conjugate. The Jurkat cells were then stimulated with PMA and Ca²⁺ ionophore at 20 h posttransfection and harvested 8 h after stimulation. The CEM cells were harvested at 20

[54704-8057/LA033020.002] -69- h posttransfection. The cells were analyzed for the luciferase reporter activities in order to evaluate the effect of the PNA_TAR-transportan on Tat-mediated transactivation of the HIN-1 LTR. Identical sets of experiments were carried out to determine the effect of the unconjugated PΝA^-., scrambled PΝA-transportan conjugate, and transportan on Tat- mediated transactivation of the HIV-1 LTR.

In a similar set of experiments, the CEM and Jurkat cells were electroporated with the plasmid pCMV-R.Luc and incubated in the presence or absence of PΝA_TAR-transportan, unconjugated PNA-.^, scrambled PNA-transportan conjugate, or transportan. The cells were harvested at 16 to 24 h posttransfection and analyzed for Renilla luciferase activity as described below.

Luciferase assays

Luciferase assays were performed by using the Promega dual luciferase assay kit.

Briefly, the cells harvested at 1,500 m for 7 min were washed once with PBS and lysed by the addition of 50 μl of the reporter lysis buffer (Promega). Following incubation at 25°C for 15 min on a rocking shaker, the lysates were centrifuged at 15,000 φm for 10 min, and the supernatant was collected in fresh tubes. Luciferase assays were performed by mixing 30 μl of the supernatant with 75 μl of the firefly luciferase substrate in a 96-well Fluorotrac plate, and the light emission was measured using a Packard Top Count luminescence counter. The firefly luciferase activity was quenched by the addition of Stop and Glo reagent, which also served as a substrate for estimating the Renilla luciferase activity. Transfection efficiencies were normalized by the expression levels of the Renilla luciferase reporter gene construct cotransfected along with the experimental plasmid. The results of at least three separate transfections were analyzed for each experiment.

[54704-8057/LA033020.002] -70- HIV-1 production in H9 cells in the presence of PNA-^-transportan conjugate

The chronically HIN-1 -infected H9 cells were centrifuged and washed extensively in

PBS to remove the previously produced virions. The cells were suspended in 1.0 ml of serum-free RPMI 1640 medium at5 X 10⁶ cells/well and incubated in a six-well culture plate at 37°C for 6 h in the absence or presence of various concentrations of PΝA_TAR-transportan conjugate, unconjugated PNA^, scrambled PNA-transportan conjugate, or transportan alone. The cells were then reconstituted to a final cell density of 106 cells/ml in complete RPMI 1640 medium and incubated at 37°C. The cells were harvested on the third day at 1,200 φm for 7 min, and the levels of p24 antigen were analyzed in the supernatants with an enzy e- linked immunosorbent assay (ELISA) p24 antigen kit (Abbott Laboratories). The cells were also analyzed for HIV-1 mRNA levels after total RNA isolation and RNase protection analysis as described below.

Total RNA isolation and RNase protection assay

The plasmids pSP-luc+, pSP-rluc, and pGEM23 were linearized with the restriction enzymes Hindi, BsaAI, and Xbal, respectively. The former two linearized plasmids were used to generate radioactively labeled riboprobes for the firefly and Renilla luciferase corresponding to 390 nucleotides and 245 nucleotides, respectively. Briefly, the digested plasmids were transcribed using T7 RNA polymerase in the presence of [α-³²P]UTP (Perkin Elmer Life Sciences, Inc.) as per the MAXIscript in vitro transcription kit (Ambion Inc., Austin, Tex.). A 195-nucleotide-long riboprobe for the TAR RNA was synthesized from linearized pGEM23 with SP6 RNA polymerase (New England Biolabs) and [α-³ P]UTP under standard reaction conditions. The DNA templates were removed by DNase I digestion, and the RNA probes were purified by gel electrophoresis.

[54704-8057/LA033020.002] -71- For the RNase protection assay, transfection and other experimental conditions as described above were maintained in the CEM, Jurkat, and H9 cells. The cells were harvested at the stated time and washed with PBS, and total RNA was isolated from the immortalized cell lines using the RNAqueous kit (Ambion Inc., Austin, Tex.). Seven micrograms of total RNA extracted from the experimental and control samples was hybridized to 7 X 10⁴ to 10 X

10⁴ cpm of the individual riboprobe and analyzed as per the RPA III kit (Ambion Inc., Austin,

Tex.). Protected fragments were separated on a 6% polyacrylamide-urea gel and detected by

Phosphorlmager analysis.

Determination of f³Hlthvmidine incorporation into cellular DNA in chronically HIV-1- infected H9 cells

Cellular proliferation of chronically HIV-1 -infected H9 cells in the presence of

conjugate was determined by estimating the levels of [³H]thymidine incoφorated in their nuclei. Briefly, freshly split cells were grown in the presence or absence of P A.^ transportan conjugate (5 μM concentration) and supplemented with 10 μCi of [H.et/ϋ -³H]thymidine/ml (83.7 Ci/mmol). The cells were withdrawn at 3, 12, 24, 36, and 48 h, and the cell number was determined using a Coulter counter. The cells were harvested, washed with PBS, and resuspended in 200 μl of lysis buffer containing 1% NP-40 in PBS. The nucleic acids were precipitated by adding cold 10% trichloroacetic acid (TCA). Precipitates were collected on GF/C glass fiber filters (Whatman, Inc., Maidstone, Kent, England) and washed extensively with ice-cold 10% TCA and once in 70% ethanol. Filters were dried and placed in scintillation vials, and radioactivity was counted in the scintillation counter. Protein content in each lysate was estimated by the Bio-Rad protein assay. Results were expressed as counts per minute per milligram of protein.

[54704-8057/LA033020.002] -72- EXPERIMENT SET #4— PNA^ CONJUGATED WITH MEMBRANE PERMEATING PEPTIDE INHIBITS HIV-1 REPLICATION

I. OVERVIEW

A number of potential drug candidates fail because of cellular uptake problems associated with them. This problem is addressed by optimizing their biophysical properties in order to enhance their cellular uptake to acceptable levels. This process often involves chemical modification and or synthesis of a number of analogs and their evaluation for therapeutic activity and biodelivery. Peptide nucleic acids have great therapeutic potential for gene-specific, nontoxic and non-immunogenic therapy. But this capacity has been limited because nucleic acid binding agents have poor cellular uptake. An emerging and highly effective strategy involves conjugation of potential PNAs to a molecular transporter that can efficiently deliver PNA molecules into cells. Peptides derived from Antennapedia, HIV-1 Tat and heφes-simplex-virus DNA binding protein VP22 have been used to transport various agents in the cell. A non-toxic 27 amino acid long chimeric peptide, transportan, derived from the neuropeptide galanin and wasp venom toxin mastoparan has been shown to be efficiently internalized into cells. Unlike membrane transducing peptide derived from HIV-1 Tat and HSV VP22 which are highly basic, transportan MTD is relatively more hydrophobic and less basic containing only four lysine residues. The cellular uptake of this peptide is nonsaturable and resistant to treatment with phenylarsine oxide or hyperosmolar sucrose solution suggesting that the mechanism of uptake is not via endocytosis. Transportan penetrates into every cell type in a rapid and efficient way and localizes mostly in membranous structures and, upon prolonged incubation, it concentrates in the nucleus. In moderate concentrations, it

[54704-8057/LA033020.002] -73- does not affect the growth of cells in culture. These unique properties of the chimeric transportan peptide make it a useful vector for the biodelivery of different proteins or non- protein substances into cells.

None of the inhibitors or drugs available to date have the ability to inactivate HIV-1 virions circulating in the plasma; currently available drugs targeting viral RT and protease work by preventing replication of HIV after it has infected a cell. Entry inhibitors have recently been shown to block HIV entry into the cells but they do not inactivate the virions circulating in the plasma.

The present invention discloses a conjugate of polyamide nucleic acid-transportan peptide targeted to the transactivation response (P -.^ element of HIV-1 LTR that is efficiently taken up by both lymphocytes and HIV-1 virion particles. The conjugate inhibits HIV-1 replication in cell culture when supplemented into the culture medium. HIV-1 virions are rendered replication incompetent upon brief exposure to the conjugate. This unique approach can be exploited to inactivate HIV-1 virions circulating in the plasma prior to their entry and infecting new cell.

II. EXPERIMENTS

To evaluate the efficiency of transportan in delivering PNA across the membrane barrier of cells and HIV-1 virion particles, a specially designed PNA targeted to the transactivation response element (PNA-,_AR) of HIV-1 genome was conjugated with transportan. For cellular uptake studies, the conjugate was tagged with fluorescein probe on the PNA molecule and the uptake efficiency was evaluated using flow cytometiy (Fig. 24). CEM and Jurkat cells (2xl0⁶ cells) were incubated with varying concentrations (50-500 nM) of fluorescein-tagged PNA^-transportan conjugate in RPMI medium with 2% FCS at room

[54704-8057/LA033020.002] -74- temperature. Uptake of the conjugate within 30 sec and 5 min was evaluated by FACS analysis.

As shown in Figure 24, uptake of PNA_TAR-transportan conjugate occurred rapidly in CEM cells and was concentration dependent. Within 30 seconds of incubation, a rapid increase in fluorescence intensity was noted as a function of conjugate concentration (Figure 24A). At concentrations ranging from 50 nM to 500 nM of the conjugate, approximately 16- 84% of the cells were fluorescence positive within 30 seconds of incubation (Table 2). Further incubation for 5 minutes at 500 nM conjugate concentration increased the fluorescence signal in CEM cells to 95%. The uptake in Jurkat cells was relatively slower than CEM cells at lower concentration of the conjugate as indicated by 4 -5% fluorescence intensity at 100 nM concentration of the conjugate (Table 2). Interestingly, at concentrations above 100 nM, the uptake in Jurkat cells was similar to CEM cells (Figure 24B). Uptake studies with Flu-tagged naked PNA.^-, revealed no significant fluorescent intensity in CEM cells even after 6 hours of incubation at 10 μM concentration. The uptake efficiency of the conjugate in human PBMC cells was similar to that observed in CEM cells.

[54704-8057/LA033020.002] -75- Table 2: Uptake of PNA-^-transportan conjugate in CEM, Jurkat, PBMC and phagocytic monocytes (U-937)

Uptake of PNA_TAR- transportan conjugate is not affected at lower temperature as similar uptake efficiency was noted at 4 °C, 25 °C and at 37 °C (Fig. 25, Table 3). Varying concentrations of anti-TAR PNA-transportan conjugate was incubated for 30 sec with 2x10⁶ cells in complete RPMI medium and the extent of uptake was analyzed by FACScan. This indicates that uptake mechanism is distinct from endocytosis, which is completely inhibited at lower temperature. Pretreatment of the Jurkat cells with 60 μM of phenylarsine oxide for 5 min in serum free medium did not inhibit the uptake of the conjugate. The phenylarsine oxide cross-links to the thiol group of membrane surface protein and blocks both endocytosis and receptor function. The uptake of the conjugate may be via formation of inverted micelles upon interaction with the membrane bilayer, which may travel across the membrane and open up on the cytoplasmic side.

[54704-8057/LA033020.002] -76- Table 3: Temperature-independent uptake of PNA-^-transportan conjugate by Jurkat cells

Kinetics In order to determine the kinetics of uptake, radiolabeled PNA-^-transportan conjugated with ¹²⁵I. Jurkat cells (2x10⁶) was incubated with varying concentration of the radiolabeled conjugate at room temperature for 30 sec at 25°C followed by rapid filtration on glass fiber filter (GF-B). The filters were washed and the radioactive incoφoration in the cells was determined on a gamma counter. [S]₀₉ /[S]_αι represents the ratio (cooperativity index) of the substrate at 0.9 Vmax and 0.1 Vmax. The results shown in Figure 26(A) indicate that the uptake kinetics of the PNA-transportan conjugate yield a sigmoidal curve suggesting a cooperative interaction between the conjugate and the cellular membrane.

Figure 26(B) is a Hill plot of uptake data shown in the left panel. Hill coefficient (nH) was determined from the slope. [S]₀. , represents the concentration of the conjugate at which velocity of the uptake is equal to 0.5 Vmax. A Hill coefficient (nH) of 0.53 was obtained from the slope of the Hill plot suggesting that the observed cooperativity is not due to multiple conjugate binding sites on the membrane. The observed cooperativity of uptake may be due to micelle formation, which requires a concentration threshold of components. The [S]₀₅ value and cooperativity index (ratio of [S] ₀₉ /[S] _0]) determined from the sigmoidal plot were 1.5 μM and 6, respectively. The [S]₀₅ value indicates the concentration of the

[54704-8057/LA033020.002] -77- conjugate at which uptake velocity is equal to 0.5 V_max while cooperativity index is the ratio of substrate (conjugate) concentration at 0.9 V_max and 0.1 V_max. A cooperativity index of 6 suggests that in order to increase the uptake velocity from 0.1 V_max to 0.9 V_max, only a 6-fold increase in the concentration of conjugate is required. In contrast, uptake kinetics following a hyperbolic curve, theoretically will requires 90 fold increase in the substrate concentration to achieve a velocity * of 0.9 V max from 0.1 V max.

Since PNA_TAR-transportan conjugate targeted to the TAR region of HIV-1 RNA genome is efficiently taken up by the lymphocytes, the next step was to examine its antiviral efficacy in activated PBMC cells infected with HIV-1. PBMCs obtained from HIV-1- seronegative donors were stimulated with phytohemagglutinin (PHA) and interleukin 2 (IL-2) two days before infection. The cells (2x10^s) preincubated with varying concentrations of PNA_TAS-transportan conjugate were infected with pseudotyped HIV-1 virions carrying the firefly luciferase reporter gene at MOI of 10. Scrambled PNA-transportan was also included as a control. The infected cells were pelleted, washed with PBS and resuspended in fresh medium supplemented with varying concentration of PNA_TAR-transportan. After 48 hours of incubation at 37 C, the cells were harvested, lysed and normalized for protein concentration. An aliquot of each lysate containing equal amounts of protein was assayed for firefly luciferase activity (Luciferase Reporter Assay System, Promega Coφoration, Madison, WI). Results shown in Figure 27 indicate that inhibition of luciferase expression was observed at all concentrations of PNA_TAR-transportan conjugate. Approximately, 75-99% inhibition was noted at concentrations ranging from 2-5 μM of the conjugate. CEM cells infected with pseudotyped HJN-1 SI strain in the presence of indicated concentration of PΝA_TAR- transportan or scrambled PNA-transportan conjugate. Cells were harvested after 48 hours of infection, lysed and assayed for luciferase activity. SC represents 5μM of scrambled PNA.

[54704-8057/LA033020.002] -78- lμM of anti-TAR PNA transportan conjugate corresponds to 7.2μg conjugate/mL. Scrambled PNA-transportan conjugate at 5μM did not exhibit any antiviral activity.

Permeation Into HIV-1 Virons

Since cells efficiently took up PNA-^-transportan, the PNA_TAr.-transportan was expected to permeate across the lipid bilayer membrane of HIV-1 virions, as these are also derived from the host cell membrane. To examine this, we first purified pseudotyped HIV-1 virions from the culture supernatant of 293T cells transfected with pHIN-l_m,_CSF.Lucenv (-) along with pVSV-G. For this, the culture supernatant was first filtered through 0.45μm pore size membrane and ultracentrifuged to pellet the virion particles. The virion pellet was resuspended in PBS and recentrifuged through 20% sucrose cushion. The purified HIV-1 virion preparations have been shown to be contaminated with cellular microvesicles of 50 - 500 nm size. To rule out any interference in the uptake of the conjugate by microvesicles, the virions were subjected to subtilisin treatment to remove the microvesicles. A mock virus sample from culture supernatant of uninfected cell was also processed similarly. The purified HIV-1 virions were incubated with 100 nM of I¹²⁵-labeled anti-TAR

PΝA-transportaα conjugate (20x10³ CPM/pmol) in complete RPMI medium for 20 minutes. The RPMI medium and mock virus sample served as negative controls. The incubation mixture was ultracentrifuged through discontinuous sucrose density gradient for 1 hour. Fractions were collected from the bottom and analyzed for radioactivity and p24 antigen levels. In Figure 28, panel A is culture medium control without HIV-1 virion. Panel B is culture medium control with HIV-1 virion. Panel C is a mock virion sample processed from uninfected cell. Radiolabeled PΝA TAR-transportan incubated in RPMI culture medium with or without HIV-1 virions was ultracentrifuged in discontinuous sucrose density gradient, fractionated from the bottom and fractions were analyzed for radioactivity and p24 antigen.

[54704-8057/LA033020.002] -79- The control samples with culture medium without HIV-1 virions or with mock virus sample, the entire radiolabeled conjugate remained at the top of the gradient (Figures 28 A and C). By contrast, in the HIV-1 virion samples, a portion of the radioactivity sedimented at the bottom corresponding to the sedimentation position of HIV-1 virions indicating that PNA-.^- transportan peptide conjugate is able to penetrate the membrane barrier of the virions. Fetal calf serum did not interfere in the permeation of the labeled conjugate into virions thus suggesting the absence of any nonspecific interaction of the conjugate with serum proteins.

Binding to the TAR Region

Because PNA_TAR-transportan could efficiently permeate across the membrane of the virion particles, the next experiment tested to see whether the conjugate penetrates through the nucleocapsid and binds to the TAR region of the packaged viral genome. In this scenario, the synthesis of (-) strand strong stop DNA should be severely impaired since it was earlier demonstrated that PNA bound to the viral RNA could not be displaced by HIV-1 RT. To examine this possibility, HIV-1 virions pretreated in the absence or presence of 1 μM and 2.5 μM of PNA_TAR-transportan conjugate or scrambled PNA-transportan were ultracentrifuged through 20%) sucrose cushion to remove external PNA^-transportan conjugate. An aliquot of the virions was then disrupted and examined for endogenous RT activity by supplementing with MgCl₂ and oι³²P-labeled dNTPs. The reaction mixture was subjected to phenol- chloroform extraction followed by alcohol precipitation and the reaction products were resolved on a denaturing polyacrylamide urea gel. In Figure 29, lanel is untreated HIV-1 virions; lanes 2 and 3 are pre-treated with 1 μM and 2.5 μM of PNA^. transportan conjugate, respectively; lanes 4 and 5 are pre-treated with scrambled PNA-transportan conjugate at 1 μM and 2.5 μM concentrations, respectively. The results indicate that endogenous reverse transcription in HIN-1 virions pretreated with PΝA_TAR-transportan conjugate is predominantly

[54704-8057/LA033020.002] -80- terminated in the TAR region (lanes 2 and 3). In the control, without the conjugate or with scrambled PNA-transportan conjugate, products larger than 400 nucleotides are seen (lanes 1, 4 and 5). The predicted size of the tRNA₃ ^Lys derived (-) ssDNA is 253 nucleotide. The two major bands migrating above the 400-nucleotides position represent products of strand transfer reaction.

Stopping HIV-1 Replication

The blockage of endogenous (-) ssDNA synthesis in disrupted virions clearly indicates that HIV-1 virions can be rendered replication incompetent by PNA-transportan peptide conjugates. CEM cells were infected with the pretreated virions at MOI 100 and the infection was monitored by determining the level of firefly luciferase expression after 48 hours of growth. Specifically, HIV-1 virions (HIV-l_m.CSF__Lu.env) were pre-incubated with 500 nM of PNA-^-transportan conjugate for 20 min in RPMI medium in the presence or absence of 10%) FCS were ultra-centrifuged through 20% sucrose. The virion pellet was resuspended in complete RPMI medium and used to infect the CEM cells. In Figure 30, Lane 1 is the virion control + FCS; Lane 2 is the virion control - FCS; Lane 3 is the virion + PNA_TAR transportan + FCS; Lane 4 is the virion + PNA,.^ transportan - FCS; Lane 5 is the virion + scrambled PNA-transportan + FCS; and Lane 6 is the virion + scrambled PNA transportan - FCS. The results shown indicate that virion particles pre-treated with PNA^-transportan conjugate were severely impaired in their ability to infect CEM cells. Since expression of firefly luciferase is a direct consequence of reverse transcription of viral RNA genome and its subsequent integration into the host genome, the results provide evidence of PNA-peptide conjugate permeating across the nucleoprotein complex to block the reverse transcription process. HIV-1 TAR element is a 59 nucleotide long RNA stem loop- bulge located at the 5' end of the HIV-1 RNA genome, which is essential for transactivation

[54704-8057/LA033020 002] -81 - of HIV-1 transcription in infected cells. The loss of infectivity of the virions upon exposure to the PNA_rAR-transportan conjugate shows that they are invalidated in the reverse transcription process, a crucial step for establishing the infection. PNA_TAR-transportan penetrating the membrane barrier of the virions sequesters the TAR region resulting in the abortion of the first crucial step [synthesis of (-) ssDNA] in the reverse transcription process. This discovery is supported by the observation that endogenous reverse transcription in virions pretreated with PNA-^-transportan conjugate is completely aborted in the TAR region (Figure 29). The present invention is unique in the sense that HIV-1 in the plasma can be rapidly inactivated prior to their entry into the cells upon exposure to a combination of potential PNA-transportan conjugates targeted against critical regions of HIV-1 genome. These conjugates have great potential as prophylactic agents to block HIV-1 infection caused due to accidental exposure.

III. METHODS AND MATERIALS

Purification of HIV-1 virions The pseudotyped HIV-1 virions were isolated from the culture supernatant of 293T cells transfected with pHTV-l_JR,CSP.Lm.env (-) and pVSV-G clones. The culture supernatant (500 ml) was filtered through 0.45μm pore size membrane and centrifuged at 70,000 g for 45 min. The pellet obtained was resuspended in 2 ml of PBS and recentrifuged through 5 ml of 20% sucrose/PBS cushion for 30 min at 100,000 g. The purified HIV-1 virions preparations have been shown to be contaminated with cellular microvesicles of 50 - 500 nm size, which may interfere with the uptake studies. To remove these microvesicles, viral pellets were resuspended in 500μL of Dulbecco's phosphate buffered saline containing 1 mM CaCl₂ and treated with subtilisin (1 mg/ml) at 37°C for 18 h. The subtilisin digestion was stopped by

[54704-8057/LA033020.002] -82- PMSF (5 μg/ml). The subtilisin-treated samples were repurified through 1 ml of 20% sucrose and resuspended in Dulbecco's phosphate buffered saline without CaCL, or MgCl₂. A mock- virion preparation from the culture supernatant of uninfected cells was also carried out simultaneously. Determination of HIV-1 virion number in the purified samples

HIV-1 virions were quantitated by determining the RNA copy number in the sample using Nuclesins HIV-1 -QT Amplification Kit (Organon Teknika, Durham, NC). The virion number was also determined from the p24 concentration as excellent correlation between HIV-1 RNA copy number and p24 concentration has been demonstrated (Nadal et al., 1999). Considering that 2000 copy of p24 are present per virion particle, the virion number estimated from the RNA copy number were in agreement with the number determined by p24 quantification (1 pg p24 per 12500 virions).

FACscan analysis of cellular uptake

CEM and Jurkat cells grown in complete RPMI 1640 medium containing 10% FCS were harvested, washed with PBS and resuspended in the same medium containing 2% FCS at a cell density of 4x10⁶ cells/ml. Cells were aliquoted in 12 well microtiter plate at 2x10⁶ cells per well and incubated at room temperature with varying concentrations of fluorescein tagged PNA_TAR-transportan conjugate (50-500 nM). At varying time points the fluorescent signal/10,000 cells were obtained using fluorescent flow cytometry (FACscan). Uptake kinetics

The PNA-.^- transportan conjugate was labeled with ¹²⁵I using the radioisotope and the chloramine-T labeling kit from ICN. For radiolabeling, 0.5 nmole of the conjugate and 0.28 nmole of ¹²⁵-I (0.5 mCi) were reacted in the presence of 2.8 nmoles of chloramine- T for 1 min according to the manufacturer's protocol and quenched by the addition of 62 nmoles of sodium metabisulfite. The labeled conjugate was purified by NAP- 10 gel filtration, followed

[54704-8057/LA033020.002] -83- by C18 disposable cartridges. The final purified product was lyophilized, dissolved in water and quantified by absorption at 260 nm. The specific radioactivity was adjusted to desired concentration by adding unlabeled conjugate. For uptake experiments, 2 x 10^δ Jurkat cells were incubated with varying concentrations of the radiolabeled conjugate at room temperature. After 30 seconds of incubation, each individual sample was filtered on glass fiber filter (GF-B), washed extensively with phosphate buffered saline solution and the amount of radiolabel internalized into the cells was determined by gamma counting.

Permeation of PNA_TAR-transportan conjugate into virion particles

The purified HIV-1 virion particles were incubated with 50 nM of I¹²⁵-labeled PNA-.^- transportan conjugate (1.8xl0⁵ CPM/pmol) in complete RPMI medium (250 μl) for 20 minutes. The RPMI medium and mock-virus sample served as negative controls. The incubation mixture was then subjected to ultracentrifugation through discontinuous sucrose density gradient (1 ml of 40% sucrose/PBS + 2mL of 20% sucrose/PBS + 2 ml of 10% sucrose/PBS) for 1 hour. Fractions were collected from the bottom and analyzed for p24 and presence of radioactivity.

Endogenous reverse transcription in HIV-1 virions pretreated with PNA_TAR-transportan

HIV-1 virions (5xl0^u/mL) were incubated with 1 - 2.5 μM concentrations of

PNA_TAR-transportan or scrambled PNA-transportan in complete RPMI medium (200 μl) for 30 min at 4 C. The incubation mixture was then layered on 3 ml of 20% sucrose and centrifuged at 100,000 g for 30 min. The virions pellet free from external PNA^-transportan conjugate was resuspended in Tris buffer containing 0.1% NP-40 and 10 mM DTT to disrupt the viral membrane. An aliquot of the disrupted virions was examined for endogenous RT activity by supplementing with 2 mM MgCL, and 5 μM of each dNTPs containing 5μCi each of ³²P-dCTP and α³²P dATP. Following a one-hour reaction at 37°C, the mixture was

[54704-8057/LA033020.002] -84- extracted with phenol-chloroform, precipitated with alcohol and lyophilized. The lyophilized material was dissolved in TE buffer and resolved on denaturing polyacrylamide-urea gel.

Infectivitv of HIV-1 virions pre-treated with PNA^-transportan conjugate

The pseudotyped HIN-1 virions containing firefly luciferase reporter (Planelles et al., 1995) were pretreated with PΝA^. transportan conjugate as described above. Control experiments without the conjugate or with scrambled PΝA-transportan conjugate were also performed simultaneously. Infection of CEM cells was carried out in 0.5 ml RPMI medium

containing 10 μg/ml polybrene at MOI of 100 at 37°C for 60 min. Infected CEM cells (10⁶

cells) were washed 2x with PBS and resuspended in 0.5 ml of complete RPMI medium and incubated in a 37 °C, CO₂ incubator. After 48 hours, cells were harvested, washed and lysed

in 100 μl of lysis buffer. After normalizing the protein concentration, an aliquot of each extract was analyzed for firefly luciferase expression as described before (Kaushik et al., 2002).

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Claims

CLAIMSWe claim:

1. A conjugate comprising a novel polyamide nucleotide acid (PNA) connected to a transportan membrane transducing (MTD) peptide, wherein the conjugate contacts a target and inhibits replication of a virus.

2. The conjugate of claim 1, wherein the PNA comprises primer binding site polyamide nucleotide acid (PNA_PBS), a variant of PNA_PBS, transactivation response element polyamide nucleotide acid (P ^, and or a variant of P -.^ wherein the variants retain the binding properties of the original PNA.

3. The conjugate of claim 2, wherein PNA_PBS has a sequence of SEQ ID NO: 1 and PNA_TARhas a sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

4. The conjugate of claim 1 , wherein the MTD is transportan.

5. The conjugate of claim 1 , wherein the target is a virus or lymphocyte and wherein the conjugate contacts the virus or lymphocyte before the virus or lymphocyte enters a cell.

6. The conjugate of claim 5, wherein the virus is a retrovirus or virion.

7. The conjugate of claim 5, wherein the conjugate inhibits replication of the retrovirus by inhibiting reverse transcription of the virus.

8. The conjugate of claim 6, wherein the retrovirus is HIV.

9. The conjugate of claim 8, wherein the conjugate binds to the transactivation response element (TAR) region or the primer binding sequence of the HIV virus.

[54704-8057/-A033020.002] - 110-

10. The conjugate of claim 1 , wherem the target is:

(a) a cell infected by a virus and viral inhibition occurs by affecting the virus within the cell; or

(b) a cell at risk of infection by a virus and viral inhibition occurs by affecting the virus before the virus infects the cell.

11. A method of treating a subject infected with a virus, comprising administering to the subject a therapeutically effective amount of a conjugate comprising a novel polyamide nucleotide acid (PNA) connected to a transportan membrane transducing (MTD) peptide, which inhibits viral infection, viral replication, or expression of a nucleic acid or protein of the virus in the subject.

12. The method of claim 11, wherein the PNA comprises primer binding site polyamide nucleotide acid (PNA_PBS), a variant of PNA_PBS, transactivation response element polyamide nucleotide acid (PNA-.^, and/or a variant of PNA-.^ wherein the variants retain the binding properties of the original PNA.

13. The method of claim 11, wherein PNA_PBS has a sequence of SEQ ID NO: 1 and P A^ has a sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

14. The method of claim 11 , wherein the MTD is transportan.

15. The method of claim 11, wherein the conjugate inhibits replication of the virus by inhibiting reverse transcription of the virus.

[54704-8057/LA033020.002] - 111 -

16. The method of claim 15 , wherein inhibiting reverse transcription of the virus results in reduction of the virus in the subject.

17. The method of claim 11 , wherein the virus is retrovirus or virion

18. The method of claim 17, wherein the retrovirus is HIN.

19. The method of claim 18, wherein inhibition effectively blocks Tat-mediated transactivation of HIV-1 LTR.