RNA aptamers derived by SELEX (systematic evolution of ligands by exponential enrichment) and specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bind at the template-primer cleft with high affinity and inhibit its activity. In order to determine the potential of such template analog RT inhibitors (TRTIs) to inhibit HIV-1 replication, 10 aptamers were expressed with flanking, self-cleaving ribozymes to generate aptamer RNA transcripts with minimal flanking sequences. From these, six aptamers (70.8,13, 70.15, 80.55,65, 70.28, 70.28t34, and 1.1) were selected based on binding constants (K(d)) and the degree of inhibition of RT in vitro (50% inhibitory concentration [IC(50)]). These six aptamers were each stably expressed in 293T cells followed by transfection of a molecular clone of HIV(R3B). Analysis of the virion particles revealed that the aptamers were encapsidated into the virions released and that the packaging of the viral genomic RNA or the cognate primer, tRNA(Lys)(3), was apparently unaffected. Infectivity of virions produced from 293T cell lines expressing the aptamers, as measured by infecting LuSIV reporter cells, was reduced by 90 to 99.5% compared to virions released from cells not expressing any aptamers. PCR analysis of newly made viral DNA upon infection with virions containing any of the three aptamers with the strongest binding affinities (70.8,13, 70.15, and 80.55,65) showed that all three were able to form the minus-strand strong-stop DNA. However, virions with the aptamers 70.8 and 70.15 were defective for first-strand transfer, suggesting an early block in viral reverse transcription. Jurkat T cells expressing each of the three aptamers, when infected with HIV(R3B), completely blocked the spread of HIV in culture. We found that the replication of nucleoside analog RT inhibitor-, nonnucleoside analog RT inhibitor-, and protease inhibitor-resistant viruses was strongly suppressed by the three aptamers. In addition, some of the HIV subtypes were severely inhibited (subtypes A, B, D, E, and F), while others were either moderately inhibited (subtypes C and O) or were naturally resistant to inhibition (chimeric A/D subtype). As virion-encapsidated TRTIs can predispose virions for inhibition immediately upon entry, they should prove to be efficacious agents in gene therapy approaches for AIDS.

Potent inhibition of human immunodeficiency virus type 1 replication by template analog reverse transcriptase inhibitors derived by SELEX (systematic evolution of ligands by exponential enrichment).

Methods for producing interfering RNA molecules in mammalian cells are provided. Therapeutic uses for the expressed molecules, including inhibiting expression of HIV, are also provided.

Methods for producing interfering RNA molecules in mammalian cells and therapeutic uses for such molecules

Combination genetic therapy to inhibit HIV-1.

The central role of endoconvertases and HIV-1 protease (HIV-1 PR) in the processing of HIV proproteins makes the design of specific inhibitors important in anti-HIV gene therapy. Accordingly, we tested native α1 antitrypsin (α1AT) delivered by a recombinant simian virus-40-based vector, SV(AT), as an inhibitor of HIV-1 proprotein maturation. Cell lines and primary human lymphocytes were transduced with SV(AT) without selection and detectable toxicity. Expression of α1AT was confirmed by Northern blotting, immunoprecipitation and immunostaining. SV(AT)-transduced cells showed no evidence of HIV-1-related cytopathic effects when challenged with high doses of HIV-1NL4-3. As measured by HIV-1 p24 assay, SV(AT)-transduced cells were protected from HIV-1NL4-3 at challenge dose of 40 000 TCID50 (MOI = 0.04). In addition, peripheral blood lymphocytes treated with SV(AT) were protected from HIV doses challenge up to 40 000 TCID50 (MOI = 0.04). By Western blot analyses, the delivered α1AT inhibited cellular processing of gp160 to gp120 and decreased HIV-1 virion gp120. SV(AT) inhibited processing of p55Gag as well. Furthermore, high levels of uncleaved p55Gag protein were detected in HIV virus particles recovered from SV(AT)-transduced cells lines and primary lymphocytes. Thus, delivering α1AT using SV(AT) to human lymphocytes strongly inhibits replication of HIV-1, most likely by inhibiting the activities both of the cellular serine proteases involved in processing gp160 and of the aspartyl protease, HIV-1 PR, which cleaves p55Gag. α1AT delivered by SV(AT) may represent a novel and effective strategy for gene therapy to interfere with HIV replication, by blocking a stage in the virus replicative cycle that has until now been inaccessible to gene therapeutic intervention.

HIV-1 proprotein processing as a target for gene therapy

anti-AIDS therapies. Tat biology has been the subject of several reviews (4-7). The aim of the present paper is to review recent studies examining how interference with HIV-Tat biology may be a key therapeutic strategy to cure AIDS. The HIV-1 Tat gene encodes for a 86-101 amino acid polypeptide that acts as the main transactivating factor of HIV (6). Tat protein can be actively released by HIVinfected cells (7) and is detected in the serum of HIV-infected individuals (8). Extracellular Tat is able to enter latently HIV-infected cells and activate the transcription of the viral genome. In AIDS patients, the reactivation of HIV by extracellular Tat may explain the burst of replication associated with early phases of HIV infection, where synchronized virion replication takes place (9). Moreover, extracellular Tat promotes HIV-1 coreceptors expression (10), thus inducing a self-perpetuating permissiveness for HIV-1 infection. Extracellular Tat also acts on different types of uninfected cells by interacting with several receptors including integrins α5β1, αvβ3 and αvβ5; the vascular endothelial growth factor (VEGF) receptors VEGFR1/Flt-1 and VEGFR2/KDR; and the chemokine receptors CCR2 and CCR3 and CD26 (also known as dipeptidyl peptidase IV) (7). More recently, Tat has been shown to also bind the chemokine receptor CXCR-4 (11), the low density lipoprotein receptor-related protein (LPR) (12) and heparan sulfate proteoglycans (HSPGs) (13). Given the variety of receptors bound by extracellular Tat, it can be expected that a complex network of signal transduction pathways must be activated by the transactivating factor in target cells (7). Also, LPR and HSPG receptors mediate the internalization of Tat inside the cell. As already mentioned, internalized Tat retains the capacity to transactivate viral and/or cellular genes. Extracellular Tat exerts a variety of biological activities some of which have been tentatively associated with distinct AIDS-associated pathologies (Fig. 1). For example, Tat is considered a neurotoxin implicated in the pathogenesis of AIDS dementia (14) and also acts on different cells of immunity contributing to the immune suppression in AIDS patients (4, 7). Tat appears to be responsible, at CONTENTS

HIV-1 Tat protein: a target for the development of anti-AIDS therapies

Vectors based on recombinant SV40 viruses (rSV40) are highly effective in delivering transgene expression driven by constitutive promoters. We tested here whether these vectors could be used with conditional promoters and promoters using RNA polymerase III transcription, with inhibition of HIV-1 by Tat activation response (TAR) decoys as a functional measure of effective transgene delivery and activity. TAR decoys inhibit HIV-1 Tat, a trans-activator of HIV-1 transcription. Tat acts early in the viral replicative cycle and is essential for efficient viral replication. We evaluated rSV40 gene delivery using two different inhibitors of Tat. One was a dual function polyTAR gene encoding 25 sequential TAR elements (TAR(25)), plus an antisense tat, driven either by HIV-1 long terminal repeat (HIV-LTR) as a conditional promoter, or by cytomegalovirus immediate-early promoter (CMV-IEP) as a constitutive promoter. The other inhibitor was a single TAR decoy, driven by the U6 small nuclear RNA promoter (U6-P). These decoys were delivered to unselected cells in two different human T lymphocyte lines and to unstimulated primary human peripheral blood mononuclear cells (pbmc). Gene delivery was confirmed by PCR, and expression by RT-PCR. By in situ hybridization analysis, >95% of cells were transduced. These transgene constructs protected all cell types tested from HIV-1, as measured by syncytia formation and p24 antigen release. Somewhat better inhibition of HIV-1 replication was achieved with HIV-1 long terminal repeat (HIV-1 LTR) as a conditional promoter than with the constitutive CMV-IEP. The U6-P was also very effective, driving a TAR(1) transcript. Cell viability was not detectably affected by TAR decoy expression. Thus, rSV40 vectors effectively deliver HIV-1-inhibitory RNAs using either constitutive or conditional pol II promoters, or using a pol III promoter. The versatility of this gene delivery system may prove to be useful in anti-HIV-1 therapeutics.

SV40-derived vectors provide effective transgene expression and inhibition of HIV-1 using constitutive, conditional, and pol III promoters

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SV40-derived vectors provide effective transgene expression and inhibition of HIV-1 using constitutive, conditional, and pol III promoters