TAR Decoy Research Articles

296. Pre-Selection of Anti-HIV Lentiviral Vector Gene Modified Hematopoietic Stem Cells Significantly Improves Protection from HIV Infection: The Basis for a Future Clinical Trial

The successful treatment and cure of HIV in the Berlin Patient has highlighted the utility of HIV-resistant hematopoietic stem cells (HSC) to offer a potential functional cure for HIV infected individuals. Stem cell gene therapy for HIV can mimic this result by genetically engineering a patient's own HSC with lentiviral vectors encoding HIV resistance genes. Clinical trials of HIV stem cell gene therapy have been unsuccessful at demonstrating complete efficacy due to the transplantation of a low percentage of anti-HIV gene expressing cells.Therefore, to bridge the gap between these previous approaches and the Berlin patient, we have developed a pre-selection process which allows for the purification of vector transduced cells prior to transplantation into patients. This pre-selective lentiviral vector (1TAX) encodes a triple combination of anti-HIV genes (a human/rhesus macaque TRIM5alpha, a CCR5 shRNA, and a TAR decoy) in addition to a truncated/mutated form of human CD25 which is expressed on the cell surface of transduced HSC and is used to enrich for the HIV-resistant cell population.Purity levels of 1TAX transduced human HSC obtained after selection for CD25+ were >94% as determined by flow cytometry. Safety of the 1TAX vector transduced/purified human HSC was demonstrated in vivo in NOD-SCID-Rag-/-IL2rgamma-/-(NRG) mice with the detection of engraftment and multi-lineage hematopoiesis into human T cells, B cells, and macrophages in the peripheral blood and lymphoid organs. Upon infection of 1TAX mice with a CCR5-tropic BaL-1 or a CXCR4-tropic NL43 strain of HIV-1, maintenance of normal human CD4+ cell levels were observed in the peripheral blood. A significant decrease (>4 logs) in HIV-1 plasma viremia was also demonstrated in 1TAX mice as compared to mice transplanted with nontransduced human HSC. Together, this work has demonstrated the safety and efficacy of this 1TAX pre-selective lentiviral vector in HSC and is the basis for a future human clinical trial of HIV stem cell gene therapy.

Inhibition of HIV‐1 transcription by a tunable chimeric tRNA(Ser)‐nucloelar localizing trans‐activation response element (TAR) decoy

In order for HIV‐1 to achieve high‐level transcription of its integrated proviral DNA, the trans‐activator of transcription (Tat) protein must bind to the TAR region of the nascent viral RNA transcript. A TAR decoy is a short RNA oligomer that inhibits HIV‐1 transcription by binding to the Tat protein, thereby competitively inhibiting the TAR‐tat interaction. Previous work has shown that nucleolar‐localization of a TAR decoy by fusion with the U16 small nucleolar RNA (snoRNA) backbone dramatically increases the TAR decoy inhibition efficiency. In this work, the nucleolar‐localized TAR decoy is expressed as the 3′ trailing sequence of a tRNA(Ser) chimeric transcript. Maturation of the tRNA chimera by tRNA processing enzymes releases the U16‐TAR element from the tRNA. Using a dual‐luciferase reporter assay with firefly luciferase under the control of the HIV‐1 long terminal repeat (LTR) promoter, we show that expression of the tRNA‐TAR decoy chimera inhibits HIV‐1 transcription in human HCT116 cells. We also show that mutations in the tRNA(Ser) acceptor stem allow for inhibition efficiency to be tunable.

Generation of an HIV-1-Resistant Immune System with CD34 + Hematopoietic Stem Cells Transduced with a Triple-Combination Anti-HIV Lentiviral Vector

HIV gene therapy has the potential to offer an alternative to the use of current small-molecule antiretroviral drugs as a treatment strategy for HIV-infected individuals. Therapies designed to administer HIV-resistant stem cells to an infected patient may also provide a functional cure, as observed in a bone marrow transplant performed with hematopoietic stem cells (HSCs) homozygous for the CCR5-Δ32-bp allele. In our current studies, preclinical evaluation of a combination anti-HIV lentiviral vector was performed, in vivo, in humanized NOD-RAG1(-/-) IL2rγ(-/-) knockout mice. This combination vector, which displays strong preintegration inhibition of HIV-1 infection in vitro, contains a human/rhesus macaque TRIM5α isoform, a CCR5 short hairpin RNA (shRNA), and a TAR decoy. Multilineage hematopoiesis from anti-HIV lentiviral vector-transduced human CD34(+) HSCs was observed in the peripheral blood and in various lymphoid organs, including the thymus, spleen, and bone marrow, of engrafted mice. Anti-HIV vector-transduced CD34(+) cells displayed normal development of immune cells, including T cells, B cells, and macrophages. The anti-HIV vector-transduced cells also displayed knockdown of cell surface CCR5 due to the expression of the CCR5 shRNA. After in vivo challenge with either an R5-tropic BaL-1 or X4-tropic NL4-3 strain of HIV-1, maintenance of human CD4(+) cell levels and a selective survival advantage of anti-HIV gene-modified cells were observed in engrafted mice. The data provided from our study confirm the safety and efficacy of this combination anti-HIV lentiviral vector in a hematopoietic stem cell gene therapy setting for HIV and validates its potential application in future clinical trials.

A dual function TAR Decoy serves as an anti-HIV siRNA delivery vehicle

The TAR RNA of HIV was engineered as an siRNA delivery vehicle to develop a combinatorial therapeutic approach. The TAR backbone was found to be a versatile backbone for expressing siRNAs. Upon expression in human cells, pronounced and specific inhibition of reporter gene expression was observed with TARmiR. The resulting TARmiR construct retained its ability to bind Tat and mediate RNAi. TARmiR was able to inhibit HIV gene expression as a TAR decoy and by RNA interference when challenged with infectious proviral DNA. The implications of this dual function therapeutic would be discussed.

Preintegration HIV-1 Inhibition by a Combination Lentiviral Vector Containing a Chimeric TRIM5α Protein, a CCR5 shRNA, and a TAR Decoy

Human immunodeficiency virus (HIV) gene therapy offers a promising alternative approach to current antiretroviral treatments to inhibit HIV-1 infection. Various stages of the HIV life cycle including pre-entry, preintegration, and postintegration can be targeted by gene therapy to block viral infection and replication. By combining multiple highly potent anti-HIV transgenes in a single gene therapy vector, HIV-1 resistance can be achieved in transduced cells while prohibiting the generation of escape mutants. Here, we describe a combination lentiviral vector that encodes three highly effective anti-HIV genes functioning at separate stages of the viral life cycle including a CCR5 short hairpin RNA (shRNA) (pre-entry), a human/rhesus macaque chimeric TRIM5 alpha (postentry/preintegration), and a transactivation response element (TAR) decoy (postintegration). The major focus on designing this anti-HIV vector was to block productive infection of HIV-1 and to inhibit any formation of provirus that would maintain the viral reservoir. Upon viral challenge, potent preintegration inhibition of HIV-1 infection was achieved in combination vector-transduced cells in both cultured and primary CD34(+) hematopoietic progenitor cell (HPC)-derived macrophages. The generation of escape mutants was also blocked as evaluated by long-term culture of challenged cells. The ability of this combination anti-HIV lentiviral vector to prevent HIV-1 infection, in vitro, warrants further evaluation of its in vivo efficacy.

Inhibition of HIV-1 replication by long-term treatment with a chimeric RNA containing shRNA and TAR decoy RNA

Combinatorial therapies for the treatment of HIV-1 infection are effective for reducing patient viral loads and slowing the progression to AIDS. Our strategy was based on an anti-HIV-1 shRNA vector system in which HIV-1 vif-shRNA was fused to a decoy TAR RNA (mini-TAR RNA) to generate vif-shRNA-decoy TAR RNA under the control of the human U6 Pol III promoter. Upon expression in human cells, the RNA molecule was cleaved into its component parts, which inhibited HIV-1 replication in a synergistic manner. This chimeric RNA expressed a dual RNA moiety and greatly enhanced the inhibition of HIV-1 replication under the production of resistant virus by short interference RNA (siRNA) in long-term culture assays. We suggest that this technique provides a practical basis for the application of siRNA-based gene therapy in the treatment of HIV/AIDS.

78. Potent Pre-Integration Inhibition of HIV-1 Infection by a Triple Combination Anti-HIV Lentiviral Vector Containing a CCR5 shRNA, a Chimeric TRIM5α Restriction Factor, and a TAR Decoy for HIV Gene Therapy

78. Potent Pre-Integration Inhibition of HIV-1 Infection by a Triple Combination Anti-HIV Lentiviral Vector Containing a CCR5 shRNA, a Chimeric TRIM5α Restriction Factor, and a TAR Decoy for HIV Gene Therapy

First in Human Engraftment of Anti-HIV Lentiviral Vector Gene Modified CD34+ Peripheral Blood Progenitor Cells in the Treatment of AIDS Related Lymphoma (ARL).

Background: Autologous stem cell transplantation (ASCT) has become an accepted treatment option for high risk or relapsed ARL. Treatment related mortality is similar to the HIV negative setting. However, ultimately further improvement in ASCT will depend on both effective anti lymphoma therapy and better control of the HIV infection. Highly active antiretroviral therapy (HAART) can lower HIV viral loads to undetectable levels in the peripheral blood, but reservoirs of HIV are still present in the tissues and acquired resistance to HAART also remains a problem. A treatment strategy that would confer intrinsic resistance to HIV could circumvent theses issues. Herein we report on one such strategy using multiplexed RNA based anti-HIV gene transfer strategies to render autologous peripheral blood progenitor cells resistant to HIV. Patients with high risk ARL deemed candidates for ASCT were eligible. Seven subjects with NHL have been enrolled. (4PR, 2 REL, 1CR2), of whom 2 failed screening phase, 1 failed product release test, 2 are pending transplant, and 2 patients have undergone successful transplantation.Median age was 43 yrs at enrollment. Four pts to date were mobilized with chemotherapy plus GCSF and cells were collected for the clinical product (Fx1) and for CD34-selection (CliniMACSª, Miltenyi) and research treatment (Fx2). (see table )UPN #Fx1 (CD34+/kg)Fx2 (CD34+/kg)Post Selection and transductionCD34+/kg3012.8X1063.5X106.26 X106 not infused3043.9X1063.6X1061.2 X1063053.4X1063.8X1061.4X1063065.6X1068.8X106pendingThree days prior to the completion of CBV (cyclophosphamide 100mg/kg, BCNU 450mg/ m2, VP16 60mg/kg) conditioning, the Fx2 cells were thawed and transduced with a lentivirus vector (LV,rHIV7-ShI-TAR-CCR5Z) encoding 3 RNA elements including short hairpin RNA (shRNA) targeted to HIV tat/rev, a nucleolar localizing TAR decoy sequence, and a ribozyme targeted to CCR5. Cell viability post transduction ranged between 52–64% in three pts. On day 0 Fx2 is given and Fx1 is given 24hrs later (day+1). UPN301 did not receive the transduced Fx2 cells due to a low cell dose. For UPN304 and 305 who received the gene modified Fx2 cells, WBC engraftment occurred at day +11, platelet engraftment at day+16, and there have been no serious adverse events.Results to date at 30 and 60 days post ASCT reveal peripheral blood marking consistent with the ratio of gene modified to unmodified cells infused. Q-PCR analysis demonstrated distribution of genetically modified cells in myeloid and lymphoid lineages, and RT-PCR evidence of shRNA in progeny cells provided further evidence of successful transduction and engraftment of progenitor cells. Follow-up data for these and subsequent patients will be presented at the meeting.Conclusion: Lentiviral vector transduction of autologous peripheral blood progenitor cells with multiplexed RNA is feasible, well tolerated, and led to successful engraftment following high dose chemotherapy for ARL.

563. In Vivo Derivation of HIV-1 Resistant Transgenic T Cells by Lentiviral Vector Transduction of a Triple Combination of Anti-HIV Genes Anti-CCR5 Ribozyme, Tat-Rev siRNA and a TAR Decoy

563. In Vivo Derivation of HIV-1 Resistant Transgenic T Cells by Lentiviral Vector Transduction of a Triple Combination of Anti-HIV Genes Anti-CCR5 Ribozyme, Tat-Rev siRNA and a TAR Decoy

RNAi in Combination with a Ribozyme and TAR Decoy for Treatment of HIV Infection in Hematopoietic Cell Gene Therapy

Combinatorial therapies for the treatment of HIV infection have changed the course of the AIDS epidemic in developed nations where the antiviral drug combinations are readily available. Despite this progress, there are many problems associated with chemotherapy for AIDS including toxicities and emergence of viral mutants resistant to the drugs. Our goal has been the development of a hematopoietic gene therapy treatment for HIV infection. Like chemotherapy, gene therapy for treatment of HIV infection should be used combinatorially. We have thus combined three different inhibitory genes for treatment of HIV infection into a single lentiviral vector backbone. The inhibitory agents engage RNAi via a short hairpin RNA targeting HIV tat/rev mRNAs, a nucleolar localizing decoy that binds and sequesters the HIV Tat protein, and a ribozyme that cleaves and downregulates the CCR5 chemokine receptor used by HIV for cellular entry. This triple combination has proven to be highly effective for inhibiting HIV replication in primary hematopoietic cells, and is currently on track for human clinical application.

Long-Term Inhibition of HIV-1 Infection in Primary Hematopoietic Cells by Lentiviral Vector Delivery of a Triple Combination of Anti-HIV shRNA, Anti-CCR5 Ribozyme, and a Nucleolar-Localizing TAR Decoy

Combinatorial therapies for the treatment of HIV-1 infection have proven to be effective in reducing patient viral loads and slowing the progression to AIDS. We have developed a series of RNA-based inhibitors for use in a gene therapy-based treatment for HIV-1 infection. The transcriptional units have been inserted into the backbone of a replication-defective lentiviral vector capable of transducing a wide array of cell types, including CD34+ hematopoietic progenitor cells. The combinatorial therapeutic RNA vector harbors a U6 Pol III promoter-driven short hairpin RNA (shRNA) targeting the rev and tat mRNAs of HIV-1, a U6 transcribed nucleolar-localizing TAR RNA decoy, and a VA1-derived Pol III cassette that expresses an anti-CCR5 ribozyme. Each of these therapeutic RNAs targets a different gene product and blocks HIV infection by a distinct mechanism. Our results demonstrate that the combinatorial vector suppresses HIV replication long term in a more-than-additive fashion relative to the single shRNA or double shRNA/ribozyme or decoy combinations. Our data demonstrate the validity and efficacy of a combinatorial RNA-based gene therapy for the treatment of HIV-1 infection.

367. Development of Lentiviral Vectors Carrying shRNA, Ribozyme and RNA Decoy for the Inhibition of HIV-1 Infection

The replication-defective lentiviral vectors are able to transduce a wide array of quiescent cell types with sustained long-term expression of the genes within the vector backbone. These properties make them favorable vectors for anti-HIV-1 therapeutic gene delivery. The recently discovered small interfering RNA(siRNA) mediated RNA interference provides a powerful new tool for gene silencing in a sequence specific manner, which suggest the potential of siRNAs for gene therapy application. A stable expression system of siRNAs and the efficient delivery vehicle of lentiviral vectors would prove an excellent match as a powerful gene therapy tool. We have developed lentiviral vectors expressing U6 pol III promoter-driven short hairpin RNAs (shRNAs) targeting the rev and tat mRNAs of HIV-1. Utilizing these shRNAs we have achieved potent inhibition of HIV-1 replication in primary cells. Up to date, the majority of HIV-1 gene therapy protocols target a single therapeutic gene. Like any therapeutic approach for inhibition of HIV infection, gene therapy is also faced with the problem of development of viral variants that are resistant to therapeutic agents. We hypothesize that if a combination of therapeutic genes targeting different viral products and steps in the viral life cycle is used, the probability of viral escape mutants will be greatly reduced. Furthermore, there is the possibility of synergy between the different gene therapy agents. Previously, we created the pol III expressing cassettes expressing an anti-CCR5 ribozyme or a nucleolar localizing TAR decoy and cloned them into a self-inactivating lentiviral vector. Each of the constructs showed marked anti-HIV-1 activities. In this study, we combined the shRNAs with the ribozyme or the RNA decoy, or all three constructs into a single vector backbone. Each of these therapeutic RNAs targets a different gene and blocks HIV infection by a distinct mechanism. After transduction with the vectors and infection with HIV-1, a potential synergistic effect was apparent in CD34+ hematopoietic stem cells and primary T cells. We have also developed the vectors expressing only the various therapeutic genes but excluding the reporter gene and WPRE sequence to eliminate any elements that potentially interfere with the expression of the therapeutic genes or have adverse effects on target cells. Our long-term goal is to develop vectors suitable for human gene therapy.

Lentiviral transduction of Tar Decoy and CCR5 ribozyme into CD34+ progenitor cells and derivation of HIV-1 resistant T cells and macrophages

BackgroundRNA based antiviral approaches against HIV-1 are among the most promising for long-term gene therapy. These include ribozymes, aptamers (decoys), and small interfering RNAs (siRNAs). Lentiviral vectors are ideal for transduction of such inhibitory RNAs into hematopoietic stem cells due to their ability to transduce non-dividing cells and their relative refractiveness to gene silencing. The objective of this study is to introduce an HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme into CD34+ hematopoietic progenitor cells via an HIV-based lentiviral vector to derive viral resistant progeny T cells and macrophages.ResultsHigh efficiency and sustained gene transfer into CD34+ cells were achieved with lentiviral vector constructs harboring either Tar decoy or Tar decoy in combination with CCR5 ribozyme. Cells transduced with these constructs differentiated normally into T-lymphocytes in vivo in thy/liv grafts of SCID-hu mice, and into macrophages in vitro in the presence of appropriate growth factors. When challenged in vitro, the differentiated T lymphocytes and macrophages showed marked resistance against HIV-1 infection.ConclusionsViral resistant transgenic T cells and macrophages that express HIV-1 Tar aptamer either alone or in combination with an anti-CCR5 ribozyme could be obtained by lentiviral gene transduction of CD34+ progenitor cells. These results showed for the first time that expression of these anti-HIV-1 transgenes in combination do not interfere with normal thymopoiesis and thus have set the stage for their application in stem cell based gene therapy for HIV/AIDS.

Inhibition of HIV-1 infection by lentiviral vectors expressing pol III-promoted anti-HIV RNAs

A primary advantage of lentiviral vectors is their ability to pass through the nuclear envelope into the cell nucleus thereby allowing transduction of nondividing cells. Using HIV-based lentiviral vectors, we delivered an anti-CCR5 ribozyme (CCR5RZ), a nucleolar localizing TAR RNA decoy, or Pol III-expressed siRNA genes into cultured and primary cells. The CCR5RZ is driven by the adenoviral VA1 Pol III promoter, while the human U6 snRNA Pol III-transcribed TAR decoy is embedded in a U16 snoRNA (designated U16TAR), and the siRNAs were expressed from the human U6 Pol III promoter. The transduction efficiencies of these vectors ranged from 96–98% in 293 cells to 15–20% in primary PBMCs. A combination of the CCR5RZ and U16TAR decoy in a single vector backbone gave enhanced protection against HIV-1 challenge in a selective survival assay in both primary T cells and CD34+-derived monocytes. The lentiviral vector backbone-expressed siRNAs also showed potent inhibition of p24 expression in PBMCs challenged with HIV-1. Overall our results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection.

689. Tar Decoy and Anti-CCR5 Ribozyme Mediated Inhibition of HIV-1 in T Cells and Macrophages Derived from Lentiviral Vector Transduced CD34+ Hematopoietic Progenitor Cells

689. Tar Decoy and Anti-CCR5 Ribozyme Mediated Inhibition of HIV-1 in T Cells and Macrophages Derived from Lentiviral Vector Transduced CD34+ Hematopoietic Progenitor Cells

Combination Genetic Therapy to Inhibit HIV-1

Compared with single agents, combination antilentiviral pharmacotherapy targets multiple HIV-1 functions simultaneously, maximizing efficacy and decreasing chances of escape mutations. Combination genetic therapy could theoretically enhance efficacy similarly, but delivery of even single genes to high percentages of hematopoietic cells or their derivatives has proven problematic. Because of their high efficiency of gene delivery, we tested recombinant SV40-derived vectors (rSV40s) for this purpose. We made six rSV40s, each carrying a different transgene that targeted a different lentiviral function. We tested the ability of these constructs, individually and in double and triple combinations, to protect SupT1 human T lymphoma cells from HIV-1 challenge. Single chain antibodies (SFv) against CXCR4 and against HIV-1 reverse transcriptase (RT) and integrase (IN) were used, as were polymeric TAR decoys (PolyTAR) and a dominant-negative mutant of HIV-1 Rev (RevM10). Immunostaining showed that virtually all doubly treated cells expressed both transgenes. All transgenes individually protected from HIV-1 but, except for anti-CXCR4 SFv, their effectiveness diminished as challenge doses increased from 40 through 2500 tissue culture infectious dose50 (TCID50)/106 cells. However, all combinations of transgenes protected target cells better than individual transgenes, even from the highest challenge doses. Thus, combination gene therapies may inhibit HIV-1 better than single agents, and rSV40s may facilitate delivery of multigene therapeutics.

High level inhibition of HIV replication with combination RNA decoys expressed from an HIV-Tat inducible vector.

Intracellular immunization, an antiviral gene therapy approach based on the introduction of DNA into cells to stably express molecules for the inhibition of viral gene expression and replication, has been suggested for inhibition of HIV infection. Since the Tat and Rev proteins play a critical role in HIV regulation, RNA decoys and ribozymes of these sequences have potential as therapeutic molecular inhibitors. In the present study, we have generated several anti-HIV molecules; a tat-ribozyme, RRE, RWZ6 and TAR decoys and combinations of decoys, and tested them for inhibition of HIV-1 replication in vitro. We used T cell specific CD2 gene elements and regulatory the HIV inducible promoter to direct high level expression and a 3' UTR sequence for mRNA stabilization. We show that HIV replication was most strongly inhibited with the combination TAR + RRE decoy when compared with the single decoys or the tat-ribozyme. We also show that the Tat-inducible HIV promoter directs a higher level of steady-state transcription of decoys and inhibitors and that higher levels of expression directly relate to increased levels of inhibition of HIV infection. Furthermore, a stabilization of the 3' end of TAR + RRE inhibitor transcripts using a beta-globin 3' UTR sequence leads to an additional 15-fold increase in steady-state RNA levels. This cassette when used to express the best combination decoy inhibitor TAR + RRE, yields high level HIV inhibition for greater than 3 weeks. Taken together, both optimization for high level expression of molecular inhibitors and use of combinations of inhibitors suggest better therapeutic application in limiting the spread of HIV.

Anti-HIV Effects of HIV Vectors

We have recently developed an HIV-1 packaging cell line, Ψ422, as an improved tool for anti-HIV gene therapy. After stable transfection with an HIV-1 or HIV-2 vector, Ψ422 has been shown to synthesize virions able to transduce CD4+T cells and macrophages. We now report that HIV vectors per se, in the absence of antiviral genes, inhibit HIV infection of transduced cells. This antiviral effect was shown to be due, at least in part, to a TAR and RRE decoy effect. These data highlight further advantages of HIV-derived gene delivery systems for HIV therapy, in addition to CD4 cell targeting and the ability to transduce nondividing cells.

TAR RNA decoys inhibit tat-activated HIV-1 transcription after preinitiation complex formation.

The ability of the HIV-1 Tat protein to trans -activate HIV-1 transcription in vitro is specifically inhibited by a circular TAR RNA decoy. This inhibition is not overcome by adding an excess of Tat to the reaction but is partially overcome by adding Tat in combination with nuclear extract, suggesting that TAR RNA might function by interacting with a complex containing Tat and cellular factor(s). A cell-free transcription system involving immobilized DNA templates was used to further define the factor(s) that interact with TAR RNA. Preinitiation complexes formed in the presence or absence of Tat were purified on immobilized templates containing the HIV-1 promoter. After washing, nucleotides and radiolabelled UTP were added and transcription was measured. The presence of Tat during preinitiation complex formation resulted in an increase in the level of full-length HIV-1 transcripts. This Tat-activated increase in HIV-1 transcription was not inhibited by circular TAR decoys added during preinitiation complex formation but was inhibited by circular TAR decoys subsequently added during the transcription reaction. These results suggest that TAR decoys inhibit Tat-activated HIV-1 transcription after preinitiation complex formation, perhaps by interacting with components of transcription complexes.

A small circular TAR RNA decoy specifically inhibits Tat-activated HIV-1 transcription.

Linear TAR RNA has previously been used as a decoy to inhibit HIV-1 transcription in vitro and HIV-1 replication in vivo. A 48 nucleotide circular RNA containing the stem, bulge and loop of the HIV-1 TAR element was synthesized using the self-splicing activity of a group I permuted intron-exon and was tested for its ability to function as a TAR decoy in vitro. This small circular TAR molecule was exceptionally stable in HeLa nuclear extracts, whereas a similar linear TAR molecule was rapidly degraded. The TAR circle bound specifically to Tfr38, a peptide containing the TAR-binding region of Tat. The ability of Tat to trans-activate transcription from the HIV-1 promoter in vitro was efficiently inhibited by circular TAR RNA but not by TAR circles that contained either bulge or loop mutations. TAR circles did not inhibit transactivation exclusively by binding to Tat since this inhibition was not reversed by adding excess Tat to the transcription reaction. Together, these data suggest that TAR circles act as decoys that inhibit transactivation by binding to Tat and at least one cellular factor. These data also demonstrate the utility of small circular RNA molecules as tools for biochemical studies.