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Biomedical subjects

F Wong-Staal

Publications and source records attributed to F Wong-Staal.

At least 91 records · Page 5Linked to original sources
PubMed1994

Activity and cleavage site specificity of an anti-HIV-1 hairpin ribozyme in human T cells.

Human CD4+ T cells (Molt-4) were transduced with retroviral vectors containing a hairpin ribozyme which targets the rev/env coding region of HIV-1 RNA (HXB2: 8629-8644). This target sequence is conserved among many HIV-1 clones, including the prototype virus HXB2, but the infectious clone SF2 contains a single nucleotide substitution at the cleavage site (from N*GUC to N*UUC). Cells stably expressing the ribozyme or its disabled counterpart were challenged with HXB2 or SF2 and the amount of p24 antigen produced was monitored. While this ribozyme was effective in inhibiting the replication of HXB2 in Molt 4 cells, it showed only marginal inhibitory effect on SF2 replication. The same level of virus production was observed with cells transduced by the disabled ribozyme, which functions essentially as an antisense molecule. Expression of the ribozyme was comparable in HXB2- or SF2-infected cells as detected by reverse transcription-polymerase chain reaction. These data provide in vivo evidence that the antiviral activity of the hairpin ribozyme is strictly dependent on the presence of the cleavage site in the target RNA and supports the conclusion that the ribozyme acts as catalytic RNA rather than as antisense RNA in vivo.

Base Sequence↗
PubMed1994

Further evaluation of soluble CD4 as an anti-HIV type 1 gene therapy: demonstration of protection of primary human peripheral blood lymphocytes from infection by HIV type 1.

We previously reported on the construction of retroviral vectors that produce a secreted form of the HIV-1 receptor, T cell antigen CD4 (Morgan et al., AIDS Res Hum Retroviruses 1990;6:183-191). In this article we test the ability of these sCD4-expressing retroviral vectors to protect human T-cell lines or primary T cells from HIV-1 infection. To demonstrate that protection from HIV-1 infection is mediated by the soluble nature of this protein, two coculture protection experiments were conducted. In these experiments, sCD4-expressing retroviral vectors were used to engineer mouse NIH 3T3 cells. In one coculture experiment the human SupT1 cell line was added directly to the culture of sCD4-producing NIH 3T3 cells, and in another experiment the two cell types were separated physically by a semipermeable membrane. In both coculture configurations, the T cell line was protected from HIV-1 challenge as measured by syncytium formation and indirect immunofluorescent assays. In addition, the SupT1 line was directly engineered with sCD4-expressing retroviral vectors and shown to be protected from HIV-1 challenge. As a prelude to further preclinical studies, we tested the ability of retroviral vectors to transduce primary human peripheral blood lymphocytes (PBLs). Conditions used to stimulate T cell growth resulted in significant shifts in the CD4/CD8 cell in favor of CD8 cells. Retroviral-mediated gene transfer under these conditions resulted in low levels of gene transfer (< 5%).(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1994

Transfer of an anti-HIV-1 ribozyme gene into primary human lymphocytes.

We reported previously that human CD4+ T cell lines stably expressing a hairpin ribozyme targeted to the human immunodeficiency virus type 1 (HIV-1) U5 leader sequence were resistant to challenge with diverse HIV-1 viral clones and clinical isolates (Yamada et al., 1994). To simulate more closely the in vivo infection process for investigations of anti-HIV-1 ribozyme gene therapy, we developed a system to transfer this ribozyme gene into freshly isolated human peripheral blood lymphocytes (PBLs) using a murine retrovirus vector. Following transduction and G418 selection, human PBLs from multiple donors expressed the ribozyme and resisted challenge by HIV-1 viral clones and clinical isolates, while control vector-transduced PBLs remained fully permissive for HIV-1 infection. No inhibition of an HIV-2 clone lacking the target was seen in ribozyme-expressing PBLs. Ribozyme expression had no effect on viability or proliferation kinetics of the primary lymphocytes. This study is the first demonstration in primary human T cells of resistance to HIV-1 infection conferred by gene transfer. A human clinical trial is in development to test further the safety and efficacy of this ribozyme in PBLs of HIV-1-infected patients in vivo.

Base Sequence↗
PubMed1994

Antiviral and anticancer ribozymes.

Molecular biologists are beginning to apply the site-specific RNase properties of ribozymes to gene therapy for HIV infection and cancer. Requirements for substrate recognition and cleavage are being finely mapped. Methods of augmenting intracellular cleavage and of dissecting observed discrepancies between in vitro and cellular activity are being explored. Antiviral efficacy against HIV type 1 has been seen in tissue culture for both hammerhead and hairpin ribozymes, and a phase I clinical trial for ex vivo T-cell gene therapy is planned. Oncogene transcripts, such as the bcr/abl fusion messenger RNA and ras, have also been targeted with ribozymes.

Base Sequence↗
PubMed1994

Progress towards gene therapy for HIV infection.

The retroviral life cycle and genetic plasticity of human immunodeficiency virus 1 (HIV-1) present unprecedented therapeutic challenges. Twelve years into the HIV epidemic, satisfactory treatment remains elusive. Our current understanding of AIDS pathogenesis calls for early intervention with antiviral agents. Although still in its infancy, human gene therapy holds considerable potential for the long-term treatment of genetic disorders, cancer and chronic infectious diseases. Gene therapy for HIV infection is receiving particularly intensive study: approaches that are in development include both immunotherapy (e.g. therapeutic vaccines and adoptive transfer of CD8+ T-cell clones) and direct antiviral therapy (intracellular immunization). The latter strategies include transdominant modifications of HIV proteins, RNA decoys, antisense RNA, ribozymes and modifications of cellular proteins (e.g. intracellular antibodies, soluble CD4). Several of these strategies are now entering clinical trials. While significant conceptual and technical hurdles remain to be overcome before the promise of gene therapy for HIV infection can be fully realized, progress in this field is likely to be rapid and to contribute to the broader applicability of human gene therapy to the treatment of other disorders.

Clinical Protocols↗
PubMed1994

Intracellular immunization of human T cells with a hairpin ribozyme against human immunodeficiency virus type 1.

T-cell lines (Jurkat and Molt-4) were transduced with retroviral vectors containing a hairpin ribozyme that targets a conserved sequence in the 5' transcribed leader sequence of human immunodeficiency virus (HIV) type 1. Stable cell lines were generated which constitutively and persistently expressed the ribozyme gene driven by either the Moloney retroviral long terminal repeat (LTR) or an internal human tRNA(val) promoter. There was no apparent deleterious effect of long-term ribozyme expression on cell proliferation or viability. Cells expressing ribozyme were resistant to challenge from diverse strains of HIV, including an uncloned clinical isolate. No reverse transcriptase activity or virus infectivity was detectable in the culture supernatants of Jurkat cells expressing the ribozyme driven by the tRNA(val) promoter up to 35 days after challenge with HIV-1/HXB2. Expression of the ribozyme also significantly decreased (by approximately 50- to 100-fold) the efficiency of incoming virus to synthesize viral DNA. These and previously reported results indicate that transfer and expression of the ribozyme gene interfere with both early and late events in the HIV replication cycle and confer long-term resistance to HIV-1 infection.

Base Sequence↗
PubMed1994

Transcellular activation of the HTLV promoter by human hematopoietic cells.

We examined the ability of hematopoietic cells to transactivate the HTLV promoter by a transcellular mechanism. HeLa cells containing a CAT reporter gene driven by the HTLV-2 promoter were cocultivated with hematopoietic cells of the B-(Raji), T-(HuT78, Jurkat) and monocyte/promyelocytic (THP-1, U937 and HL60) lineages. Cocultivation with U937 and HuT78 cells constitutively and significantly transactivated the HTLV-2 promoter, while no effect was observed with the other lines. However, activation of other T-cell lines (CEM, Jurkat, Molt-3 and MT-4) with a combination of phorbolester and phytohemagglutinin also resulted in potent transactivation. Supernatant from HuT78 cells exhibited detectable transactivating activity, suggesting that the activation is mediated by a secreted factor(s). This factor also transactivates the HTLV-1 promoter. We used a panel of HTLV-1 LTR deletion mutants to map the responsive elements to this factor(s). Unlike the response element to the HTLV transactivator protein, Tax, which can be mapped to a small region in the enhancer, maximal transactivation by the cellular factor(s) required the complete U3 sequence. Transcellular activation of the HTLV promoter by activated T-cells may play a role in the development of leukemia in HTLV infected individuals.

Deltaretrovirus↗
PubMed1993

Exogenous tat protein activates human endothelial cells.

tat protein, a human immunodeficiency virus (HIV) gene product that functions as a transactivator for HIV replication, is known to be secreted extracellularly by infected cells. To determine the potential role of tat in the dissemination of HIV into extravascular tissue, this protein was examined for its ability to activate human endothelial cells. The results show that tat does indeed stimulate endothelial cells. This is evidenced by their expression of the endothelial-leukocyte adhesion molecules, E-selectin, critical for the initial binding of leukocytes to the blood vessel wall, and their increased synthesis of interleukin-6 (IL-6), a cytokine known to enhance endothelial cell permeability. Furthermore, tat acts synergistically with low concentrations of tumor necrosis factor-alpha to enhance IL-6 secretion. These data suggest that extracellular tat protein secreted or released into the microenvironment may contribute significantly to the determination of specific sites of leukocyte binding to blood vessels, to transmigration into tissue, and to eventual dissemination of HIV-infected cells or free virions into tissue.

Cell Adhesion Molecules↗
PubMed1993

A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1.

Ribozymes have enormous potential as antiviral agents. We have previously reported that a hairpin ribozyme expressed under the control of the beta-actin promoter that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence can inhibit HIV-1 (pHXB2gpt) expression. For such a ribozyme in a retroviral vector delivery system to be useful in gene therapy for the treatment of HIV-1 infection, it must be able to inhibit the expression of multiple HIV-1 strains. We have now cloned this ribozyme into various regular expression vectors (including retroviral vectors) by using various gene expression control strategies. Here we show by transient transfection that inhibition of expression of diverse strains of HIV-1 can be achieved by this ribozyme expressed in the proper vectors. These data further support the potential of this hairpin ribozyme as a therapeutic agent for HIV-1.

Antiviral Agents↗
PubMed1993

Mapping the determinants of human immunodeficiency virus 2 for infectivity, replication efficiency, and cytopathicity.

Human immunodeficiency virus 2 (HIV-2) ISY and the newly derived HIV-2KR are infectious molecular clones that yield viruses differing markedly in their abilities to infect and/or induce syncytia in various T- and monocytoid-cell lines. Chimeric viruses were constructed from these two viral genomes to localize the genetic determinants of some of these properties. Envelope sequences, particularly those spanning the CD4 binding site, appear to be critical for the ability of HIV-2KR to infect MOLT-4 clone 8 and SupT1 cells and to efficiently infect the H9 cell line. On the other hand, multiple determinants may contribute to cytopathicity (gp41 and nef) in H9 cells and replication efficiency in monocytic (THP-1) cells.

Acquired Immunodeficiency Syndrome↗
PubMed1993

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin.

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD-containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Inhibition of Rev activity and human immunodeficiency virus type 1 replication by antisense oligodeoxynucleotide phosphorothioate analogs directed against the Rev-responsive element.

The interaction between the Rev protein of human immunodeficiency virus type 1 and its highly structured and conserved RNA target, the Rev-responsive element, is required for virus replication. We demonstrate that antisense oligodeoxynucleotide phosphorothioate analogs directed against the Rev-responsive element effectively inhibit Rev activity, as well as human immunodeficiency virus type 1 replication, and are candidates for antiviral therapy.

Base Sequence↗
PubMed1993

Transdominant inhibition of wild-type human immunodeficiency virus type 2 replication by an envelope deletion mutant.

The envelope glycoprotein of human immunodeficiency virus type 2 (HIV-2) is primarily responsible for virus attachment and entry into the target cell population. We constructed an HIV-2 mutant virus containing an in-frame deletion within the putative CD4-binding sequences of the envelope glycoprotein and confirmed that the mutant envelope is unable to bind CD4 and that the mutant virus is noninfectious. To investigate whether this mutant could dominantly interfere with wild-type replication, we coexpressed proviral DNAs of both wild-type and mutant viruses in cells and assayed the production of infectious HIV-2 virions. Interference with virus replication was indeed observed with mutant DNA, and a maximal effect was achieved with 10-fold excess mutant DNA over wild-type DNA in the cotransfection experiments. The transdominant effect on virus replication does not appear to be at the level of wild-type envelope expression or gp120-CD4 interaction. Rather, the interference may be at the level of mixed-oligomer formation during progeny virus assembly and may occur by either destabilizing the multimeric structure of gp120 or forming a defective mixed multimeric gp120 which is unable to complete the receptor binding and/or postbinding events needed for infection.

Amino Acid Sequence↗
PubMed1992

Inhibition of human immunodeficiency virus type 1 expression by a hairpin ribozyme.

Ribozymes are RNAs that possess the dual properties of RNA sequence-specific recognition, analogous to conventional antisense molecules, and RNA substrate destruction via site-specific cleavage. The cleavage reaction is catalytic in that more than one substrate molecule is processed per ribozyme molecule. We have designed a hairpin ribozyme that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence (at nucleotides +111/112 relative to the transcription initiation site). The ribozyme was tested in vitro and gave efficient and specific cleavage of RNA containing the leader sequence. To test the antiviral efficacy of this ribozyme, we have cotransfected into HeLa cells HIV-1 proviral DNA and a plasmid expressing the ribozyme from the human beta-actin promoter. HIV-1 expression was inhibited as measured by p24 antigen levels and reduced Tat activity. The antiviral effect of the ribozyme appears to be specific and results from directed RNA cleavage; activity requires both a target sequence and a functional RNA catalytic center. These results suggest that this HIV-1-directed hairpin ribozyme may be useful as a therapeutic agent.

Base Sequence↗
PubMed1992

Biological characterization of noninfectious HIV-1 particles lacking the envelope protein.

To understand the role of the HIV-1 envelope protein in the assembly of virus, we constructed a proviral clone of HIV-1 where the methionine initiator codon of the env gene was substituted with a translational stop codon. Upon DNA transfection into permissive cells in culture, this clone produces virus-like particles similar in size to parental virus but are noninfectious in human T-cells, promonocytic cells, and primary macrophages. This mutant readily recombines with a deletion mutant provirus lacking the entire gag-pol region producing a recombinant virus that is infectious. Substitution of the same initiator methionine codon with valine results in a leaky missense mutant provirus capable of a low level of Env protein synthesis that leads to a productive infection. Thus, the prototype initiation codon AUG is dispensable for virus infectivity. Further, the expression of the envelope protein is not a prerequisite for the assembly of the virus particles in the HIV-1 system. These noninfectious envelope-less particles revert readily to wild-type phenotype upon cotransfection with Env-producing plasmid DNAs.

Animals↗