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Biomedical subjects

F Wong-Staal

Publications and source records attributed to F Wong-Staal.

At least 73 records · Page 4Linked to original sources
PubMed1996

Efficient gene transfer by a human immunodeficiency virus type 1 (HIV-1)-derived vector utilizing a stable HIV packaging cell line.

By transfecting fibroblast cells with an HIV-1-MN molecular clone with a deletion of the major packaging sequence, we have developed a stable HIV-1 packaging cell line, psi 422, psi 422 cells form syncytia with CD4-positive cells, correctly express HIV-1 structural proteins, and produce a large amount of mature particles with normal reverse transcriptase activity. Yet these particles, in which RNA was not detected by reverse transcriptase-PCR, are not infectious. When stably transfected with an HIV-1-based retroviral vector, the psi 422 cell line produces virions capable of transducing CD4-positive cells with high efficiency (up to 10(5) cells per ml). The availability of this stable noninfectious HIV-1 packaging cell line capable of generating high-titer HIV-1 vectors represents a new step toward the use of an HIV-1 delivery system in gene therapy.

Base Sequence↗
PubMed1996

Development of HIV vectors for anti-HIV gene therapy.

Current gene therapy protocols for HIV infection use transfection or murine retrovirus mediated transfer of antiviral genes into CD4+ T cells or CD34+ progenitor cells ex vivo, followed by infusion of the gene altered cells into autologous or syngeneic/allogeneic recipients. While these studies are essential for safety and feasibility testing, several limitations remain: long-term reconstitution of the immune system is not effected for lack of access to the macrophage reservoir or the pluripotent stem cell population, which is usually quiescent, and ex vivo manipulation of the target cells will be too expensive and impractical for global application. In these regards, the lentivirus-specific biologic properties of the HIVs, which underlie their pathogenetic mechanisms, are also advantageous as vectors for gene therapy. The ability of HIV to specifically target CD4+ cells, as well as non-cycling cells, makes it a promising candidate for in vivo gene transfer vector on one hand, and for transduction of non-cycling stem cells on the other. Here we report the use of replication-defective vectors and stable vector packaging cell lines derived from both HIV-1 and HIV-2. Both HIV envelopes and vesicular stomatitis virus glycoprotein G were effective in mediating high-titer gene transfer, and an HIV-2 vector could be cross-packaged by HIV-1. Both HIV-1 and HIV-2 vectors were able to transduce primary human macrophages, a property not shared by murine retroviruses. Vesicular stomatitis virus glycoprotein G-pseudotyped HIV vectors have the potential to mediate gene transfer into non-cycling hematopoietic stem cells. If so, HIV or other lentivirus-based vectors will have applications beyond HIV infection.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Conservation of a hairpin ribozyme sequence in HIV-1 is required for efficient viral replication.

We have previously described a hairpin ribozyme that targets a highly conserved sequence in the U5 region of HIV-1. To determine if escape mutations would compromise virus replication, we introduced critical mutations into the ribozyme target site of an infectious molecular clone of HIV-1MN. HIV-1 MNA has a substitution of A for G immediately 3' to the cleavage site and HIV-1 MNGC has two substitutions in the flanking sequences that are complementary to the ribozyme. In vitro studies confirmed that neither the MNA-nor the MNGC-mutated target sequence was cleaved by the ribozyme, and furthermore, the MNGC-mutated target sequence failed to bind the ribozyme. Compensatory GC substitutions in the substrate recognition domain of the ribozyme resulted in a switch of binding and cleavage specificity. Replication of both the MNA and MNGC mutant viruses was initially two to three logs lower than that of wild-type virus, but after 3 weeks, virus production rose sharply in both cultures. Nucleotide sequence of RT-PCR-amplified viral sequences obtained from virus produced at later time points revealed complete reversion of MNA or partial reversion of MNGC to wild-type genotypes. No additional mutations within the ribozyme target sequence were observed. These results indicate that mutations in this conserved ribozyme target sequence led to significant attenuation of HIV-1MN.

Animals↗
PubMed1996

Sequence analysis of the V3 loop in brain and spleen of patients with HIV encephalitis.

Infection with a particularly neurovirulent strain of HIV has been hypothesized to explain why only a subset of patients develops HIV encephalitis. We studied the third hypervariable region (V3) of multiple clones from both brains and spleens of three patients who died with HIV encephalitis, to see if there was a molecular signature associated with neurological disease. Clones from the spleen and brain of individual patients showed significant nucleic acid homology and had envelope sequences characteristic of macrophage-tropic viruses. No brain-specific unique sequences were observed, suggesting that while CNS virus is macrophage tropic there is no evidence in the V3 envelope region studied to suggest a specific neurotropic variant.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Intracellular immunization against SIVmac utilizing a hairpin ribozyme.

A hairpin ribozyme targeting the 3' LTR region (9456) of SIVmac238 was cloned into a murine retroviral vector. This target sequence is conserved among various SIV, as well as most HIV-2, strains. The ribozyme cassette is driven from a polymerase III promoter, that of the human tRNAval gene. Hybrid human B-/T-cell lines (CEM/174) were transduced with the retroviral constructs and selected for G418 resistance. Cells stably expressing the 9456 ribozyme exhibited long-term resistance to infection by a pathogenic molecular clone of SIV and two strains of HIV-2. The ribozyme was also able to effectively reduce the proviral DNA burden. Its efficient protection against SIV/HIV-2 infection constitutes an important step toward evaluating ribozyme gene therapy in a primate model.

Animals↗
PubMed1996

A chimeric human immunodeficiency virus type 1 (HIV-1) minimal Rev response element-ribozyme molecule exhibits dual antiviral function and inhibits cell-cell transmission of HIV-1.

We have previously shown that hairpin ribozymes targeting the human immunodeficiency virus (HIV) genome can effectively inhibit virus replication in a variety of primary and cultured hematopoietic cells. To further increase antiviral potency and minimize the chance of viral resistance, we have now cloned the stem-loop II sequences of the HIV type 1 Rev response element into ribozyme transcription cassettes. Fusion RNA molecules were shown to function both as RNA decoys and ribozymes. Stable Molt-4/8 cell lines expressing fusion RNA of stem-loop II and a ribozyme directed at the HIV-type 1 U5 sequence (MSLMJT) or its disabled counterpart (MSLdMJT) were generated. The expression of fusion RNA was persistent for at least 6 months without apparent cytotoxicity. When virus inhibition was examined after the cocultivation of transduced cells with chronically infected Jurkat cells, much greater protection was observed in MSLMJT cells than in MSLdMJT or MMJT (expressing only the ribozyme) cells. Furthermore, to specifically compare the ribozyme activities in various transduced cells, we determined the quantitative levels of proviral DNA in the first round of virus replication (7 h after infection with HXB2). By competitive PCR, the proviral DNA levels in MSLMJT and MMJT cells were found to be reduced to 1/7 and 1/3, respectively, compared with those in MSLdMJT and MdMJT cells. These results suggest not only that the greater inhibition afforded by this fusion RNA was due to its function both as decoy and ribozyme but also that the ribozyme activity may be facilitated.

Base Sequence↗
PubMed1996

Ex vivo transduction and expansion of CD4+ lymphocytes from HIV + donors: prelude to a ribozyme gene therapy trial.

Preparations for a phase I trial of ex vivo, anti-HIV ribozyme gene therapy have included optimization of transduction and expansion of CD4+ lymphocytes from HIV-1 infected donors, using reagents suitable for production of cell products for human infusion. We also determined whether transduction by the ribozyme vector would inhibit replication and spread of endogenous HIV-1, and result in preferential survival of ribozyme-transduced CD4+ cells during lymphocyte expansion. Transduction efficiency, as estimated by DNA quantitative competitive (QC)-PCR, was similar for both control (LNL6) and ribozyme expressing (MJT) murine retroviral vectors (approximately 20%.) In the absence of antiviral agents, cells transduced with MJT exhibited three-fold greater numbers of CD4+ cells 2 weeks after transduction than did LNL6 transduced cells. In addition, viral replication was delayed 2-3 weeks in MJT transduced cultures. Both transduced cell populations expanded by 2-3 logs within 2 weeks. The clinical protocol involves infusion of both ribozyme and control vector transduced cells, making identification of agents capable of suppressing replication and spread of endogenous virus during ex vivo expansion necessary. The combination of nevirapine (100 nM) and CD4-PE40 (100 nM) completely suppressed endogenous virus replication in cultures transduced with either vector. At reduced concentrations of nevirapine, virus replication was suppressed only in MJT transduced cells.

Animals↗
PubMed1995

Intracellular immunization of human fetal cord blood stem/progenitor cells with a ribozyme against human immunodeficiency virus type 1.

Successful treatment of human immunodeficiency virus infection may ultimately require targeting of hematopoietic stem cells. Here we used retroviral vectors carrying the ribozyme gene to transduce CD34+ cells from human fetal cord blood. Transduction and ribozyme expression had no apparent adverse effect on cell differentiation and/or proliferation. The macrophage-like cells, differentiated from the stem/progenitor cells in vitro, expressed the ribozyme gene and resisted infection by a macrophage tropic human immunodeficiency virus type 1. These results suggest the feasibility of stem cell gene therapy for human immunodeficiency virus-infected patients.

Antigens, CD↗
PubMed1995

In vitro and in vivo characterization of a second functional hairpin ribozyme against HIV-1.

We have constructed a hairpin ribozyme targeted to cleave a conserved sequence in the HIV-1 pol gene. The ribozyme was modified to include a structure-stabilizing tetraloop. In vitro studies revealed a cleavage efficiency unprecedented for hairpin ribozymes (Kcat/Km = 75 min-1 microM-1). Stable retroviral vector transduction of this ribozyme gene in T-cell lines resulted in long-term ribozyme expression. As compared to control vector transduced T-cells, the pol ribozyme-transduced cells exhibited significant inhibition of different strains of HIV-1 virus production; this protection was greater when ribozyme expression was driven from an internal pol III promoter (adenovirus VA1) than when driven by a pol II promoter (the MMLV LTR). These results further demonstrate the potential of hairpin ribozymes as anti-HIV gene therapy agents and suggest possibilities for employing combinations of independently targeted hairpin ribozymes.

Base Sequence↗
PubMed1995

Stem cells as vehicles for gene therapy: novel strategy for HIV infection.

Restoration of bone marrow and immune function by means of allogeneic bone marrow transplantation has been attempted in AIDS patients but has not been successful as the donor-derived cells, or their progeny, inevitably became infected. A hairpin ribozyme that specifically cleaves HIV-1 RNA has been developed by F. Wong-Staal et al. and has been demonstrated to confer resistance against HIV-1 infection. Allogeneic transplantation of CD34+ cells or their pluripotent subsets, transduced by vectors bearing this ribozyme gene, can protect the stem cells and their progeny from HIV-1 infection and eventually restores immune function. We have provided evidence that long-term repopulating stem cells can be mobilized into peripheral blood by growth factors. The combination of G-CSF and GM-CSF seems to yield a high frequency of pluripotent stem cells with a CD34+ subset profile that is similar to placental and umbilical cord blood (PUCB). We have then demonstrated a highly efficient transduction of CD34+ cells from PUCB and mobilized leukapheresis products by retroviral vectors bearing the ribozyme gene. Expression of the ribozyme gene, as shown by reverse transcriptase-polymerase chain reaction, was of similar magnitude (70%-90% of cells that grow into colonies). Challenge of the progeny macrophages from such transduced CD34+ cells with monocyte-trophic strains of HIV-1 showed that they were resistant to infection. Thus allogeneic transplantation of CD34+ cells or their pluripotent subsets, transduced with ribozyme gene, can be a promising strategy for the treatment of HIV infection.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Molecular cloning and characterization of a TAR-binding nuclear factor from T cells.

The TAt protein of the human immunodeficiency virus type 1 (HIV-1) activates the expression of viral mRNA through a cis-acting element in the LTR termed TAR. TAR RNA forms a stable stem-loop structure. Mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and three unpaired bases in the stem (bulge) are important for Tat activation. Using the in vitro-transcribed TAR RNA as a probe, we have cloned a gene (TARBP-b) that encodes a TAR-binding protein from a cDNA expression library derived from Hut-78 cells. Expression of the 1.4-kb TARBP-b mRNA was observed in all mammalian cell lines tested. TARBP-b binds specifically to the bulge region of TAR RNA and trans-activates the HIV-1 long terminal repeat in the presence of ptat and prev expression plasmids. These results suggest that TARBP-b contributes to tat-mediated trans-activation.

Amino Acid Sequence↗
PubMed1995

Localization of human immunodeficiency virus Rev in transfected and virus-infected cells.

The rev gene product of human immunodeficiency virus (HIV) is obligatory for viral replication. Rev interacts specifically with a structured RNA sequence within the viral genome termed the REV response element (RRE). Although the importance of Rev for the expression of viral proteins is well documented, its functional mechanism remains unresolved. Previous studies identified Rev in the absence of RRE to be a nuclear protein localized primarily within the nucleoli. To extend our understanding of the role of Rev in viral replication, immunolocalization studies of Rev and other nuclear components were carried out in transfected cells expressing both the Rev protein and RRE-containing mRNA and in cells infected with HIV. In both types of cells, Rev-like immunoreactivity was distributed both in the nucleoplasm and cytoplasm. Within the nucleus, Rev immunoreactivity was not evenly distributed but was present within focal concentrations. In transfected cells that were double labeled for Rev and SC-35, which labels a known component of spliceosomes, the foci of Rev labeling were distinct from the "speckles" labeled by SC-35, although Rev foci and speckles were often juxtaposed. In addition, morphological changes in the three-dimensional network of speckles were observed in both transfected cells expressing both the Rev protein and RRE-containing mRNA and in cells infected with HIV-1 and HIV-2. Our observations are consistent with the proposed dual role of Rev in mRNA transport and splicing.

Base Sequence↗
PubMed1995

An infectious chimeric human immunodeficiency virus type 2 (HIV-2) expressing the HIV-1 principal neutralizing determinant.

The human immunodeficiency virus type 1 strain MN (HIV-1MN) principal neutralizing determinant (PND, V3 loop) was introduced into infectious molecular clones HIV-2KR and simian immunodeficiency virus mm239 (SIVmm239) by hybridization PCR, replacing the corresponding HIV-2 or SIV envelope cysteine loops with the HIV-1 coding sequence. The HIV-2 chimera (HIV-2KR-MNV3) was found to be capable of infecting a number of T-cell lymphoblastic cell lines as well as primary peripheral blood mononuclear cells. In contrast, the SIV chimera (SIV239MNV3) was not replication competent. Envelope produced by HIV-2KR-MNV3 but not the parental HIV-2KR was recognized by V3-specific and HIV-1-specific polyclonal antisera in radioimmunoprecipitation assays. HIV-2-specific antisera recognized both the chimeric and parental virus but not HIV-1MN. The chimeric HIV-2KR-MNV3 virus proved to be exquisitely susceptible to neutralization by HIV-1-specific and V3-specific antisera, suggesting the potential for use in animal models designed to test HIV-1 vaccine candidates which target the PND.

Base Sequence↗
PubMed1995

Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.

Amino Acid Sequence↗
PubMed1995

Identification and mapping of inhibitory sequences in the human immunodeficiency virus type 2 vif gene.

Human immunodeficiency virus (HIV) regulates the expression of its genes temporally at the mRNA processing step. A subset of the mRNA species which encode the structural and some accessory genes contains inhibitory sequences (INS or CRS elements) which prevent nuclear export of the RNA or its utilization in the cytoplasm. Such inhibition is overridden by the interaction of a viral protein, Rev, with its RNA target sequence, RRE. The vif gene product, which is essential for virus replication in vivo, is encoded by a singly spliced mRNA, and its expression is dependent on rev in infected cells. However, INS elements have not been found in the HIV-1 vif gene itself, although such elements have been observed in Gag, Pol, and Env coding sequences. We have now identified an INS within the 5' half of HIV-2 vif which does not show any homology with cellular mRNAs or other previously identified INS and CRS elements of HIV. These results suggest that retroviral mRNAs have novel labile sequences different from those of cellular mRNAs.

Base Sequence↗
PubMed1995

Tat-independent replication of human immunodeficiency viruses.

The replication of human immunodeficiency retroviruses involves a complex series of events that is regulated at both transcriptional and posttranscriptional levels. The tat gene product is a potent trans-activator of viral transcription and therefore an attractive target for the development of antiviral drugs. Tat-defective HIV-1 proviral DNA clones have been shown previously to be replication defective. In this study, we report that tat-defective HIV-1 and HIV-2 viral DNA transfected into U937 cells can direct efficient viral replication in the presence of transcriptional stimulators such as TNF-alpha and PMA. In MT-4 cells, tat-defective HIV-1 can replicate without any stimulation. The viruses recovered from MT-4 cells remained tat defective defined by their inability to infect T cell lines (e.g., Molt 4/8) although replication could be rescued with cytokines. Limited replication was observed in primary mononuclear cells. Furthermore, we showed that Ro 24-7429, a potent tat antagonist and antiviral compound, failed to suppress HIV-1 replication in TNF-alpha-stimulated T cells. These results have important implications for targeting tat as a therapeutic strategy for AIDS.

Benzodiazepines↗
PubMed1995

Increased titer of recombinant AAV vectors by gene transfer with adenovirus coupled to DNA-polylysine complexes.

One of the principal problems with using recombinant adeno-associated virus vectors (rAAV) as vehicles for gene delivery has been the difficulty in obtaining high-titer virus stocks after the initial transfection into producer cells. In this report we describe a method for transfecting cells at extremely high efficiency with rAAV vector DNA and complementation plasmid while simultaneously infecting those cells with replication competent adenovirus using adenovirus-polylysine-DNA complexes. We further show that this technique results in an increase in rAAV transducing titer by two orders of magnitude over what is typically achieved by standard calcium phosphate transfection.

Adenoviridae↗