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Biomedical subjects

F Wong-Staal

Publications and source records attributed to F Wong-Staal.

At least 55 records · Page 3Linked to original sources
PubMed1998

A minimally replicative HIV-2 live-virus vaccine protects M. nemestrina from disease after HIV-2(287) challenge.

M. nemestrina immunized with an apathogenic HIV-2 molecular clone (HIV-2KR) were protected from CD4 decline and disease upon challenge with HIV-2(287), after any immunizing virus could be detected. Higher but not lower inocula of HIV-2KR were protective against intravenous inoculation of either 10(5) or 10(1) TCID50 of HIV-2(287). Protected animals displayed substantial reductions in PBMC proviral burden (1-3 logs), viral titers (1-2 logs), and plasma viral RNA (2-4 logs) compared to unprotected or naive animals as early as 1 week postinfection. Plasma viral RNA became undetectable after 24 weeks in protected animals, but remained high in unprotected animals. No viral RNA was present in the spleen of the protected animal necropsied more than a year after challenge (though viral DNA was still present). No neutralizing responses could be demonstrated, but CTL activity was detected sooner and at higher levels after challenge in protected than in unprotected macaques. In this novel HIV-2 vaccine model, protection was clearly dose-dependent, and clearance of challenge virus RNA from the plasma did not require detectable ongoing replication of the immunizing virus at the time of challenge.

AIDS Vaccines↗
PubMed1998

Specific interaction of HTLV tax protein and a human type IV neuronal intermediate filament protein.

The human T-cell leukemia virus (HTLV) is associated with adult T cell leukemia and neurological disorders (TSP/HAM). The HTLV transcriptional transactivator, Tax, is known to exert its effect through protein-protein interaction with several transcription factors that activate genes in T cell proliferation. The pathogenic mechanism in the CNS is less defined. Using the yeast two-hybrid system, we have identified a specific Tax-binding protein as the neuronal specific intermediate filament protein, alpha-internexin. Tax binds to the domain corresponding to the rod region of alpha-internexin, which is essential for neurofilament assembly. The Tax domains involved in binding are separable from those involved in transactivation. TxBP-1/alpha-internexin and Tax are expressed in the cytoplasm and nucleus, respectively, when expressed alone, but in coexpressing cells, colocalization of both proteins was observed in a perinuclear, punctate distribution. This in vivo interaction also resulted in a dramatic reduction in Tax transactivation and the network formation by alpha-internexin. The specific interaction of Tax and a neuronal specific intermediate filament protein may provide a clue to the pathogenesis of TSP/HAM.

Amino Acid Sequence↗
PubMed1998

Characterization of a molecular clone of HIV type 2 infectious for Macaca nemestrina.

A lambda phage clone containing a full-length HIV-2 provirus, designated HIV-2KR, was obtained from the genomic DNA of Molt4 clone 8 (Molt4/8) lymphoblastic cells infected with the HIV-2PEI2 strain. HIV-2KR is genetically distinct from known HIV-2 isolates, possessing both a unique deletion in the LTR promoter region, and a long rev reading frame. It is replication competent in vitro after transfection into Molt4/8 cells, replicates in a variety of established human T lymphoblastic (Molt-3, Molt4/8, SupT1, H9, C8166) and myelomonocytic (U937) cell lines, and displays prominent cytopathic effects on infection of Molt4/8 cells, reflecting usage of both CCR5 and CXCR4 coreceptors. In addition, HIV-2KR was found to be infectious for human and Macaca nemestrina peripheral blood lymphocytes, and primary human monocyte-macrophage cultures. Intravenous inoculation of cell-free virus into M. nemestrina resulted in infection characterized by transient, low-level viremia and modest temporary decline in CD4 lymphocyte numbers, making HIV-2KR the first HIV-2 molecular clone reported to be infectious for this primate species.

Amino Acid Sequence↗
PubMed1998

Expression of ribozymes in gene transfer systems to modulate target RNA levels.

The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, they have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation.

Animals↗
PubMed1998

Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors.

The molecular bases for species barriers to lentiviral replication are not well understood, but are of interest for explaining lentiviral pathogenesis, devising therapeutic strategies, and adapting lentiviruses to gene therapy. HIV-1-based lentiviral vectors efficiently transduce nondividing cells, but present complex safety concerns. Nonprimate (ungulate or feline) lentiviruses might provide safer alternatives, but these viruses display highly restricted tropisms, and their potential for adaptation as replication-defective vectors capable of transducing human cells is unknown. Feline immunodeficiency virus (FIV) does not infect humans or other non-Felidae despite prevalent natural exposure. Although long terminal repeat (LTR)-directed FIV expression was found to be negligible in human cells, promoter substitution enabled an env-deleted, three-plasmid, human cell-FIV lentiviral vector system to express high levels of FIV proteins and FIV vectors in human cells, thus bypassing the hazards of feline vector producer cells. Pseudotyped FIV vectors efficiently transduced dividing, growth-arrested, and postmitotic human targets. The experiments delineate mechanisms involved in species-restricted replication of this lentivirus and show that human cells support both productive- and infective-phase mechanisms of the FIV life cycle needed for efficient lentiviral vector transduction. Nonprimate lentiviral vectors may offer safety advantages, and FIV vectors provide unique experimental opportunities.

Animals↗
PubMed1998

Transduction of human macrophages using a stable HIV-1/HIV-2-derived gene delivery system.

We have previously established a stable HIV-1 packaging cell line, psi 422, which yielded high titers of an HIV-1 vector capable of efficiently transducing CD4+ cells. In order to increase the safety of this gene delivery system, we have now replaced the HIV-1 vector with an HIV-2 vector to abolish any risk of homologous recombination between the packaging cells and the vector. The HIV-2 vector was also modified by insertion of a cis-acting constitutive transport element which confers Rev independence. The supernatant of psi 422 cells stably transfected with this new vector was capable of transducing CD4+ cells with a titer of 10(4) transducing units per milliliter. This result shows that cross-packaging of HIV-2 vectors with the HIV-1 packaging cells is quite efficient. Using this new stable HIV-1/HIV-2 gene delivery system, we were able to transduce human monocyte-derived primary macrophages, which are refractory to murine retrovirus-mediated transduction. The availability of a stable HIV-based gene delivery system for macrophages, a key target cell in HIV infection; is an important advance in gene therapy for AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Gene therapy targeting cord blood-derived CD34+ cells from HIV-exposed infants: preclinical studies.

Hematopoietic CD34+ cells from placental and umbilical cord blood (PUCB) can be valuable vehicles for gene therapy of immunodeficiencies and genetic disorders. We have conducted preclinical studies towards the treatment of HIV-1-infected infants with genetically 'immunized' CD34+ cells derived from PUCB using anti-HIV-1 hairpin ribozyme genes. PUCB was collected from 10 newborns of HIV-1-positive mothers. CD34+ cells were enriched with a modified procedure using Dynal immunomagnetic beads and chymopapain, stimulated with stem cell factor (SCF), IL-3 and IL-6, and transduced using cell-free recombinant retroviral vector (MJT) expressing a ribozyme against the U5 region of HIV-1. No significant differences were observed in the growth pattern of CD34+ cells from normal infants, HIV-1 exposed infants or infants confirmed to be infected by HIV-1. The transduction efficiency of the CD34+ cells from all the infants was also comparable. MJT-transduced CD34+ cells from an HIV-1-infected infant were maintained in a liquid culture system for 4 weeks, and the progeny macrophage cells were challenged with the monocyte-tropic laboratory strain, HIV-Bal, or the HIV-1 isolate from the infant's mother. Significant inhibition of virus replication was observed in ribozyme-transduced cells. Thus, we have demonstrated efficient and stable gene transfer into progenitor cells from the cord blood of HIV-1-exposed or -infected infants and shown that protection from HIV-1 infection was conferred to the progeny cells produced by CD34+ cells transduced with the anti-HIV ribozyme gene construct.

Antigens, CD34↗
PubMed1998

Genetic variation in a human immunodeficiency virus type 2 live-virus Macaca nemestrina vaccine model.

Four pigtailed macaques were inoculated with an infectious, apathogenic human immunodeficiency virus type 2 (HIV-2) molecular clone (HIV-2KR) and subsequently challenged with a highly pathogenic strain, HIV-2287, together with two naive control animals. After challenge, two animals inoculated with a high dose of the immunizing strain were protected from CD4 decline and immunodeficiency. To examine the role of genetic heterogeneity in protection, fragments of the env gene were amplified from peripheral blood mononuclear cell DNA and plasma RNA of challenged animals by PCR, examined by using a heteroduplex tracking assay (HTA), and sequenced. By HTA, variation was detected principally within the V1 and V2 regions of envelope. Extent of variation in viral DNA clones as assessed by HTA correlated with inoculum size, as did the degree of variation in sequences of clones derived from viral DNA. Conversely, a rapid reduction in the number of plasma viral RNA variants was noted by HTA at 8 weeks postinfection in protected animals; this reduction was not present in naive or unprotected macaques. Sequences derived from plasma viral RNA were found to be more closely related than corresponding viral DNA sequences, and protection correlated with a significant reduction in variation in plasma RNA sequences in animals given the identical inocula of HIV-2287. Nonsynonymous mutations were significantly less prevalent in the protected animals. An additional potential glycosylation site was predicted to be present in the V2 region in all but one clone, and amino acid signatures related to protection were identified in viral DNA and RNA clones within both the V1 and V2 regions. Examination of the role of viral variation in this HIV-2 live-virus vaccine model may provide valuable insights into immunopathogenesis.

Amino Acid Sequence↗
PubMed1998

Identification of a human immunodeficiency virus type 2 (HIV-2) encapsidation determinant and transduction of nondividing human cells by HIV-2-based lentivirus vectors.

Although previous lentivirus vector systems have used human immunodeficiency virus type 1 (HIV-1), HIV-2 is less pathogenic in humans and is amenable to pathogenicity testing in a primate model. In this study, an HIV-2 molecular clone that is infectious but apathogenic in macaques was used to first define cis-acting regions that can be deleted to prevent HIV-2 genomic encapsidation and replication without inhibiting viral gene expression. Lentivirus encapsidation determinants are complex and incompletely defined; for HIV-2, some deletions between the major 5' splice donor and the gag open reading frame have been shown to minimally affect encapsidation and replication. We find that a larger deletion (61 to 75 nucleotides) abrogates encapsidation and replication but does not diminish mRNA expression. This deletion was incorporated into a replication-defective, envelope-pseudotyped, three-plasmid HIV-2 lentivirus vector system that supplies HIV-2 Gag/Pol and accessory proteins in trans from an HIV-2 packaging plasmid. The HIV-2 vectors efficiently transduced marker genes into human T and monocytoid cell lines and, in contrast to a murine leukemia virus-based vector, into growth-arrested HeLa cells and terminally differentiated human macrophages and NTN2 neurons. Vector DNA could be detected in HIV-2 vector-transduced nondividing CD34(+) CD38(-) human hematopoietic progenitor cells but not in those cells transduced with murine vectors. However, stable integration and expression of the reporter gene could not be detected in these hematopoietic progenitors, leaving open the question of the accessibility of these cells to stable lentivirus transduction.

Antigens, CD34↗
PubMed1997

Intracellular immunization of rhesus CD34+ hematopoietic progenitor cells with a hairpin ribozyme protects T cells and macrophages from simian immunodeficiency virus infection.

Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Gene therapy targeting peripheral blood CD34+ hematopoietic stem cells of HIV-infected individuals.

Gene therapy is a promising treatment modality for acquired immunodeficiency syndrome (AIDS). Autologous transplantation with genetically altered pluripotent hematopoietic stem cells encoding anti-human immunodeficiency virus (HIV) genes could in theory completely and permanently reconstitute all blood lineages and immune functions with cells resistant to HIV. Recent studies showed that CD34+ stem cell can be mobilized in HIV-infected individuals after granulocyte colony-stimulating factor (G-CSF) administration without major side effects or increase of viral load. In this study, peripheral blood CD34+ cells of five HIV-infected individuals were mobilized with G-CSF and after leukapheresis and enrichment, subjected to retroviral transduction with genes encoding anti-HIV ribozyme-decoy fusion molecules. These cells were tested for the ability to give rise to progeny cells, for retroviral transduction efficiency, and for expression of the transgene. CD34+-derived macrophage-like cells were also challenged with HIV. Results showed that CD34+ cells from HIV-infected individuals gave rise to similar numbers of progeny colonies as cells from healthy donors. The transduction efficiency of these cells varied from 68.8 to 100% as assessed by DNA polymerase chain reaction (PCR) of the transgene in individual colonies. CD34+-derived macrophages expressed anti-HIV genes and displayed a substantial and sustained inhibition of HIV replication as compared to untransduced cells. Furthermore, we showed that after thawing, cryopreserved CD34+ cells from these individuals have survival, proliferation, and transduction parameters comparable to fresh cells. Thus, CD34+ cells from HIV-infected patients can be stored for further genetic manipulations with improved vectors or anti-HIV genes as they become available.

Animals↗
PubMed1997

Functional interaction of the HTLV-1 transactivator Tax with activating transcription factor-4 (ATF4).

The Tax protein of the Human T-cell Leukemia Virus (HTLV) activates the expression of viral mRNA through a three 21 bp repeat enhancer located within the HTLV-1 LTR. Since Tax does not bind to the 21 bp DNA repeats directly, it has been speculated that Tax interacts with cellular protein(s) which mediate binding to the enhancer. We employed the yeast two hybrid system to identify host proteins that are potentially relevant to Tax transactivation. We identified a Tax binding protein encoded from a cDNA expression library derived from peripheral blood lymphocytes. The corresponding cDNA has sequence identity with a known transcription factor, activating factor-4 (ATF-4). ATF-4 also binds to GST-Tax fusion protein in vitro. Tax mutants that did not transactivate the HTLV-1 LTR also failed to bind ATF-4. The critical domain for Tax binding resides in a 85 amino acid stretch in the C-terminus of ATF-4, which contains the basic domain and leucine zipper. We further demonstrated that both full length and N-terminal truncated ATF-4 were able to enhance Tax transactivation. Thus, ATF-4 may act as an adapter between Tax and the TRE (Tax responsive element), and play an important role in Tax-mediated transactivation.

Activating Transcription Factor 4↗
PubMed1997

A cellular cofactor for the constitutive transport element of type D retrovirus.

A human nuclear protein that specifically interacts with the constitutive transport element (CTE) of simian retrovirus was identified as adenosine 5'-triphosphate-dependent RNA helicase A. This protein could bind to functional CTE but not to inactive CTE mutants. The interaction of helicase A with CTE was distinct from previously described helicase activity of this protein. Helicase A shuttled from the nucleus to the cytoplasm in the presence of a transcription inhibitor or in cells transiently overexpressing CTE-containing RNA. In vivo colocalization of helicase A and CTE was observed in experiments that combined in situ hybridization and immunostaining. These results suggest that helicase A plays a role in the nuclear export of CTE-containing RNA.

Adenosine Triphosphate↗
PubMed1997

A new reporter cell line to monitor HIV infection and drug susceptibility in vitro.

Determination of HIV infectivity in vitro and its inhibition by antiretroviral drugs by monitoring reduction of production of p24 antigen is expensive and time consuming. Such assays also do not allow accurate quantitation of the number of infected cells over time. To develop a simple, rapid, and direct method for monitoring HIV infection, we generated a stable T-cell line (CEM) containing a plasmid encoding the green fluorescent protein (humanized S65T GFP) driven by the HIV-1 long terminal repeat. Clones were selected that displayed low constitutive background fluorescence, but a high level of GFP expression upon infection with HIV. HIV-1 infection induced a 100- to 1,000-fold increase in relative fluorescence of cells over 2 to 4 days as monitored by fluorescence microscopy, cytofluorimetry, and flow cytometry. Addition of inhibitors of reverse transcriptase, protease, and other targets at different multiplicities of infection permitted the accurate determination of drug susceptibility. This technique also permitted quantitation of infectivity of viral preparations by assessment of number of cells infected in the first round of infection. In conclusion, the CEM-GFP reporter cell line provides a simple, rapid, and direct method for monitoring HIV infectivity titers and antiretroviral drug susceptibility of syncytium-inducing strains.

Anti-HIV Agents↗
PubMed1997

Severe combined immunodeficiency mice engrafted with human T cells, B cells, and myeloid cells after transplantation with human fetal bone marrow or liver cells and implanted with human fetal thymus: a model for studying human gene therapy.

To develop an in vivo model wherein human hematopoiesis occurs, we transplanted severe combined immunodeficiency (SCID) mice with either human fetal bone marrow (HFBM) or human fetal liver (HFL). After transplantation of SCID mice with cultured HFBM (BM-SCID-hu mice) or HFL cells (Liv-SCID-hu mice), significant engraftment of the mouse bone marrow (BM) and population of the peripheral blood with human leukocytes was detected. Human colony-forming unit-granulocyte macrophage and burst forming unit-erythroid were detected in the BM of the BM-SCID-hu and Liv-SCID-hu mice up to 8 months after transplantation. When the HFBM or HFL cells were transduced with a retroviral vector before transplantation, integrated retroviral sequences were detected in human precursor cells present in the SCID mouse BM and in leukocytes circulating in the peripheral blood (PB) up to 7 months after transplantation. The PB of the BM-SCID-hu mice also became populated with human T cells after implantation with human thymic tissue, which provided a human microenvironment wherein human pre-T cells from the BM could mature. When the HFBM was retrovirally transduced before transplantation, integrated retrovirus was detected in sorted CD4+CD8+ double positive and CD4+ single positive cells from the thymic implant and CD4+ cells from the PB. Taken together, these data indicated that the BM of our BM-SCID-hu and Liv-SCID-hu mice became engrafted with retrovirally transduced human hematopoietic precursors that undergo the normal human hematopoietic program and populate the mouse PB with human cells containing integrated retroviral sequences. In addition to being a model for studying in vivo human hematopoiesis, these mice should also prove to be a useful model for investigating in vivo gene therapy using human stem/precursor cells.

Animals↗
PubMed1997

Identification and purification of cellular proteins that specifically interact with the RNA constitutive transport elements from retrovirus D.

Human immunodeficiency virus (HIV) encodes a transacting protein, Rev, which interacts with an RNA element (RRE) to mediate nuclear export of unspliced viral mRNA. Recently, the RNA constitutive transport elements (CTE) from Mason-Pfizer monkey virus (MPMV) and simian retrovirus type I (SRV-1) were shown to render Rev-independent expression of gag, pol, or env genes in subgenomic constructs of HIV-1 and to support replication of HIV-1 mutants lacking RRE and Rev. Since CTEs act in cis, in the absence of any viral regulatory proteins, it is widely believed that they interact directly with the cellular export machinery by means of RNA-protein interaction. In this report, Electrophoretic mobility shift and UV-crosslinking assays were carried out to identify nuclear proteins that interact specifically with CTEs from both MPMV and SRV-1. Two of the four proteins (65 and 40 kDa, respectively) that bound CTE RNA did not interact in the same assays with RNA of a nonfunctional CTE mutant generated by site-directed mutagenesis. Both proteins have been partially purified. The nature of these proteins and their roles in RNA intracellular trafficking are currently under investigation.

Animals↗
PubMed1997

Multigene antiviral vectors inhibit diverse human immunodeficiency virus type 1 clades.

The chronicity of infection by the human immunodeficiency virus (HIV) calls for therapeutic regimens that offer sustained antiviral effects, such as gene therapy. Recent studies have demonstrated that expression of HIV mutant transdominant proteins, RNA decoys, and ribozymes efficiently inhibited HIV replication. We have previously shown that an RNA decoy (stem-loop II of the Rev response element of HIV type 1 [HIV-1], named SL2) and a ribozyme (Rz) targeting the U5 region of the HIV-1 5' long terminal repeat (LTR), combined in a fusion molecule, was more efficient in inhibiting HIV-1 replication than the ribozyme or the decoy alone. In this study, we expressed this fusion molecule in a retrovirus-based double-copy vector to obtain higher expression of this molecule. Furthermore, we inserted a sequence internally to drive expression of another fusion molecule with a ribozyme targeting the env/rev region linked to SL2 to obtain a triple-copy vector. These multigene antiviral vectors were subsequently transduced or transfected into human CD4+ T cells (Molt-4). Results showed that the translocation of the SL2-Rz cassette from the 3' to the 5' LTR occurred in 80% of the transduced cells. The numbers of ribozyme RNA transcripts, estimated by competitive-quantitative reverse transcription (RT)-PCR, were 1.2 x 10(5), 1.2 x 10(4), and 1.5 x 10(3) copies per cell for the triple-copy, double-copy, and single-copy vectors, respectively. Cell challenge with multiple subtypes of HIV-1 (clades A to E) showed commensurate levels of virus inhibition for the three vectors. This study suggests that the combination of multiple anti-HIV genes, such as ribozymes and decoys, targeting multiple sites of HIV RNA and expressed at high levels are promising for the treatment of HIV-1 infection.

Animals↗