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Biomedical subjects

E Gilboa

Publications and source records attributed to E Gilboa.

At least 73 records · Page 4Linked to original sources
PubMed1992

Comparison of the expression of a mutant dihydrofolate reductase under control of different internal promoters in retroviral vectors.

To determine the effect of different promoters on the expression of an altered dihydrofolate reductase (DHFR) gene conferring methotrexate (MTX) resistance in different cell types, double-copy retroviral vectors were constructed carrying a murine mutant DHFR under the control of five different promoters, i.e., human adenosine deaminase (ADA), simian virus 40 (SV40), thymidine kinase (TK), human beta-actin, and cytomegalovirus (CMV). Their expression was compared in NIH-3T3 cells, three human leukemia cell lines, and mouse bone marrow. The variant DHFR is readily expressed from these various promoters in retroviral vectors at a selectable level. In 3T3 cells, the DHFR constructs containing the SV40 promoter conferred the highest levels of resistance to MTX. In K562 and Raji cells, the construct with the TK promoter produced the highest level of resistance. However granulocyte-macrophage colony-forming unit (CFU-GM) colonies from mouse marrow were more resistant to MTX when infected with vectors containing the SV40 promoter and ADA promoter as compared to the other promoter constructs. These studies show that mouse fibroblast cell lines such as NIH-3T3 do not predict the effectiveness of retroviral-mediated gene transfer for marrow progenitor cells, and that the activity of retroviral vector-encoded promoters vary in an unpredictable manner from cell type to cell type. Possible implications for basic gene transfer studies and clinical applications are discussed.

3T3 Cells↗
PubMed1992

Overexpression of RRE-derived sequences inhibits HIV-1 replication in CEM cells.

Overexpression of sequences corresponding to the major Rev-binding site in the Rev response element of human immunodeficiency virus type 1 (HIV-1) (RRE decoys) was used to render cells resistant to HIV-1 replication. This was accomplished by the use of a chimeric tRNA-RRE transcription unit in a double-copy murine retroviral vector to express high levels of HIV-1 RRE-containing transcripts in CEM SS cells. Replication of HIV-1 was inhibited more than 90% in cells expressing chimeric tRNA-RRE transcripts, as determined by in situ immunofluorescence analysis and a p24 antigen ELISA test. Analysis of RNA from HIV-1-infected cells suggests that expression of RRE-containing sequences in CEM SS cells inhibits HIV-1 replication by interfering with Rev function, presumably by competing for Rev binding to its physiological target. The use of a subfragment of RRE as decoy RNA reduces the likelihood that essential cellular factors will be sequestered in cells expressing the decoy RNA. Thus, use of RRE-based decoy RNA to inhibit HIV-1 replication may represent a safer alternative to the use of TAR decoy RNA.

Base Sequence↗
PubMed1991

A frameshift mutation at the NH2 terminus of the nucleoprotein gene does not affect generation of cytotoxic T lymphocyte epitopes.

BALB/3T3 cells infected with a retroviral vector encoding the influenza virus nucleoprotein (NP) gene are efficiently lysed by CTL generated in BALB/c mice (H-2d background). Cells transduced with a mutant form of NP which contains a frameshift mutation at its NH2 terminus (NPm) do not express biochemically detectable levels of protein but nevertheless present Ag to CTL with high efficiency. Cold target inhibition studies indicate that the same CTL epitope(s) are recognized in cells harboring NP or NPm. L929 cells transduced with the NPm gene also present Ag efficiently to CTL raised in C3H mice (H-2k background). Cells engineered to express 5- to 15-fold lower levels of wild-type NP were not capable of presenting Ag to CTL, arguing against the notion that CTL are able to lyse cells expressing very low levels of Ag which might have resulted from suppression of the frameshift mutation in NPm. Implications to the mechanism of epitope generation in class I MHC-restricted immune responses are considered.

Animals↗
PubMed1991

An in vivo model of somatic cell gene therapy for human severe combined immunodeficiency.

Deficiency of adenosine deaminase (ADA) results in severe combined immunodeficiency (SCID), a candidate genetic disorder for somatic cell gene therapy. Peripheral blood lymphocytes from patients affected by ADA- SCID were transduced with a retroviral vector for human ADA and injected into immunodeficient mice. Long-term survival of vector-transduced human cells was demonstrated in recipient animals. Expression of vector-derived ADA restored immune functions, as indicated by the presence in reconstituted animals of human immunoglobulin and antigen-specific T cells. Retroviral vector gene transfer, therefore, is necessary and sufficient for development of specific immune functions in vivo and has therapeutic potential to correct this lethal immunodeficiency.

Adenosine Deaminase↗
PubMed1991

Expression of functional epidermal growth factor receptors in a human hematopoietic cell line.

To test the feasibility of using the human epidermal growth factor receptor (EGFR) as a model for growth factor receptor action in human hematopoietic cells, we infected Burkitt lymphoma cells (Namalwa) with a recombinant amphotrophic retrovirus containing a thymidine kinase promoter-driven human EGFR complementary DNA and the neomycin resistance gene. Neomycin-resistant cells expressing surface EGFR were selected by cell sorting using anti-EGFR monoclonal antibody 225. The selected cells expressed a Mr 170,000 protein immunoprecipitated by monoclonal antibody 225 and apparently identical to EGFR from A431 carcinoma cells. Infected Namalwa cells expressed 42,000 epidermal growth factor (EGF) binding sites/cell, and Scatchard analysis showed two affinities (Kd approximately 5 nM and approximately 0.5 nM). EGFR autophosphorylation was detected using antiphosphotyrosine antibodies after 5 min exposure to EGF. EGF binding induced rapid EGFR internalization (t1/2 = 9 min) and mobilization of transferrin receptors to the cell surface within 1 min. In fetal bovine serum-containing and serum-free cultures, EGF did not stimulate Namalwa cell proliferation. However, in the presence of 1.25% dimethyl sulfoxide (DMSO), EGF caused a dose-dependent increase in DNA synthesis. DMSO induced a cell cycle block in G1, which was partially reversed by EGF. DMSO induced changes in some B-cell markers suggesting cellular differentiation and increased surface EGF receptor number. Cells grown in DMSO and EGF were established as an EGF-dependent cell line for greater than 12 weeks, whereas cells grown in DMSO without EGF died within 1-2 weeks. Namalwa cells expressing EGFR demonstrated more rapid tumor growth in athymic mice. These studies demonstrate expression of functional EGFR mediating early biochemical and growth responses in a human hematopoietic cell, and indicate that EGFR can be used as an effective model in human hematopoietic cells. Results using DMSO are consistent with studies in other human leukemia cells indicating that agents inducing differentiation can restore growth factor dependence in previously factor-independent leukemia cells.

Animals↗
PubMed1991

Analysis of trans-acting response decoy RNA-mediated inhibition of human immunodeficiency virus type 1 transactivation.

Overexpression of trans-acting response element (TAR)-containing sequences (TAR decoys) in CEM SS cells renders cells resistant to human immunodeficiency type 1 (HIV-1) replication. Mutagenesis of TAR was used to investigate the molecular mechanism underlying the observed inhibition. A nucleotide change which disrupts the stem structure of TAR or sequence alterations in the loop abolish the ability of the corresponding TAR decoy RNAs to inhibit HIV replication. A compensatory mutation which restores the stem structure also restores TAR decoy RNA function. Synthesis of viral RNA is drastically reduced in cells expressing a functional TAR decoy RNA, but it is unaffected in cells expressing a mutant form of TAR decoy RNA. It is therefore concluded that overexpression of TAR-containing sequences in CEM SS cells interferes with the process of Tat-mediated transactivation of viral gene expression. However, the phenotype of several mutations suggests that TAR decoy RNA does not inhibit HIV-1 gene expression by simply sequestering Tat but rather does so by sequestering a transactivation protein complex, implying that transactivation requires the cooperative binding of both Tat and a loop-binding cellular factor(s) to TAR. Expression of wild-type or mutant forms of TAR had no discernible effects on cell viability, thus reducing concerns about using TAR decoy RNAs as part of an intracellular immunization protocol for the treatment of AIDS.

Base Sequence↗
PubMed1990

Retroviral vector-mediated gamma-interferon gene transfer into tumor cells generates potent and long lasting antitumor immunity.

Retroviral vectors were used to introduce the gamma-interferon (IFN-gamma) gene into CMS-5 cells, a weakly immunogenic tumor of BALB/c origin. After selection in G418-containing medium, colonies were isolated, cloned, and expanded to cell lines. IFN-gamma secretion was assessed using a bioassay and enzyme-linked immunosorbent assay, and high (25 units/ml) and low (5 units/ml) IFN-gamma producers were isolated. Tumor growth was followed after intradermal injection, and spleen cells were isolated at different time points. IFN-gamma secretion by tumor cells abrogated their tumorigenicity and induced a persistent and specific antitumor immunity. In contrast to the normally observed cellular immunosuppression in unmodified CMS-5 tumor-bearing mice, IFN-gamma-producing tumors induced a long lasting state of T-cell immunity, as judged by rejection of a CMS-5 tumor challenge and persistence of specific cytotoxic activity in the spleen cell population. High levels of tumor-specific cytotoxic activity could also be detected if IFN-gamma-secreting tumor cells, but not unmodified CMS-5 cells, were used as targets at a time point when immunosuppression was usually seen. These studies highlight the potential advantages of localized IFN-gamma secretion to induce potent antitumor immune responses.

Animals↗
PubMed1990

Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication.

Overexpression of TAR-containing sequences (TAR decoys) was used to render cells resistant to HIV replication. A chimeric tRNA(meti)-TAR transcription unit contained in a double copy murine retroviral vector was used to express high levels of HIV-1 TAR-containing transcripts in CEM SS cells. Replication of HIV-1 was inhibited over 99% in cells expressing chimeric tRNA-TAR transcripts, but an amphotropic murine retrovirus replicated normally in these cells. Expression of TAR sequences in CEM SS cells had no adverse effects on cell viability, indicating that essential cellular factors are not being sequestered in these cells. TAR decoy RNA-mediated HIV inhibition may also be effective against natural HIV isolates in spite of their hypervariable nature, as suggested by the fact that replication of SIVmac was also inhibited in cells expressing HIV-1 TAR decoys.

Base Sequence↗
PubMed1990

Interleukin 2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity.

To study the effects of localized secretion of cytokines on tumor progression, the gene for human interleukin 2 (IL-2) was introduced via retroviral vectors into CMS-5 cells, a weakly immunogenic mouse fibrosarcoma cell line of BALB/c origin. Secretion of low levels of IL-2 from the tumor cells abrogated their tumorigenicity and induced a long-lasting protective immune response against a challenge with a tumorigenic dose of parental CMS-5 cells. Co-injection of IL-2-producing CMS-5 cells with unmodified tumor cells inhibited tumor formation even when highly tumorigenic doses of CMS-5 cells were used. Cytolytic activity in mice injected with parental CMS-5 cells was transient and was greatly diminished 3 wk after injection, as commonly observed in tumor-bearing animals. However, in mice injected with IL-2-producing cells, tumor-specific cytolytic activity persisted at high levels for the duration of the observation period (at least 75 d). High levels of tumor-specific cytolytic activity could also be detected in parental CMS-5 tumor-bearing animals 18 d after inoculation with tumor cells, if IL-2-producing CMS-5 cells but not unmodified parental tumor cells were used as targets. These studies highlight the potential advantages of localized secretion of cytokines mediated via gene transfer to induce potent anti-tumor immune responses.

Animals↗
PubMed1990

Restoration of normal lysosomal function in mucopolysaccharidosis type VII cells by retroviral vector-mediated gene transfer.

Retroviral vectors were constructed containing a rat beta-glucuronidase cDNA driven by heterologous promoters. Vector-mediated gene transfer into human and canine beta-glucuronidase-deficient mucopolysaccharidosis type VII fibroblasts completely corrected the deficiency in beta-glucuronidase enzymatic activity. In primary cultures of canine mucopolysaccharidosis type VII retinal pigment epithelial cells, which contain large amounts of undegraded glycosaminoglycan substrates, vector correction restored normal processing of specific glycosaminoglycans in the lysosomal compartment. In canine mucopolysaccharidosis type VII bone marrow cells, beta-glucuronidase was expressed at high levels in transduced cells. Thus, the vector-encoded beta-glucuronidase was expressed at therapeutic levels in the appropriate organelle and corrected the metabolic defect in cells exhibiting the characteristic pathology of this lysosomal storage disorder.

Animals↗
PubMed1990

Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.

NIH 3T3 cells infected with Moloney murine leukemia virus (MoMLV) express high levels of virus-specific RNA. To inhibit replication of the virus, we stably introduced chimeric tRNA genes encoding antisense templates into NIH 3T3 cells via a retroviral vector. Efficient expression of hybrid tRNA-MoMLV antisense transcripts and inhibition of MoMLV replication were dependent on the use of a particular type of retroviral vector, the double-copy vector, in which the chimeric tRNA gene was inserted in the 3' long terminal repeat. MoMLV replication was inhibited up to 97% in cells expressing antisense RNA corresponding to the gag gene and less than twofold in cells expressing antisense RNA corresponding to the pol gene. RNA and protein analyses suggest that inhibition was exerted at the level of translation. These results suggest that RNA polymerase III-based antisense inhibition systems can be used to inhibit highly expressed viral genes and render cells resistant to viral replication via intracellular immunization strategies.

Animals↗
PubMed1989

Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector.

This study describes a type of retroviral vector called double-copy (DC) vector that was designed to improve the expression of transduced genes. The unique feature of DC vectors is that the transduced gene is inserted within the U3 region of the 3' long terminal repeat (LTR). Consequently, in the infected cell the gene is duplicated and transferred to the 5' LTR. The important result is that in its new position the gene is placed outside the retroviral transcriptional unit, eliminating or at least reducing the negative effects of the retroviral transcriptional unit. The utility of the DC vector design was tested by using a 2.1-kilobase-pair (kbp)-long adenosine deaminase (ADA; EC 3.5.4.4) minigene that was inserted into the 3' LTR of the N2 retroviral vector, generating a 2.7-kbp-long chimeric LTR. DNA blot analysis was used to show that the chimeric LTR was faithfully duplicated in cells infected with the corresponding virus, generating two copies of the ADA minigene, one copy in each LTR. Insertion of the ADA minigene into the 3' LTR of the N2 vector led to a 10- to 20-fold increase in ADA transcripts and human ADA isozyme synthesized in NIH 3T3 cells as compared to cells harboring the same vector in which the ADA minigene was inserted between the two LTRs. A similar increase in ADA expression was observed in two human lymphoid cell lines tested, HUT 78 and Raji. These results are consistent with previous observations that upstream promoters exert an inhibitory effect on promoters placed downstream and bear out the predictions used in the design of DC vectors. The use of DC vectors may contribute to the solution of the problems encountered in expressing retrovirally transduced genes in cultured cells and, in particular, when introduced into the live animal.

Adenosine Deaminase↗
PubMed1989

Retroviral vector-mediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADA-deficient marrow cells.

Two recombinant retroviral vectors encoding the cDNA of the human adenosine deaminase (ADA; EC 3.5.4.4) gene and the bacterial neomycin resistance (Neo) gene have been used to transduce bone marrow cells obtained from four patients affected by the ADA-deficient variant of severe combined immunodeficiency. By utilizing the long-term marrow culture system, freshly isolated bone marrow cells were subjected to multiple infection cycles with cell-free supernatants containing high titers of viral vector and then maintained in long-term marrow culture in the absence of any overt selection pressure. By using this experimental protocol, about 30-40% of the hematopoietic progenitors were productively transduced with the viral vector, as judged by the appearance of G418-resistant colonies derived from granulocyte/macrophage and multipotent hematopoietic progenitor cells. The vector-encoded human ADA gene was expressed efficiently in both the myeloid and lymphoid progeny of the cultured bone marrow cells, reaching levels between 15% and 100% as compared to the levels of ADA in normal bone marrow cells. The efficiency of gene transfer and ADA production was proportional to the number of infection cycles. Furthermore, transduction of the ADA vectors into the bone marrow cells derived from an ADA-deficient patient restored the capacity of the cells to respond to phytohemagglutinin and interleukin 2.

Adenosine Deaminase↗
PubMed1989

Inhibition of human T-cell leukemia virus type I replication in primary human T cells that express antisense RNA.

The human T-cell leukemia virus type I is associated with adult T-cell leukemia-lymphoma in humans, a disease which is induced by a malignant transformation of T lymphocytes. Retrovirus vectors carrying human T-cell leukemia virus type I-derived sequences in reversed transcriptional orientation were used to express antisense RNA transcripts in primary human leukocytes. Human T-cell leukemia virus type I replication and virus-mediated immortalization were inhibited in cells harboring antisense constructs. This study suggests that retrovirus-mediated antisense RNA inhibition can be used to protect primary human T-lymphocytes from human T-cell leukemia virus type I-mediated cell transformation.

Cell Division↗
PubMed1989

In utero gene transfer and expression: a sheep transplantation model.

Retroviral-mediated gene transfer was used to insert a Neo R gene into fetal sheep hematopoietic cells obtained by exchange transfusion from lambs in utero. After gene transfer the cells were returned to the donor fetus. The lambs were examined after birth for the presence of a functioning Neo R gene. Of ten analyzable animals, six were positive for G418 resistant progenitor cells (CFU-Mix, CFU-C, BFU-E, CFU-E). Two animals were studied for extended periods of time: 8 and 24 months. Each has demonstrated a pattern wherein positive periods are interspersed with times when there were no detectable G418-resistant cells. We conclude that retroviral-mediated gene transfer can be used to insert genes into early progenitor cells of fetal sheep in utero and that the animals can continue to demonstrate blood cells expressing the gene for more than 2 years after birth. This is a US government work. There are no restrictions on its use.

Adenosine Deaminase↗