Characterization and quality control of autologous gene transfer therapeutics.
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Animals↗
Biomedical subjects
D J Jolly
Publications and source records attributed to D J Jolly.
Cross-reactive lysis of human targets infected with prototypic and clinical human immunodeficiency virus type 1 (HIV-1) strains by murine anti-HIV-1 IIIB env-specific cytotoxic T lymphocytes.
To evaluate the ability of murine anti-human immunodeficiency virus type 1 (HIV-1) IIIB env cytotoxic T lymphocytes (CTL) to recognize and lyse HIV-1-infected cells, we have constructed a human cell line (Hu/Dd) expressing both the CD4 receptor and the murine H-2Dd major histocompatibility complex (MHC) class I protein. This cell line can be productively infected with HIV-1 and can also function as a target for murine CD8+, class I MHC-restricted CTL directed against the envelope glycoprotein of HIV-1 IIIB. The ability of BALB/c anti-HIV-1 IIIB env CTL to specifically recognize and lyse Hu/Dd target cells infected with divergent HIV-1 strains was tested by using both prototypic and clinical HIV-1 strains. CTL generated by immunization of mice with syngeneic cells expressing either the native or V3 loop-deleted (delta V3) envelope glycoprotein from HIV-1 IIIB were able to recognize and specifically lyse Hu/Dd target cells infected with the HIV-1 prototypic isolates IIIB, MN, WMJ II, SF2, and CC as well as several HIV-1 clinical isolates. These results demonstrate that CTL determinants for HIV-1 env exist outside the hypervariable V3 region, anti-HIV-1 IIIB env CTL appear to recognize common determinants on diverse HIV-1 strains, and classification of HIV-1 strains based on neutralizing antibody reactivities does not appear to correspond to CTL recognition and lysis. The results suggest that the cell-mediated components of the immune system may have a broader recognition of divergent HIV-1 strains than do the humoral components.
Animals↗
Induction of HIV-specific CTL and antibody responses in mice using retroviral vector-transduced cells.
Recombinant retroviral vectors can efficiently transduce and express foreign genes in mammalian cells. We have examined the utility of retroviral vector-mediated gene transfer to deliver genes which encode human immunodeficiency virus type I (HIV) antigens capable of stimulating specific immune responses. Murine fibroblast cell lines were transduced with a nonreplicating murine retroviral vector carrying the gene encoding the HIV-IIIB envelope protein and were shown to express the gp160/120 protein. Mice immunized with syngeneic vector-transduced cells developed CD8+, class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) specific for targets expressing the HIV envelope protein. The CTL also exhibited lytic activity on target cells coated with synthetic peptides derived from the gp120 V3 hypervariable region of both the HIV-IIIB and HIV(MN) isolates. Following adoptive transfer in a murine tumor model, these CTL were shown to be effective in vivo by their ability to eliminate established tumor cells expressing the HIV protein. Vector-transduced syngeneic cells were also capable of eliciting HIV envelope-specific antibody responses in immunized mice. Sera obtained from these mice were found to bind to the HIV-IIIB gp160 protein as well as a peptide-defined neutralizing antibody epitope contained within the V3 domain of gp120. These sera exhibited virus-neutralizing activity in that they markedly reduced the ability of HIV to infect and form syncytia of a human T-cell line. This is the first demonstration that cells transduced with a retroviral vector encoding the HIV-IIIB envelope protein are capable of inducing effective HIV-specific cellular and humoral immune responses in mice.
Amino Acid Sequence↗
HIV infection and gene transfer therapy.
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Animals↗
Induction of anti-HIV-1 immune responses by retroviral vectors.
Retroviral vectors encoding HIV-1 proteins, in particular, the envelope from HIV-1 IIIB, have been constructed and used to generate infectious vector particles. Murine cells transduced with these vectors express HIV proteins. Vector-transduced cells, when injected into syngeneic BALB/c mice, induce potent CD8+, class I MHC-restricted cytotoxic T-lymphocyte responses and elicit the production of neutralizing antibody specific for HIV-1. The induction of similar responses in primates may provide the basis for considering the use of these vectors as immunostimulants in humans. The retroviral vectors or vector-transduced cells would probably be first employed as an immunotherapeutic for HIV-infected individuals.
Animals↗
Retroviral vectors as vaccines and immunotherapeutics.
Retroviral vectors have been tested in mice for their ability to induce immune response to the proteins which their genomes encode. Using HIV encoded proteins as a model, potent cytotoxic T lymphocyte (CTL) and antibody responses are seen to these proteins. This apparent efficacy of induction may be due to the relative simplicity of the vector and the manner in which antigens are presented to the immune system. Safety issues and other proposed methods of CTL induction are discussed. The potential application of retroviral vectors as immune stimuli in man seems quite broad (viral diseases and cancer), and the first attempted applications in man are likely to be therapeutic rather than prophylactic.
Genetic Vectors↗
A region in the steroid binding domain determines formation of the non-DNA-binding, 9 S glucocorticoid receptor complex.
This work was initiated to determine if a specific region of the glucocorticoid receptor determines the formation of the inactive (i.e. non-DNA-binding) 9 S form of the receptor recovered in cytosol preparations. It is known that the murine glucocorticoid receptor of the nti phenotype, which consists of only the carboxyl-terminal 40-kDa peptide containing the DNA-binding and steroid-binding domains separated by a short linker region, is recovered in hypotonic lysates as a 9 S heteromeric complex (Gehring, U., and Arndt, H. (1985) FEBS Lett. 179, 138-142). To further localize the domain required for formation of the 9 S complex, we have determined the sedimentation coefficients of receptors produced in COS-7 cells transfected with several mutants of the human glucocorticoid receptor gene. Deletion of the DNA-binding domain results in a 9 S complex that is somewhat less stable than the wild type receptor during sucrose gradient centrifugation. Deletion of the linker region yields a molybdate-stabilized 9 S complex, but deletion of the entire steroid-binding domain or internal deletion of the amino-terminal two-thirds of this domain yields receptors that are constitutive transcriptional activators and are present in cytosol only in the 4 S form. Taken together, these observations demonstrate that the steroid-binding domain contains the features required for formation of the 9 S heteromeric complex, and they are consistent with the proposal that the steroid-binding domain normally represses receptor function.
Amino Acid Sequence↗
Cellular distribution and hormonal regulation of h-SBP in human hepatoma cells.
The cellular distribution of human Sex Steroid Binding Plasma Protein (h-SBP) was studied in human cells and tissues by indirect immunofluorescence. h-SBP was detected in the cytoplasm of hepatocytes, of prostate and epididymis epithelial cells and in endometrium. Sexual and non-sexual skin, intestine epithelium, striated muscle and some rodent organs were not labelled. The intracellular localization of h-SBP indicate that h-SBP could be taken up from the extracellular compartment or synthesized in situ in sex steroid target organs, where it may play a role in hormone uptake. The hormonal regulation of h-SBP secretion by a human hepatoma cell line, H5A, showed that tri-iodothyronine was more potent than estradiol or tamoxifen, which acted as estrogen agonist, in increasing secreted h-SBP and the combined effect of both thyroid and estrogen hormones resulted in an additive stimulation of h-SBP secretion. As shown by Northern blot analysis, oligonucleotides synthesized from the known sequence of h-SBP hybridized with a RNA of approximately 2 kb which was more represented in H5A cells than in normal human liver, and was increased 2-3 times after hormonal stimulation of the cells. The presence of a poly(A+)RNA coding for h-SBP in the human liver indicated the hepatic synthesis of this protein.
Animals↗
High-efficiency gene transfer into cells.
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Adenine Phosphoribosyltransferase↗
Epitope insertion into the human hypoxanthine phosphoribosyltransferase protein and detection of the mutant protein by an anti-peptide antibody.
The translational stop codon TAA of the human hypoxanthine phosphoribosyltransferase (HPRT) cDNA has been changed to GAA by site-specific mutagenesis. This modification extends the open reading frame to a downstream stop codon and results in the addition of a unique negatively charged hexapeptide to the C terminus of human HPRT protein. The mutated cDNA was transferred into HPRT-deficient rodent cells by retroviral vector infection, and the expressed enzyme was found to be fully active. An antibody against a synthetic octapeptide corresponding to the mutated HPRT C terminus precipitated the HPRT protein specifically from cells infected with the mutant virus and not infected with the wild-type HPRT virus. The technique of inserting a novel epitope into a protein by site-directed mutagenesis should be generally applicable in studies of the regulation of gene expression in vitro and in vivo.
Epitopes↗
Gene expression from transcriptionally disabled retroviral vectors.
Retroviral vectors are used for the efficient transfer of foreign genes into mammalian cells. We report here the construction of murine retrovirus-based vectors carrying the full-length cDNA for human hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) and from which the enhancer sequences, the "CAAT box," and the "TATA box" in the long terminal repeats (LTRs) have been deleted. After infection of HPRT-deficient rat cells by the vectors, transcriptional activity from the 5' LTR was undetectable and expression of the HPRT cDNA was dependent on an internal promoter. Removal of the LTR regulatory elements increased HPRT gene expression from an internal promoter, indicating interference between the two sets of transcriptional signals. Such disabled vectors may reduce the likelihood of undesirable genetic changes through insertional mutagenesis in cells infected with retroviral vectors.
Base Sequence↗
The organization of the human HPRT gene.
The organization of the X-linked gene for human hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8.) has been determined by a combination of restriction endonuclease mapping, heteroduplex analysis and DNA sequence analysis of overlapping genomic clones. The entire gene is 42 kilobases in length and split into 9 exons. The sizes of the 7 internal exons and the exon-intron boundaries are identical to those of mouse HPRT gene. The 5' end of the gene lacks the prototypical 5' transcriptional regulatory sequence elements but contains extremely GC-rich sequences and five GC hexanucleotide motifs (5'-GGCGGG-3'). These structural features are very similar to those found in the mouse HPRT gene and to some of the regulatory signals common to a class of constitutively expressed "housekeeping" genes. Several transcriptional start sites have been identified by nuclease protection studies. Extensive sequence homology between the mouse and human genes is found in the 3' non-coding portion of the gene.
Animals↗
Gene expression from a transcriptionally disabled retroviral vector.
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Animals↗
Variable stability of a selectable provirus after retroviral vector gene transfer into human cells.
Human lymphoblasts deficient in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) were infected with an amphotropic helper-free retroviral vector expressing human HPRT cDNA. The stability and expression of the HPRT provirus in five cell lines with different proviral integration sites were examined by determining HPRT mutation and reversion frequencies and by blot hybridization studies. Mutation to the HPRT-negative phenotype occurred at frequencies of approximately 4 X 10(-5) to 3 X 10(-6) per generation. Most mutations in each of the five cell lines were associated with partial or complete deletions or rearrangements of the provirus. Several mutants retained a grossly intact HPRT provirus, and in one such mutant HPRT shutdown resulted from a revertible epigenetic mechanism that was not associated with global changes in proviral methylation. Therefore, mutation and shutdown of the HPRT provirus in human lymphoblasts result from mechanisms similar to those reported for several other avian and mammalian replication-competent retroviruses.
Cell Line↗
The role of the HPRT gene in human disease.
Human HPRT deficiency leads to two major forms of human disease. Partial enzyme deficiency results in gouty arthritis, while an almost complete deficiency leads to the Lesch-Nyhan disease. The latter is characterized by severe neurological dysfunction in addition to gouty arthritis, including retardation, choreoathetosis and aggressive and compulsive self-mutilation. The biochemical basis for the neurological symptoms is not understood. The human and mouse cDNA (RNA copy) genes have been isolated and sequenced. In addition, the amino acid sequence of the human protein has been directly determined. The human and mouse proteins differ at 7 amino acids out of the total, (including the N terminal methionine, which is processed off during maturation) of 218. There are 42 out of 654 nucleotide differences between the human and mouse genes in the amino acid coding region. The mouse genomic structure has been determined. It has 9 exons and 8 introns with a total size of approximately 36 kb. The human gene is very similar with identical intron-exon junction points and approximately the same total gene size. Both mouse and human presumed promotor region at the 5' end, lack a recognizable promotor in the form of a "TATAA" box and are very G-C rich, though not the same. This may be a feature of most "housekeeping" genes. HPRT gene point mutations in three gouty arthritis and one Lesch-Nyhan patient have been identified by peptide sequencing. Six gross gene rearrangements have been identified in Lesch-Nyhan HPRT genes. However it is likely that most mutations are point mutations or small deletions. So far all gene mutations identified are different from all others. The gene has been engineered into retrovirus vehicles which allows its efficient introduction into a wide variety of cells, including mouse marrow stem cells. This may allow treatment of Lesch-Nyhan patients as a model of gene therapy.
Amino Acid Sequence↗
Retroviral vector-mediated gene transfer into human hematopoietic progenitor cells.
The transfer of the human gene for hypoxanthine phosphoribosyltransferase (HPRT) into human bone marrow cells was accomplished by use of a retroviral vector. The cells were infected in vitro with a replication-incompetent murine retroviral vector that carried and expressed a mutant HPRT complementary DNA. The infected cells were superinfected with a helper virus and maintained in long-term culture. The production of progeny HPRT virus by the bone marrow cells was demonstrated with a colony formation assay on cultured HPRT-deficient, ouabain-resistant murine fibroblasts. Hematopoietic progenitor cells able to form colonies of granulocytes or macrophages (or both) in semisolid medium in the presence of colony stimulating factor were present in the nonadherent cell population. Colony forming units cloned in agar and subsequently cultured in liquid medium produced progeny HPRT virus, indicating infection of this class of hematopoietic progenitor cell.
Animals↗
Expression of a retrovirus encoding human HPRT in mice.
Transmissible retroviruses encoding human hypoxanthine phosphoribosyltransferase (HPRT) were used to infect mouse bone marrow cells in vitro, and the infected cells were transplanted into mice. Both active human HPRT-protein and chronic HPRT-virus production were detected in hematopoietic tissue of the mice, showing transfer of the gene. These results indicate the possible use of retroviruses for somatic cell therapy.
Animals↗
Partial phenotypic correction of human Lesch-Nyhan (hypoxanthine-guanine phosphoribosyltransferase-deficient) lymphoblasts with a transmissible retroviral vector.
A human Lesch-Nyhan (hereditary, severe hypoxanthine-guanine phosphoribosyltransferase (HPR transferase) deficiency) B-lymphoblast line was infected with an amphotropic retroviral vector containing human HPR transferase cDNA under transcriptional control of viral long terminal repeat sequences. Of 17 clones isolated, 12 integration groups were defined by analysis of restriction enzyme digests of their genomic DNA with HPR transferase and viral long terminal repeat probes. These groups had HPR transferase activity restored to levels of 4 to 23% of normal values. Aberrant metabolic parameters associated with severe deficiency of HPR transferase activity, i.e. elevated rates of purine excretion, increased accumulation of hypoxanthine, elevated 5-phosphoribosyl-1-pyrophosphate contents, altered nucleoside triphosphate pools, resistance to toxic effects of 6-thioguanine, were partially to nearly completely corrected; the degree of correction generally corresponded to the degree of restoration of HPR transferase activity. The integration of the HPR transferase gene was found to be variably stable during 9 months of culture of the virally transformed lymphoblasts under nonselective conditions. The HPR transferase gene-infected lines reverted to resistance to 20 microM 6-thioguanine, i.e. severe HPR transferase deficiency, at frequencies of 10(-6) to in excess of 10(-5) per generation. The reversions were accompanied by either a loss or rearrangement of the integrated HPR transferase sequences or by retention of the sequences in an unaltered form.
Animals↗