[The liver and gene therapy. Experimental or revolutionary procedure?].
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
J M Heard
Publications and source records attributed to J M Heard.
Disappearance of lysosomal storage in spleen and liver of mucopolysaccharidosis VII mice after transplantation of genetically modified bone marrow cells.
Mice homozygous for the gusmps allele lack beta-glucuronidase activity and provide a useful model for human Mucopolysaccharidosis type VII (MPS VII), also known as Sly syndrome. Bone marrow (BM) transplantation was shown to correct the metabolic defect and to increase the life span of diseased animals. We have used this murine model in a preclinical study aimed at evaluating whether the techniques currently available for gene transfer into large mammalian and human BM cells will provide efficient enzyme replacement therapy in MPS patients. Autologous BM was transplanted into deficient mice after retrovirus-mediated transfer of the human beta-glucuronidase cDNA. Conditioning of recipients was performed by a single sublethal irradiation of 4.5 Gy, giving rise to low donor engraftment. In recipient mice analyzed until 145 days after gene transfer, the percentage of genetically modified hematopoietic cells was less than 5%. Nevertheless, beta-glucuronidase enzyme activity was detectable in various organs, including the brain, and disappearance of lysosomal storage was obvious in the liver and spleen. These results show that the autologous transplantation of genetically engineered BM cells could be beneficial in MPS patients.
Animals↗
Macrophage colony-stimulating factor: serum levels and cDNA structure in malignant osteopetrosis.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Correction of lysosomal storage in the liver and spleen of MPS VII mice by implantation of genetically modified skin fibroblasts.
Genetic defects of lysosomal hydrolases result in severe storage diseases and treatments based on enzyme replacement have been proposed. In mice lacking beta-glucuronidase, which develop a disease homologous to human mucopolysaccharidosis type VII (Sly syndrome), we have used autologous implants of genetically-modified skin fibroblasts for the continuous in vivo production of the enzyme. The human beta-glucuronidase cDNA was introduced with a retroviral vector into mutant mice skin fibroblasts grown in primary culture. Fourteen mutant mice were implanted intraperitoneally with these modified cells embedded into collagen lattices. All animals expressed beta-glucuronidase from the vascularized neo-organs that developed after implantation and accumulated the enzyme in their tissues. A complete disappearance of the lysosomal storage lesions was observed in their liver and spleen.
Animals↗
In situ retrovirus-mediated gene transfer into dog liver.
Dogs were used as a large animal model to assess the feasibility and safety of a surgical method for gene transfer into hepatocytes in vivo. This method, which we previously described in rats, consists of a partial hepatectomy aimed at inducing liver regeneration, followed by the selective in situ perfusion of the remnant liver parenchyma with a retrovirus preparation. Isolation of the liver was obtained by clamping the afferent and efferent blood vessels, a procedure that prevented retroviral vector dissemination and genetic modification of nonhepatic organs. A helper-free retrovirus vector encoding beta-galactosidase targeted to the nucleus was perfused in the liver of 5 golden retriever dogs. Volumes up to 1,650 ml of fresh or concentrated vector stocks were perfused and the procedure was well tolerated. Gene transfer, observed in 3 of 5 treated dogs when documented on liver biopsy fragments obtained at day 4, involved 0.15-0.6% hepatocytes and persisted at equivalent levels at the time of sacrifice, 6 weeks later. No propagation of the vector to other tissues was detected. These observations suggest that the selective perfusion of the regenerating liver might be considered an alternative to liver transplantation for the treatment of certain severe genetic liver disorders, or for the delivery of a therapeutic protein into the serum.
Animals↗
Continuous systemic secretion of a lysosomal enzyme by genetically modified mouse skin fibroblasts.
Lysosomal enzymes secreted or externally supplied into the extracellular medium can be internalized by cells and targeted to lysosomes after binding to specific membrane receptors. This process allows for the replacement of the missing enzyme activity in deficient cells. Using a retroviral vector, we have introduced the human beta-glucuronidase cDNA into primary mouse skin fibroblasts. The genetically modified cells were then engrafted into neo-organs that had been previously implanted into the peritoneal cavity of syngeneic recipient mice. The hypervascularized structures, made of collagen and basic fibroblast growth factor-coated synthetic fibers embedded into extracellular matrix gel, allowed in vivo survival of engrafted fibroblasts that expressed the human beta-glucuronidase cDNA for at least 3 months. The human enzyme was detected in the liver, lung, and spleen of experimental animals, but became undetectable after removal of the neo-organ. This observation indicated that the human enzyme was secreted into the serum and then captured by distant organs. The use of genetically modified fibroblasts implanted into neo-organs may, therefore, represent a convenient approach to enzyme replacement therapy in lysosomal storage diseases.
3T3 Cells↗
Reduced cell surface expression of processed human immunodeficiency virus type 1 envelope glycoprotein in the presence of Nef.
nef genes from two laboratory grown human immunodeficiency virus type 1 (HIV-1) strains and from two proviruses that had not been propagated in vitro were introduced into CD4+ lymphoblastoid CEM cells. The stable expression of all four Nef proteins was associated with an almost complete abrogation of CD4 cell surface localization. The consequences of the presence of Nef on gp160 cleavage, gp120 surface localization, and envelope-induced cytopathic effect were examined in CEM cells in which the HIV-1 env gene was expressed from a vaccinia virus vector. The presence of Nef did not modify the processing of gp160 into its subunits but resulted in a significant decrease of cell surface levels of gp120, associated with a dramatic reduction of the fusion-mediated cell death. Surface levels of mutant envelope glycoproteins unable to bind CD4 were not altered in Nef-expressing cells, suggesting that the phenomenon was CD4 dependent. The intracellular accumulation of fully processed envelope glycoproteins could significantly delay the cytopathic effect associated with envelope surface expression in HIV-infected cells and may be relevant to the selective advantage associated with Nef during the in vivo infectious process.
Acquired Immunodeficiency Syndrome↗
Activation pathways and human immunodeficiency virus type 1 replication are not altered in CD4+ T cells expressing the nef protein.
While recent studies in Rhesus monkeys have pointed out the importance of an intact nef gene for the development of acquired immunodeficiency syndrome (AIDS), no biological function has been so far unambiguously attributed to its product. Since Nef has been described to possess GTP-binding properties and to down-regulate CD4 cell surface expression, we looked for evidences of Nef interfering with the transduction of activating signals in human CD4+ T cells. We used a murine leukemia retroviral vector to express the HIV-1BRU nef gene in two permanent tumoral T-cell lines (CEM and Jurkat) and in two nonimmortalized, interleukin-2 (IL2)-dependent, T-cell clones. The single copy recombinant provirus integrated in the genome of these cells directed the synthesis of a 27-kD protein with a half-life greater than 5 h. The levels of expression of cell surface molecules involved in T-cell functions (CD4, CD3, CD28, CD29, IL-2 receptor) were not modified in cell populations expressing Nef. In immunocompetent T-cell clones, cell proliferation and lymphokine production in response to activating stimuli (IL-2, alloantigens, phorbol esters, or antibodies directed against CD2, CD3, CD4, CD28) remained unmodified. Moreover, the presence of Nef did not change the kinetics of human immunodeficiency virus (HIV) infection.
3T3 Cells↗
Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses.
The envelope glycoproteins (SU) of mammalian type C retroviruses possess an amino-terminal domain of about 200 residues, which is involved in binding a cell surface receptor. In this domain, highly conserved amino acid sequences are interrupted by two segments of variable length and sequence, VRA and VRB. We have studied the role of these variable regions in receptor recognition and binding by constructing chimeric molecules in which portions of the amino-terminal domains from amphotropic (4070A), xenotropic (NZB), and polytropic (MCF 247) murine leukemia virus SU proteins were permuted. These chimeras, which exchanged either one or two variable regions, were expressed at the surface of replication-defective viral particles by a pseudotyping assay. Wild-type or recombinant env genes were transfected into a cell line producing Moloney murine leukemia virus particles devoid of envelope glycoproteins in which a retrovirus vector genome carrying an Escherichia coli lacZ gene was packaged. The host range and sensitivity to interference of pseudotyped virions were assayed, and we observed which permutations resulted in receptor switch or loss of function. Our results indicate that the determinants of receptor choice are found within the just 120 amino acids of SU proteins. Downstream sequences contribute to the stabilization of the receptor-specific structure.
3T3 Cells↗
Recombinant retroviruses as tools for gene transfer to somatic cells.
Explore the source record for details and available documents.
Animals↗
Intra-arterial calcium infusion for hydrofluoric acid burns.
Between March 3, 1987 and Sept. 8, 1989, intra-arterial calcium infusions were used to treat 28 patients (38 extremities) suffering hydrofluoric acid (HF) exposures that failed to respond to topical treatments. Although 18 of the injuries occurred at work, only 1 patient was using HF in a concentration greater than 12%. Many of these products were available over the counter. Most patients did not wear protective equipment and had no concept of the danger of injury from the product. Onset of symptoms (pain, erythema and edema) was delayed from 1 to 6 h after exposure. Arterial catheters were placed and the patient was begun on a protocol using 10 ml of 10% calcium chloride diluted with 40 ml normal saline and infused over a 4-h period. Each patient was allowed a 4-8 h rest period before assessing the need for additional infusions. Blood calcium, magnesium, phosphorus, PT, and PTT were monitored. Infusions were repeated until there was no tenderness. Using tenderness as an end-point increased the number of infusions compared to previous reports. The mean number of infusions was 4.1 (range, 1-10). Success (complete healing) was 100%. There was a significant rise in serum calcium, but not to dangerous levels (range 9.3-12.8). There was also a significant fall in magnesium, which reached levels requiring magnesium intravenously. There were no significant changes in phosphorus, PT, and PTT.
Adolescent↗
Retroviral-mediated gene transfer into hepatocytes in vivo.
Stable gene transfer into hepatocytes might be used to compensate for a genetic deficiency affecting liver function or to deliver diffusible factors into the blood stream. In rats, we have combined retroviral-mediated gene transfer with a surgical procedure in which the liver is temporarily excluded from the circulation and infected in vivo. Partial hepatectomy was performed 24-48 hr before perfusion with virus to induce hepatocyte division and facilitate viral integration. A helper-free recombinant retrovirus coding for beta-galactosidase with nuclear localization was used to score cells that expressed the transgene. For at least 3 months after gene transfer, up to 5% of hepatocytes expressed nuclear beta-galactosidase. Whereas in vitro reimplantation of genetically modified hepatocytes has proved to be inefficient in stably transferring genes into the liver, our approach provides a feasible alternative.
Animals↗
An amino-terminal fragment of the Friend murine leukemia virus envelope glycoprotein binds the ecotropic receptor.
Retrovirus entry into cells is mediated by specific binding of the envelope glycoprotein to a cell membrane receptor. Constitutive envelope gene expression prevents infection by interfering with the binding of viruses which recognize the same receptor. We have used this property to investigate the receptor binding capacities of deleted or truncated murine leukemia virus ecotropic envelope glycoproteins. Friend murine leukemia virus envelope glycoproteins bearing internal amino-terminal deletions, or a soluble 245-amino-acid gp70 amino-terminal fragment, were expressed in NIH 3T3 cells. The susceptibility of these cells to ecotropic and amphotropic virus infection was determined. We observed that both membrane-bound and soluble forms of the gp70 245-amino-acid amino-terminal domain induced resistance to ecotropic virus, indicating that this fragment binds the ecotropic receptor. Binding occurs both at the cell surface and in the endoplasmic reticulum, as shown by the use of soluble envelope fragments either secreted in the culture supernatants or retained in the endoplasmic reticulum lumen by a KDEL sequence. These results suggest that the gp70 amino-terminal domain folds into a structure which recognizes the ecotropic receptor regardless of the carboxy-terminal part of the molecule.
Amino Acid Sequence↗
Gene transfer to somatic tissues using retroviral vectors.
Retroviral vectors have successfully been used for stable gene transfer in a number of in vivo situation. Before this approach can reallistically be applied in the treatment of human genetic diseases, the correction of pathological symptoms have to be documented in an animal model. In this perspective, we have chosen to use a strain of mice carrying the gusmps mutation, where homozygotes are deficient for beta-glucuronidase and develop a mucopolysaccharidosis. Two methods which result in the stable introduction of the human beta-glucuronidase cDNA into skin fibroblasts or hematopoietic stem cells are presented. A third method, whereby genes can be stably transferred to the liver is also discussed.
Animals↗
A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors.
The myeloproliferative leukemia virus (MPLV) is an acute leukemogenic murine replication-defective retrovirus. By sequencing the envelope gene of a biologically active MPLV clone, we found that this region comprises a novel oncogene named v-mpl in phase with two parts of the Friend murine leukemia virus envelope gene. The MPLV env region could encode an env-mpl fusion polypeptide that presents the characteristics of a transmembrane protein. We show that in vitro infection of bone marrow cells with helper-free MPLV readily yields immortalized factor-independent hematopoietic cell lines of different lineages. In mice, the c-mpl proto-oncogene is expressed in hematopoietic tissues as a 3 kb mRNA. Since v-mpl shares strong structural analogies with the hematopoietin receptor superfamily, it is likely that MPLV has transduced a truncated form of an as yet unidentified hematopoietic growth factor receptor.
Amino Acid Sequence↗
Multilineage hematopoietic disorders induced by transplantation of bone marrow cells expressing the v-fms oncogene.
Mouse bone marrow cells infected with a helper-free retrovirus containing v-fms were engrafted into lethally irradiated mice. Dominant provirus-positive clones emerged in the spleens of some recipients within 1 month. When spleen cells were transplanted into lethally irradiated secondary recipients, clonal erythroleukemias or B cell lymphomas expressing the v-fms-coded glycoprotein developed. Other secondary recipients repopulated by "unmarked" progenitor cells or by cryptic provirus-positive precursors present in the spleens of the same donor mice did not develop disease; thus cells expressing v-fms did not invariably have a proliferative advantage after transplantation. Several primary engrafted recipients developed myeloproliferative disorders that were provirus-positive without evidence of clonality. Although expression of the c-fms product (CSF-1 receptor) is normally restricted to cells of the mononuclear phagocyte series, the v-fms-coded glycoprotein can contribute to proliferative abnormalities of multiple hematopoietic lineages.
Animals↗
Characterization of a transcriptional promoter of human papillomavirus 18 and modulation of its expression by simian virus 40 and adenovirus early antigens.
RNA present in cells derived from cervical carcinoma that contained human papillomavirus 18 genomes was initiated in the 1.053-kilobase BamHI fragment that covered the complete noncoding region of this virus. When cloned upstream of the chloramphenicol acetyltransferase gene, this viral fragment directed the expression of the bacterial enzyme only in the sense orientation. Initiation sites were mapped around the ATG of open reading frame E6. This promoter was active in some human and simian cell lines, and its expression was modulated positively by simian virus 40 large T antigen and negatively by adenovirus type 5 E1a antigen.
Acetyltransferases↗
Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.
The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription.
Acetyltransferases↗