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Biomedical subjects

J M Heard

Publications and source records attributed to J M Heard.

At least 55 records · Page 3Linked to original sources
PubMed1996

Retroviral vectors for the transduction of the PML-RARalpha fusion product of acute promyelocytic leukemia.

We have designed several retroviral constructs to transduce the PML-RA Ralpha fusion product of human acute promyelocytic leukemia. Our aim to generate high-titer stable vector-producing cell lines was hindered by a toxic effect of PML-RARalpha expression on packaging cells. To circumvent this, we tested retroviral vectors expressing the transgene from several internal promoters including inducible and myelospecific promoters. To compare efficiency of these constructs in their ability to generate protein expression in the appropriate target cells and optimal viral titers, we used the BOSC23 transient packaging cell line. We found that the direct-oriented vector did not ensure tissue-specificity of PML-RARalpha expression, while the reverse-oriented retroviral vector did. The latter construct, however, failed to generate high-titer recombinant virus. This study exemplifies the unpredictable behavior of retroviral constructs and the superiority of transient systems for transduction of a toxic product.

3T3 Cells↗
PubMed1996

[Gene therapy in lysosomal diseases].

The study of the mechanisms of secretion and recapture of lysosomal enzymes has lead to the proposal of a treatment of lysosomal diseases by enzyme replacement. Autologous implants of genetically modified cells which secrete enzymes ensure systemic distribution of the lacking enzyme. A procedure which permits reimplantation of genetically modified fibroblasts is described. The stable secretion of human glucuronidase by autologous fibroblasts was thus obtained in animal species. This approach should by applicable to the treatment of Hurler's syndrome by obtaining the production and distribution of alpha-L-iduronidase in patients lacking this enzyme by retroviral transfer of the human alpha-L-iduronidase gene to cultured fibroblasts and by preparation of implants.

Animals↗
PubMed1995

Sustained delivery of erythropoietin in mice by genetically modified skin fibroblasts.

We have examined whether the secretion of erythropoietin (Epo) from genetically modified cells could represent an alternative to repeated injections of the recombinant hormone for treating chronic anemias responsive to Epo. Primary mouse skin fibroblasts were transduced with a retroviral vector in which the murine Epo cDNA is expressed under the control of the murine phosphoglycerate kinase promoter. "Neo-organs" containing the genetically modified fibroblasts embedded into collagen lattices were implanted into the peritoneal cavity of mice. Increased hematocrit (> 80%) and elevated serum Epo concentration (ranging from 60 to 408 milliunits/ml) were observed in recipient animals over a 10-month observation period. Hematocrit values measured in recipient mice varied according to the number of implanted Epo-secreting fibroblasts (ranging from 2.5 to 20 x 10(6)). The implantation of neo-organs containing Epo-secreting fibroblasts appeared, therefore, as a convenient method to achieve permanent in vivo delivery of the hormone. We estimated that the biological efficacy of the approach may be relevant for the treatment of human hemoglobinopathies.

Animals↗
PubMed1995

Long-term delivery of a lysosomal enzyme by genetically modified fibroblasts in dogs.

We have evaluated the feasibility and efficacy of intraperitoneal implants (neo-organs) for protein delivery in large animals. Skin biopsies were taken from four healthy dogs. Primary fibroblast cultures were transduced with a retroviral vector coding for the human beta-glucuronidase. One to six lattices each containing 10(9) skin fibroblasts were implanted into the omentum of the donor animal. Laparotomies performed at regular intervals showed vascularized neo-organs without local inflammation. Human beta-glucuronidase levels equivalent to 0.8 to 3.1% of the endogenous canine activity were detected for up to 340 days on liver biopsy samples. These results indicate that neo-organs can be considered for the long-term delivery of therapeutic proteins or enzymes in humans.

Animals↗
PubMed1995

Functional retroviral vector for gene therapy of xeroderma pigmentosum group D patients.

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder characterized by an increased frequency of skin cancer following minimal sunlight exposure. Cells isolated from XP patients are also hypersensitive to UV rays and UV-like chemicals. This sensitivity is directly related to a defect in the early steps of nucleotide excision repair (NER) of damaged DNA. No efficient treatment is available for this disease and skin cancer prevention can only be achieved by strict avoidance of sunlight exposure. Thus, we are developing a model for gene therapy in XP, particularly for patients belonging to group D. We report here the construction of a retroviral vector (LXPDSN) containing the XPD (ERCC2) cDNA, which fully complements the DNA repair deficiency of primary skin fibroblasts. Efficient integration, mRNA synthesis, and protein expression of the XPD gene were obtained in all LXPDSN-transduced XP-D fibroblasts tested. Full correction of the DNA repair defect was observed with all DNA repair assays used, such as an increased survival after UV-radiation of the transduced cells, a normal level of DNA repair synthesis (UDS), and the reactivation of a UV-irradiated reporter vector. This retroviral vector will be used to modify keratinocytes genetically to produce repair proficient reconstituted skin for engraftment to XP patients.

Cell Survival↗
PubMed1995

In vivo delivery of human alpha-L-iduronidase in mice implanted with neo-organs.

Mucopolysaccharidose type I is a lysosomal storage disease caused by a deficiency in the enzyme alpha-L-iduronidase (IDUA). The existence of a secretory pathway for lysosomal enzymes and the capture of secreted molecules by distant cells through binding to mannose-6-phosphate receptors have provided a rationale for enzyme replacement therapy in lysosomal storage diseases. We have used genetically modified fibroblasts implanted into neo-organs as an in vivo delivery system for IDUA. The human IDUA cDNA was isolated and inserted into a retroviral vector where it was expressed from the phosphoglycerate kinase 1 gene promoter. MPS I fibroblasts transduced with this vector showed high levels of IDUA activity and secreted phosphorylated molecules that could be internalized by naive deficient cells. Neo-organs containing 2 x 10(7) IDUA-secreting cells were implanted into nude mice. Human and murine IDUA activities were measured in the liver and spleen of animals sacrificed 35-77 days after implantation. Human IDUA activity corresponded to 0.6-2.3% of the murine enzyme activity in the liver and to 0.1-0.3% in the spleen. These data indicated that human IDUA was secreted from neo-organs and internalized in distant tissues.

Animals↗
PubMed1995

Gene therapy of lysosomal storage disorders.

Lysosomal storage disorders (LSD) result from deficiencies in enzymes normally implicated in the catabolism of macromolecules inside the lysosome. Many of these enzymes can reach the lysosome after being secreted in the extracellular medium and recaptured by specific cell surface receptors. This has suggested a rationale for therapeutic approaches in LSD, in which the missing enzyme is provided by an external source. Current therapies based on this concept, including the administration of purified enzyme and bone marrow transplantation, have been shown to result in clinical improvements in both animal models and patients. Although considerable difficulties must be surmounted, LSD present a favourable situation for gene therapy. The gene corresponding to the affected enzyme has been identified in most diseases and cDNAs are available. Low and unregulated levels of enzyme activity should be sufficient for correction. Importantly, a variety of gene transfer strategies can be carefully evaluated in animal models.

Animals↗
PubMed1995

Human immunodeficiency virus type 1 Nef induces accumulation of CD4 in early endosomes.

We have studied the fate of CD4 in CEM T cells expressing a human immunodeficiency virus type 1 HIV-1 Nef protein. Nef triggered a rapid endocytosis and a degradation of CD4, while most of the p56lck was upheld at the cell membrane. In the presence of Nef, CD4 accumulated in acidic intracellular vesicles that were not stained by antibodies against rab6, a marker of the Golgi apparatus complex. Detection of transferrin in CD4-containing vesicles showed that CD4 was trapped in early endosomes, without significant accumulation of CD4 in late endocytic compartments. Internalization pathways taken by CD4 in Nef+ cells may therefore be different from those observed after treatment with phorbol esters.

CD4 Antigens↗
PubMed1995

Receptor-binding domain of murine leukemia virus envelope glycoproteins.

The surface glycoprotein (SU) of murine leukemia viruses (MuLVs) comprises two domains connected by a proline-rich hinge. The interaction of MuLV particles with subgroup-specific cell surface receptors depends primarily on two variable regions (VRA and VRB) located in the amino-terminal domain. To delineate the minimal receptor-binding domains, we examined the capacity of soluble envelope fragments to compete with the entry of virus particles. Amphotropic, ecotropic, polytropic, and xenotropic truncated SUs were produced by inserting stop codons in the env gene of the 4070A, Friend, MCF247 and NZB MuLVs, respectively. These fragments, as well as full-length envelope glycoproteins, were stably expressed in cells bearing the corresponding receptor. Synthesis, posttranslational modifications, transport, and secretion of the env gene products were monitored by immunoprecipitation. Cells expressing the modified SUs or naive cells preincubated with SU-containing conditioned media were infected with different pseudotypes of a retroviral vector carrying a beta-galactosidase marker gene. Reduction of cell susceptibility to infection in the presence of SU was used as a measure of receptor occupancy. The results indicated that the amphotropic and ecotropic envelope amino-terminal domains contain all of the determinants required for receptor binding. In contrast, additional sequences in the proline-rich region were needed for efficient interaction of the polytropic and xenotropic amino-terminal domains with the receptors.

3T3 Cells↗
PubMed1995

Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell.

We have analyzed the replication of Nef+ and Nef- isogenic human immunodeficiency virus in CEM, HUT78, MT4 lymphoid, and U937 monocytic cell lines. At each passage of infected cells, we have assessed the relative infectivity of the virus particles released in culture media by measuring the number of infections units per nanogram of p24 protein. Values appeared to be 3- to 10-fold higher for the Nef+ virus than for the Nef- number The positive effect of Nef was observed regardless of the cell line, the multiplicity of infection, and the number of virus replication cycles achieved. We showed, by using cells expressing glycosylphosphatidylinositol-linked CD4, that the enhancement of virion infectivity could be dissociated from the down-regulation of cell surface CD4 also induced by Nef. The gp120-to-p24 ratio and the RNA content of virus particles produced in the presence or in the absence of Nef were equivalent. Virions bound to cell surface CD4 receptors with equal efficiencies. Equivalent reverse transcriptase activities were measured both on exogenous substrate and on particle genomic RNAs. In contrast, reverse transcription in infected cells generated 5- to 10-fold less DNA when the virions were produced in the absence of Nef, indicating that these particles performed reverse transcription in a suboptimal environment. These data suggest that the expression of Nef in virus-producing cells is required for efficient processing of the early stages of virus replication in target cells, including the internalization in an appropriate cell compartment and the uncoating of the particle.

Cell Line↗
PubMed1995

Continuous secretion of human soluble CD4 in mice transplanted with genetically modified cells.

Somatic transgenesis can be used to confer endogenous production of proteins with therapeutic properties. One such product, recombinant soluble human CD4 (sCD4), has been shown to be an efficient inhibitor of human immunodeficiency virus 1 (HIV-1) in vitro, but its too short half-life in vivo has impaired long-term clinical trials in AIDS patients. Using a retroviral vector, we introduced the cDNA of sCD4 into primary mouse fibroblasts. The cells were enclosed in a lattice of collagen and synthetic fibers coated with basic fibroblast growth factor, and implanted in the peritoneal cavity of syngeneic mice. Implantation of such sCD4-secreting organoids into cyclosporin A-treated C3H mice elicited a strong antibody response against sCD4. Implantation of sCD4-secreting organoids into immunotolerant mice (transgenic for transmembrane human CD4) resulted in continuous sCD4 production, detected during 60 days in animal sera. The serum levels obtained were significant, but too limited as yet for anti-HIV purposes. Nevertheless, this model may be of interest in various fields, as it provides the first demonstration that one potentially therapeutic protein, despite its half-life of a few hours, could remain present in vivo 2 months after a single somatic transgenesis.

Animals↗
PubMed1994

Impairment of T cell receptor-dependent stimulation in CD4+ lymphocytes after contact with membrane-bound HIV-1 envelope glycoprotein.

A CD4+ human T cell clone (SPB21) or primary blood mononuclear cells were grown in the presence of HeLa cells stably expressing functional human immunodeficiency virus type 1 envelope glycoprotein complexes at their surface. After a short cocultivation, SPB21 cells lost their ability to proliferate in response to T cell receptor (TCR) stimulations and died by apoptosis, whereas interleukin-2 stimulation was still effective. Incubation with soluble monomeric gp120 did not alter TCR responsiveness. A selective decrease in the proportion of CD4+ cells was also observed among primary lymphocytes after cocultivation and OKT3 stimulation. We propose that binding of oligomeric membrane-bound envelope glycoprotein induces a multimerization of CD4 molecules that impairs normal TCR stimulation.

Apoptosis↗
PubMed1994

Role of N-linked glycosylation in the activity of the Friend murine leukemia virus SU protein receptor-binding domain.

The 243 N-terminal residues of Friend Murine Leukemia Virus envelope glycoprotein (SU) fold into a structurally and functionally autonomous domain which contains the determinants for binding to the ecotropic virus receptor. The two N-linked glycosylation sites present in this N-terminal portion of the viral SU were removed by site-directed mutagenesis without disturbing its biosynthesis and incorporation into infectious virions. A truncated version of the mutant protein which included only the N-terminal domain was poorly transported but still able to interact with the receptor. Interference assays indicated that the interaction between the mutated protein and the virus receptor was weaker. We conclude that the elimination of N-linked oligosaccharide chains in the envelope N-terminal domain do not prevent receptor interaction but results in subtle conformational changes that may alter recognition and binding.

3T3 Cells↗
PubMed1994

Modifications in the binding domain of avian retrovirus envelope protein to redirect the host range of retroviral vectors.

On the basis of theoretical structural and comparative studies of various avian leukosis virus SU (surface) envelope proteins, we have identified four small regions (I, II, III, and IV) in their receptor-binding domains that could potentially be involved in binding to receptors. From the envelope gene of an avian leukosis virus of subgroup A, we have constructed a set of SU mutants in which these regions were replaced by the coding sequence of FLA16, a 16-amino-acid RGD-containing peptide known to be the target for several cellular integrin receptors. Helper-free retroviral particles carrying a neo-lacZ retroviral vector were produced with the mutant envelopes. SU mutants in which regions III and IV were substituted yielded normal levels of envelope precursors but were not detectably processed or incorporated in viral particles. In contrast, substitutions in regions I and II did not affect the processing and the viral incorporation of SU mutants. When FLA16 was inserted in region II, it could be detected with antibodies against FLA16 synthetic peptide, but only when viral particles were deglycosylated. Viral particles with envelopes mutated in region I or II were able to infect avian cells through the subgroup A receptor at levels similar to those of the wild type. When viruses with envelopes containing FLA16 peptide in region II were applied to plastic dishes, they were found to promote binding of mammalian cells resistant to infection by subgroup A avian leukosis viruses but expressing the integrins recognized by FLA16. Deglycosylated helper-free viruses obtained by mild treatment with N-glycosidase F have been used to infect these mammalian cells, and infections have been monitored by neomycin selection. No neomycin-resistant clones could be obtained after infection by viruses with wild-type envelopes. Conversely, colonies were obtained after infection by viruses with envelopes bearing FLA16 in region II, and the genome of the retroviral vector was found correctly integrated in cell DNA of these colonies. By using a blocking peptide containing the minimal adhesive RGD sequence contained in FLA16, we have shown that preincubation of target cells could specifically inhibit infection by viruses with FLA16.

Amino Acid Sequence↗
PubMed1994

Gene therapy for lysosomal disorders.

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 (MPS VII, sly syndrome), we have used autologous implants of genetically-modified cells for the continuous in vivo production of the enzyme. A retroviral vector containing the human beta-glucuronidase cDNA under the control of the mouse phosphoglycerate kinase promoter was used to infect primary skin fibroblasts, bone marrow cells, or myoblasts from mutant MPS VII animals. The fibroblasts were embedded into collagen lattices and reimplanted into the peritoneal cavity of recipient MPS VII mice. All animals, when analysed 10 to 155 days later, expressed beta-glucuronidase from the vascularised 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. This procedure has been scaled up for long term lysosomal enzyme delivery in dogs. The bone marrow cells were used for partial hematopoietic reconstruction of sublethally irradiated MPS VII mice. Five months after gene transfer, animals in which under 5% of genetically-modified hematopoietic cells were detected in the spleen showed a drastic reduction of lysosomal storage lesions in the liver and spleen. Genetically-modified myoblasts were transplanted into injured muscles, where they participated in the regeneration of a significant proportion of muscle fibers. Enzyme secretion and liver uptake were observed for at least one month.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗