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J A Barranger

Publications and source records attributed to J A Barranger.

At least 37 records · Page 2Linked to original sources
PubMed1996

Retroviral gene transfer and sustained expression of human arylsulfatase A.

Transduction of mouse hematopoietic stem cells and their progeny was studied using a recombinant retroviral vector (MFG-ASA) which incorporates the human arylsulfatase A gene (ASA; EC 3.1.6.8). Successful transduction was demonstrated in spleen colonies of mice that received bone marrow transplantation, cultured bone marrow-derived macrophages, visceral tissues and brain of long-term reconstituted mice, and also the spleen colonies of secondarily transplanted mice. The efficiency of transduction in primary spleen colonies was 90%. Expression of the ASA transgene exceeded endogenous levels in spleen colonies and in cultured macrophages by 50-100%. Enzyme activity in the visceral tissues of long-term reconstituted mice consistently showed elevated ASA activity, greater than three-fold in the spleen and lung of one animal. Increased activity of ASA also could be detected in secondary spleen colonies. These data demonstrate the usefulness of the MFG-ASA vector for efficient gene transfer and expression in mouse hematopoietic stem cells and their differentiated progeny. The presence of vector DNA in the brain 4 months after transplantation suggests a role for gene transfer and stem cell transplantation in the treatment strategies for metachromatic leukodystrophy.

Animals↗
PubMed1995

Identification of the binding and activating sites of the sphingolipid activator protein, saposin C, with glucocerebrosidase.

Saposin C is a sphingolipid activator protein of 8.5 kDa that activates lysosomal glucocerebrosidase. Previously, we synthesized and characterized a synthetic full-length human saposin C protein that displays 85% of the activity of the native saposin C. In this study we use shorter synthetic peptides derived from the saposin C sequence to map binding and activation sites. By determining the activity and kinetic constant (Kact) values of these peptides, we have identified two functional domains, each comprising a binding site adjacent to or partially overlapping with an activation site. Domains 1 and 2 are located within amino acid positions 6-34 and 41-60, respectively. The activation sites span residues 27-34 and 41-49, whereas binding sites encompass residues 6-27 and 45-60. Peptides containing the sequences of either domain displayed 90% of the activity of the full-length synthetic saposin C. Domain 2, however, bound to glucocerebrosidase by at least an order of magnitude more strongly than domain 1. Binding sites within these domains contain sequences that are excellent candidates for forming amphipathic helical structures. Competition assays demonstrated that the binding of one domain to glucocerebrosidase prevents binding of the other domain, and that saposin A and saposin C bind to the same sites on glucocerebrosidase. A model predicting a saposin C:glucocerebrosidase complex with a stoichiometry of 4:2, respectively, is presented.

Amino Acid Sequence↗
PubMed1995

Position of the sulfhydryl group and the disulfide bonds of human glucocerebrosidase.

Purified human glucocerebrosidase isolated from placenta was modified with [14C]-iodoacetic acid without reduction and digested with both protease-V8 at pH 4.0 followed by alpha-chymotrypsin at pH 7.5. The majority of radioactivity was found in a peptide that contained the [14C]-carboxymethylated-cysteine identified as CM-Cys18. Direct sequencing of the N-terminus of the intact labeled protein confirmed the modification of Cys18. For identification of disulfide bond-containing peptides, another portion of glucocerebrosidase was alkylated with nonlabeled iodoacetic acid and then digested with protease V8 and alpha-chymotrypsin as before. Twenty-eight HPLC fragments were collected. These purified peaks were then reduced with beta-mercaptoethanol followed by S-carboxymethylation with [14C]-iodoacetic acid. Three peptides among these 28 peptides generated two radioactive daughter peptides. These peptides were sequenced and the position of the radioactive CM-cysteines identified. The locations of these disulfides are Cys4-Cys16, Cys23-Cys342, and Cys126-Cys248. Attempts to reproduce the free sulfhydryl labeling experiments using the glucocerebrosidase isolated from Ceredase proved unsuccessful. No label was incorporated by this enzyme prior to reduction. This result suggests that the form of the protein used in the clinic differs from the native protein.

Amino Acid Sequence↗
PubMed1995

Centrifugal enhancement of retroviral mediated gene transfer.

Centrifugation has been used for many years to enhance infection of cultured cells with a variety of different types of viruses, but it has only recently been demonstrated to be effective for retroviruses (Ho et al. (1993) J. Leukocyte Biol. 53, 208-212; Kotani et al. (1994) Hum. Gene Ther. 5, 19-28). Centrifugation was investigated as a means of increasing the transduction of a retroviral vector for gene transfer into cells with the potential for transplantation and engraftment in human patients suffering from genetic disease, i.e., gene therapy. It was found that centrifugation significantly increased the rate of transduction into adherent murine fibroblasts and into non-adherent human hematopoietic cells, including primary CD34+ enriched cells. The latter samples include cells capable of reconstitution of hematopoiesis in myeloablated patients. As a step toward optimization of this method, it was shown that effective transduction is: (1) achieved at room temperature; (2) directly related to time of centrifugation and to relative centrifugal force up to 10,000 g; (3) independent of volume of supernatant for volumes > or = 0.5 ml using non-adherent cell targets in test tubes, but dependent upon volume for coverage of adherent cell targets in flat bottom plates; and (4) inversely related to cell numbers per tube using non-adherent cells. The results support the proposal that centrifugation increases the reversible binding of virus to the cells, and together with results reported by Hodgkin et al. (Hodgkin et al. (1988) J. Virol. Methods 22, 215-230), these data support a model in which the centrifugal field counteracts forces of diffusion which lead to dissociation during the reversible phase of binding.

3T3 Cells↗
PubMed1995

Identification of the neurotrophic factor sequence of prosaposin.

Prosaposin, recently identified as a neurotrophic factor (1), is the precursor of saposins A, B, C, and D. The neurotrophic activity of prosaposin resides in the saposin C domain. We have pinpointed the active sequence to a linear 12-mer located in the NH2-terminal sequence of saposin C (LIDNNKTEKEIL). Nanomolar concentrations of a 22-mer peptide encompassing this region stimulated neurite outgrowth and choline acetyltransferase activity, and prevented cell death in neuroblastoma cells. In primary cerebellar granule cells, the 22-mer also stimulated neurite outgroth. Studies of the neuroblastoma line NS20Y using a radiolabeled 18-mer from the neurotrophic region identified a high-affinity (Kd = 70 pM) binding site indicative of receptor-ligand interaction. The 22-mer stimulated protein phosphorylation of several proteins, some of which were tyrosine-phosphorylated after brief exposure similar to saposin C. Circular dichroism studies demonstrated that the 22-mer was converted from a random to a helical structure by addition of ganglioside GM1. The results are consistent with receptor-ligand binding by the peptide initiating a signal transduction cascade and resulting in neuronal differentiation.

Amino Acid Sequence↗
PubMed1995

Long-term expression of the glucocerebrosidase gene in mouse and human hematopoietic progenitors.

Gaucher disease (GD), one of the most common inherited metabolic disorders, is an excellent candidate for gene therapy using hematopoietic stem cells as targets. Animal models have demonstrated the feasibility of introducing the human glucocerebrosidase (GC) gene into hematopoietic progenitors with long term expression using a variety of retroviral vectors. We have previously demonstrated the expression and integration of the human GC gene in mouse hematopoietic progenitors and their progeny 4-8 months post transplant in primary recipients using the retroviral vector MFG-GC. We now demonstrate enzyme expression in peripheral blood lymphocytes of secondary recipients more than 12 months post transplantation. We also show a transduction efficiency of up to 95% in colony forming unit-granulocyte macrophage (CFU-GM) colonies generated from transduced CD34+ cells from a variety of sources, using a centrifugation promoted infection protocol. Transduction has also been documented in long term culture initiating cells (LTCIC) from the same transduced CD34+ cells. These data indicate efficient transduction of mouse hematopoietic progenitors as well as human CD34+ cells using the retroviral vector MFG-GC.

Animals↗
PubMed1995

Overexpression of arylsulfatase A gene in fibroblasts from metachromatic leukodystrophy patients does not induce a new phenotype.

We tested the influence of overexpression of arylsulfatase A (ASA) on the activity of other sulfatases in fibroblasts from patients with metachromatic leukodystrophy (MLD). We demonstrated that the overexpression of ASA reduces the activity of various sulfatases by a small amount but does not induce an accumulation of glycosaminoglycan. Our results indicate that influence of ASA overexpression on other sulfatases is different from that of N-acetyl-galactosamine-4-sulfatase overexpression reported by Anson et al. We conclude that gene therapy for MLD based on the transfer of a normal ASA gene to mutant cells will be feasible because the overexpression of ASA peptides in cells does not lead to profound deficiency of other sulfatases or result in a new phenotype.

Cells, Cultured↗
PubMed1995

Successful transduction of oligodendrocytes and restoration of arylsulfatase A deficiency in metachromatic leukodystrophy fibroblasts using an adenovirus vector.

Metachromatic leukodystrophy (MLD) is an inherited metabolic disease which is characterized by deficient activity of arylsulfatase A (ASA). This deficiency causes progressive accumulation of cerebroside sulfate (CS) in oligodendrocytes (OLs) in the brain resulting in dysmyelination. We are developing approaches for treating MLD based on direct delivery of ASA genes to brain. In this paper, we report a recombinant adenovirus (Adex1SRLacZL) that efficiently transduces OLs. Moreover, transduction and expression of the human transgene was efficient in primary fibroblasts from MLD patients exposed to similar recombinant adenovirus carrying the ASA gene (Adex1SRASA) In addition, the expressed ASA protein was correctly targeted to lysosomes and was immunoreactive to anti-ASA antibody. The enzymatic activity of ASA in MLD patient cells is corrected from the deficient state to 2.3 to 5.0 times the activity of normal control cells. These results demonstrate the correction of ASA deficiency by a recombinant adenovirus that is potentially useful for transferring the gene to brain.

Adenoviridae↗
PubMed1995

Efficient retroviral mediated transfer of the glucocerebrosidase gene in CD34+ enriched umbilical cord blood human hematopoietic progenitors.

Obtaining efficient transfer of a normal gene and its sustained expression in self-renewing hematopoietic stem cell populations is a central concern for gene therapy initiatives. Potentially, 10(8) to 10(9) CD34+ enriched cells per patient will be required for transduction and subsequent reimplantation. These studies present an efficient method for the transduction of human CD34+ cells that can be used in a clinical study of gene transfer. The method uses a centrifugation-enhanced technique for the retroviral-mediated transfer of the normal human glucocerebrosidase (GC) gene to human CD34+ enriched umbilical cord blood cells (CB). Previous studies had described high expression of GC in CD34+ enriched cells but had not reported transduction efficiency in the progenitor population specifically. The data demonstrate an average transduction efficiency in the progenitor cell population of 50% as measured by polymerase chain reaction (PCR) for the integrated GC-cDNA in clonogenic cells. Measurements of enzyme activity comparing transduced and nontransduced fractions at 6 days posttransduction indicate an average enzyme increase of six-fold over normal background levels. PCR of colony forming units-granulocyte/macrophage (CFU-GM) plated at 6 weeks from long-term culture-initiating cell (LTC-IC) cultures also indicates transfer of the transgene to early progenitor cells. Finally, experiments were carried out with the human erythroleukemia cell line, TF-1, to estimate the durable expression of the transgene. Enzymatic activities in transduced TF-1 cultures remained at 30-fold above the activity of nontransduced controls. The expression persisted for 6 weeks in culture. These studies demonstrate efficient transduction of early progenitor cells and sustained expression of the transgene in cell cultures.

Antigens, CD34↗
PubMed1995

Enzymatic and molecular diagnosis of Gaucher disease.

Advances in the knowledge of the molecular genetics of Gaucher disease has made diagnosis more certain. Carrier detection in kindreds in which the responsible mutation has been defined is completely reliable now. Coupled with enzymatic assays, the diagnostic capabilities are greater than before. Use of these methods provides important information to individuals at risk and allows them to make critical decisions. The new, simplified methods reviewed in this article permit the molecular diagnosis of the disease and carrier stage of large numbers of samples within 1 week.

Base Sequence↗
PubMed1995

Molecular biology of glucocerebrosidase and the treatment of Gaucher disease.

The inherited deficiency of glucocerebrosidase results in a group of sphingolipid storage disorders referred to collectively as Gaucher disease. Study of the biochemistry and cell biology of glucocerebrosidase has made possible an effective enzyme replacement therapy for the disease. Definition of the molecular genetics of glucocerebrosidase has improved diagnostic capabilities and presents the exciting possibility of a cure through gene therapy.

Animals↗
PubMed1994

A new diagnostic test for Gaucher disease suitable for population screening.

A new test for the diagnosis of Gaucher disease is described. The test is designed to screen large numbers of clinical specimens from high-risk populations. It consists of duplex PCR amplification of genomic DNA followed by hybridization to alkaline phosphatase-conjugated allele-specific oligonucleotide probes (ASOs). High melting temperature PCR primers were used to increase specificity and eliminate the need for a separate annealing step. All hybridization and washing steps were performed at one temperature. Chemiluminescent detection of signals is fast, and results are easily interpreted directly from x-ray films. Currently, the test is being used in our laboratories to screen Ashkenazi Jewish populations in whom Gaucher disease is common.

Gaucher Disease↗
PubMed1994

Transduction of CD34+ enriched cord blood and Gaucher bone marrow cells by a retroviral vector carrying the glucocerebrosidase gene.

One promising strategy for gene therapy of Gaucher disease involves ex vivo retroviral transduction of autologous hematopoietic stem cells. Studies in small animals have demonstrated that this approach provides a life-long supply of the glucocerebrosidase (GC) enzyme. Human application has developed to the stage of a clinical trial. In this study, we describe development of a high titer amphotropic producer line for the vector, MFG-GC, and explore transduction of CD34+ cells from various human sources. Higher than three times the normal levels of glucocerebrosidase activity in non-transduced cells were achieved following transduction of CD34+ cells obtained from bone marrow or cord blood from normal donors. The improvement in enzyme activity in Gaucher marrow was about 40-fold above deficient levels. We examined the timing and stepwise effect of multiple rounds of infection and evaluated post-infection expansion of cells in two different cytokine mixtures. Transduction efficiency was determined using immunocytochemistry and Southern blot hybridization.

Antigens, CD34↗
PubMed1994

Transduction of mobilized peripheral blood CD34+ cells with the glucocerebrosidase cDNA.

Gaucher disease (GD), the most common human lysosomal storage disorder, results from a genetic deficiency of the enzyme glucocerebrosidase (GC). The cloning of human GC cDNA, the benefits of allogeneic bone marrow transplantation and the success of enzyme replacement therapy support the feasibility of gene therapy as an approach to a cure for GD. We report the transfer of the GC gene to mobilized human peripheral blood (PB) CD34+ cells obtained from patients primed with granulocyte colony-stimulating factor and/or chemotherapy. A tenfold enrichment of CD34+ cells was achieved using an avidin-biotin immunoadsorption technique. Prestimulation of the CD34+ cells with cytokines, followed by infection for 5 days with a supernatant containing the MFG-GC retroviral vector, resulted in enzyme activity up to 2.5-times greater than non-infected and lac-Z infected controls. Southern blot hybridization of DNA from these cells demonstrated a transduction efficiency of 10-30%. These studies show that the GC gene is transferred efficiently to mobilized PB CD34+ cells by the MFG-GC retroviral vector and results in expression of enzyme activity in the population of cells capable of bone marrow reconstitution. These results advance the development of gene therapy for GD.

Antigens, CD34↗
PubMed1994

Expression of the human glucocerebrosidase and arylsulfatase A genes in murine and patient primary fibroblasts transduced by an adeno-associated virus vector.

We have constructed two recombinant adeno-associated virus (AAV) vectors (pJJ-3GC and pJJ-3ASA) which contained either the human glucocerebrosidase (GC) or arylsulfatase A (ASA) cDNA under the control of an SV40 promoter. These plasmids were co-transfected to 293 cells with a helper plasmid containing trans-acting AAV genes required for packaging the vectors. The two recombinant vectors successfully infected murine and patient fibroblasts. The human glucocerebrosidase and arylsulfatase A genes were expressed at high levels in the cells as determined by Western blot analysis, enzyme assay and immunochemical staining. GC enzyme activity in Gaucher patient fibroblasts (GM-0877) infected by AAV-GC was 15-fold higher than in non-infected cells. ASA enzyme activity in MLD 557g cells infected by AAV-ASA was up to 500-fold higher than in the metachromatic leukodystrophy (MLD) control cells. Southern blotting results showed that the vector integrated 1-2 copies of pJJ-3GC and ASA in the targeted cell genome. These two vectors will be useful in studying AAV-mediated transfer of the GC and ASA genes in cells and animals.

Animals↗