Showing papers by "John W. Belmont published in 1986"

Expression of human adenosine deaminase in murine haematopoietic progenitor cells following retroviral transfer.

Abstract: Adenosine deaminase (ADA) deficiency, an autosomal recessive inborn error of metabolism, leads to severe combined immune deficiency in man. This enzyme, although constitutively expressed in most tissues, is expressed at high level in immature T cells, and study of the pathophysiology of the disorder indicates that increased deoxyadenosine or altered methylation capacity have toxic effects on T-cell maturation. Although bone marrow transplantation can correct the immune deficiency, this therapy is associated with graft-versus-host disease and incomplete immune restoration, and so our laboratory and others have sought to develop a method of gene replacement as a possible treatment for the disease. Moreover, characterization of the complementary DNA of the human ADA gene and some of its mutants makes it possible to design gene transfer strategies. We have now subcloned a human adenosine deaminase cDNA into the retrovirus shuttle vector pZIP-SV(B), and in this way have isolated a cell line, 4.2T, which produces high titres of replication-defective retrovirus which have been used to transfer the gene for human ADA to mouse bone marrow cells. Transfer and expression of the neomycin-resistance gene (neo) and the ADA gene in murine bone marrow colony-forming units (CFU) was demonstrated by in vitro colony formation in the presence of the antibiotic G418 or 9-xylofuranosyladenine plus deoxycoformycin, respectively. Isoenzyme analysis also showed human ADA expression in the cultured mouse bone marrow.

Developments Leading to Human Gene Therapy

Abstract: The development of recombinant-DNA technology has provided an exciting research stimulus to the field of human genetics. The building of the human gene map, the establishment of new linkage methods, the isolation of many disease-related genes, and the application of molecular genetics to the problems of Huntington’s chorea and Duchenne muscular dystrophy all indicate the power of this technology. Currently, several groups are exploring the use of these methods for a new therapeutic purpose—development of gene-replacement therapy (Cline, 1982; Anderson, 1981, 1984). The purpose of this review is to examine the elements needed for in vivo gene transfer and to assess the potential for human gene therapy.

Gene replacement therapy for inborn errors of purine metabolism.

Abstract: Effective retroviral vectors carrying the human HPRT and ADA genes have been described. Initial characterization of the retroviral gene transfer system using the HPRT vector allowed the delineation of several parameters important in viral titer, expression, and stability. Using the HPRT and ADA vectors, we have initiated experiments designed to insert these human genes into various tissues of the mouse and have demonstrated expression of both transduced genes in mouse bone marrow cells. Further work with these and other vector constructions is underway in the hope that this technique may allow safe and effective treatment of ADA and HPRT deficiencies, paving the way for treatments of other inborn errors of metabolism through somatic gene replacement therapy.