Expression of human adenosine deaminase in mice after transplantation of genetically-modified bone marrow

Abstract

A high titer retroviral vector was used to transfer a human adenosine deaminase (h-ADA) cDNA into murine bone marrow cells in vitro. The h- ADA cDNA was linked to the retroviral promoter, and the vector also contained a neomycin phosphotransferase gene as a selectable marker. Infected marrow was transplanted into syngeneic W/Wv recipients, and h- ADA expression was monitored for 5.5 months. Several weeks after transplantation, h-ADA was detected in the erythrocytes of all nine recipients, eight of which expressed levels equal to the endogenous enzyme. This level of expression persisted in two of six surviving mice, while expression in three others stabilized at lower, but readily detectable, levels. Only one mouse had no detectable h-ADA after 5.5 months. Vector DNA sequences with common integration sites were found in hematopoietic and lymphoid tissues of the mice at 5.5 months, providing evidence that hematopoietic stem cells had been infected. Furthermore, all mice transplanted with marrow that had been selected in G418 before infusion had multiple vector copies per genome. While this category included the two highest h-ADA expressors, it also included the negative mouse. Thus, multiple copies of the vector were not sufficient to guarantee long-term h-ADA expression. Mice were monitored for “helper virus” infections with an assay designed to detect a wide range of replication-competent retroviruses, including those endogenous to the mouse genome. No helper virus was detected in the two highest h-ADA expressors, ruling out helper-assisted vector spread as a cause of the high h-ADA expression. These results help provide a foundation for the development of somatic gene therapy techniques to be used in the treatment of human disease.

Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.

“…As a control that the labeling of host fibers with/%gal arose from the fusion of implanted myoblasts and not from the direct transfer of genetic material (Kaleko et al, 1990), we performed syngeneic transplants of fibroblast enriched cultures that were/~-gal labeled by retroviral transduction. Muscles were examined two days to six weeks after transplantation of 1@ -4 x 105 fibroblasts.…”

Retroviral mediated gene transfer into bone marrow progenitor cells: use of beta-galactosidase as a selectable marker

“…An intensively studied model system for gene therapy utilizes the hematopoietic reconstitution of irradiated mice with genetically modified syngenic bone marrow (1)(2)(3)(4)(5). For this approach to be successful very efficient gene transfer into bone marrow stem cells must occur.…”

Complementation of a primer binding site-impaired murine leukemia virus-derived retroviral vector by a genetically engineered tRNA-like primer

“…The freedom to manipulate PBS and primer sequences may address two critical points in relation to the advancement of retroviral gene transfer technology and its clinical usage. Firstly, a number of current studies have shown the wt PBS of MLV proviruses to be involved in transcriptional down-regulation of vectors in important target cells (4,7,16,24). Secondly, the risk for generation of replication-competent retroviruses has traditionally been reduced by manipulation of the viral packaging construct, by partition of the gag and pol genes and the env gene, and by the use of heterologous promoters and termination signals (21).…”