Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease.

Abstract

Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, we developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma virus enhancer/herpesvirus thymidine kinase gene (tk) promoter (Py+/Htk). NTG normalized GCase activity in transduced Gaucher fibroblasts and efficiently infected human monocytic and erythroleukemic cell lines. RNA blot-hybridization (Northern blot) analysis of these hematopoietic cell lines showed unexpectedly high-level expression from the Moloney murine leukemia virus long terminal repeat (Mo-MLV LTR) and levels of Py+/Htk enhancer/promoter-initiated human GCase RNA that approximated endogenous GCase RNA levels. Furthermore, NTG efficiently infected human hematopoietic progenitor cells. Detection (by means of the polymerase chain reaction) of the provirus in approximately one-third of NTG-infected progenitor colonies that had not been selected in G418-containing medium indicates that relative resistance to G418 underestimated the actual gene transfer efficiency. Northern blot analysis of NTG-infected, progenitor-derived cells showed expression from both the Mo-MLV LTR and the Py+/Htk enhancer/promoter. NTG-transduced hematopoietic progenitor cells from patients with Gaucher disease generated progeny in which GCase activity had been normalized.

Retroviral-mediated transfer of the human glucocerebrosidase gene into cultured Gaucher bone marrow.

“…5). The LN-transduced Gaucher marrow had only 16.9% (±3.3, SEM) of normal GC activity, which is in the range that has previously been observed for cells from patients with type 3 Gaucher disease (29). After transduction by the L-GC vector, the Gaucher marrow contained increased levels of enzymatic activity (58.…”

“…The insertion of a normal GC gene into a Gaucher patient's bone marrow stem cells followed by an autologous BMT (gene therapy) could potentially restore macrophage GC activity without the risk of immunologic complications from an allogeneic transplant. Retroviral-mediated transfer of the human GC cDNA has been shown to produce physiological levels of GC in murine bone marrow (1 1-13), Gaucher fibroblasts (14,15), and Gaucher bone marrow cells ( 16).…”

β-Glucosidase 2 (GBA2) Activity and Imino Sugar Pharmacology

“…To evaluate GBA in a more native context, we used membranes prepared from GBA2-KO mouse testes, which we assume to contain only one ␤-glucosidase, GBA. When assayed under conventional conditions for glucocerebrosidase (pH 5.5, detergents) (72)(73)(74)(75)(76), NB-DGJ reduced the glucocerebrosidase activity in wild-type membranes by 41% but did not have a significant effect in GBA2-KO membranes (Fig. 2B).…”

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

“…Many selectable marker genes have been used in retroviral vector systems for selection of transduced cells-for example, the resistance genes for neomycin (42,43) and hygromycin (44). Although these are used widely in many gene transfer experiments, these systems have many disadvantages, including exposure to nonspecific drug toxicity of target cells, long duration of the selection procedure, and difficulties in quantitating expression levels for the selection of transduced cells.…”