Gaucher disease results form deficiency of the lysosomal enzyme glucocerebrosidase (GC) which catabolizes glycolipids. Deficiency of GC leads to accumulation of glycolipid in the reticuloendothelial (RE) cells of the liver, spleen, and bone marrow. Allogeneic bone marrow transplantation (BMT) of Gaucher patients has led to improvement in clinical symptoms, presumably by engraftment of donor marrow-derived RE cells. Transduction of a normal GC gene into the pluripotent hematopoietic stem cells (PP-HSC) of a Gaucher patient, followed by autologous BMT, may produce similar clinical benefit. Retrovirus vectors are currently the most effective method for gene transduction of PP-HSC. We will examine retroviral vector-mediated transduction of the human GC gene, using both a murine gene transfer/BMT model and human marrow in long-term bone marrow culture (LTBMC). We will define methods to achieve high rates of gene transfer into PP-HSC and hematopoietic progenitor cells, using high titer vectors and stimulation of marrow with hematopoietic growth factors and growth in LTBMC. Expression of human GC by a series of GC gene retroviral vectors in progeny hematopoietic cells will be quantitated. Additionally, we will use the human GC protein, expressed by the vector, to identify donor-derived cells in the RE system of murine gene transfer/BMT recipients. These studies will define the time-course of engraftment of the RE cells and will seek to determine whether CNS microglial cells are similarly replaced after BMT. In all, the goal of these studies is to identify GC vector constructs and gene transfer protocols which reproducibly produce human GC in hematopoietic cells at levels expected to correct the genetic GC deficiency of Gaucher disease.
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