RETROVIRAL VECTOR-MEDIATED TRANSFER OF THE GALACTOCEREBROSIDASE (GALC) CDNA LEADS TO OVEREXPRESSION AND TRANSFER OF GALC ACTIVITY TO NEIGHBORING CELLS

Galactocerebrosidase (GALC) is responsible for the lysosomal catabolis m of certain galactolipids, including galactosylceramide and psychosin e. Patients with GALC deficiency have an autosomal recessive disorder known as globoid cell leukodystrophy (GLD) or Krabbe disease. Storage of undegraded glycolipids results in defective myelin and the characte ristic globoid cells observed on pathological examination of the centr al and peripheral nervous systems. Most patients have the infantile fo rm of GLD, although older individuals are also diagnosed. Recently the human, mouse, and canine GALC genes were cloned, and mutations causin g GLD have been identified. We now describe the construction of a vect or containing human GALC cDNA (MFG-GALC), and the transduction of cult ured skin fibroblasts from molecularly characterized Krabbe disease pa tients, as well as rat brain astrocytes and human CD34(+) hematopoieti c cells, using retrovirus produced by the psi-CRIP amphotropic packagi ng cell line. The transduced fibroblasts showed extremely high GALC ac tivity (up to 20,000 times pretreatment levels, about 100 times normal ). GALC was secreted into the media and was taken up by untransduced f ibroblasts from the same or a different patient. Mannose-6-phosphate r eceptor-mediated uptake was only partially responsible for the efficie nt transfer of GALC to neighboring cells. Additional studies confirmed the presence of normal GALC cDNA and mRNA in the transduced cells. Th e GALC produced by the transduced cells and donated to neighboring unt ransduced cells was localized to lysosomes as demonstrated by the norm al metabolism of [C-14]stearic acid-labeled galactosylceramide produce d from endocytosed [C-14]sulfatide, (C) 1996 Academic Press, Inc.