EXPRESSION OF HUMAN GLUCOCEREBROSIDASE IN MURINE MACROPHAGES - IDENTIFICATION OF EFFICIENT RETROVIRAL VECTORS

Gaucher's disease is an autosomal recessive disorder characterized by a functional deficiency in beta-glucocerebrosidase enzymatic activity and the resultant accumulation of the glycolipid glucocerebroside in m acrophages. Due to the nature of the affected cells, Gaucher's disease is an excellent candidate for gene therapy of hematopoietic stem cell s and autologous bone marrow transplantation of transduced cells using retroviral vectors containing the glucocerebrosidase (GC) gene. In or der to identify a retroviral vector capable of high levels of expressi on of the GC gene in macrophages, we have used the murine myeloid leuk emia cell line, M1, a cell line that can be differentiated with interl eukin-6 (IL-6) from blasts to macrophages. Two vectors use the Moloney murine leukemia virus (MoMLV) enhancer/promoter (LG vector) or the my eloproliferative sarcoma virus (MPSV) enhancer/MoMLV promoter (MG vect or), both located in the viral long-terminal repeat (LTR); the third v ector uses the phosphoglycerate kinase (PGK) promoter located internal ly in the vector (PG vector). The amphotropic PA317 and GP+am12 packag ing cell lines were used as virus producer cells, and the GP+am12 cell line demonstrated higher titers, higher levels of GC protein expressi on, and specific GC enzymatic activity as well as higher transduction efficiencies for all three vectors. The LG retroviral vector was the m ost efficient in transducing the M1 cells. On average, higher levels o f RNA and protein expression were seen in the M1 clones transduced wit h the LG vector, and these levels increased after differentiation. Thu s, the LG retroviral vector in which the expression of the GC gene is driven by the MoMLV LTR enhancer/promoter is the best vector of the th ree studied for future studies for gene therapy of Gaucher's disease a nd other hematopoietic disorders that involve macrophages.