CORRECTION OF THE ENZYME DEFICIENCY IN HEMATOPOIETIC-CELLS OF GAUCHERPATIENTS USING A CLINICALLY ACCEPTABLE RETROVIRAL SUPERNATANT TRANSDUCTION PROTOCOL

Gaucher disease is a lysosomal storage disorder caused by a deficiency of the enzyme glucocerebrosidase (GC), and is an excellent candidate for gene replacement therapy. To develop a clinically acceptable proto col for this purpose, we created two amplified (A) high-titer retrovir al vector-producer cell lines to efficiently transduce hematopoietic s tem and progenitor cells. GP+envAm12/A-LGSN (A-LGSN), contained the GC cDNA driven by the retroviral long terminal repeat (LTR) and the neom ycin phosphotransferase gene expressed from the simian virus 40 early promoter. GP+envAm12/A-LG4 (A-LG4) contained only the GC gene driven b y the LTR. Both A-LGSN and A-LG4 contained multiple proviral copies an d gave approximately 10-fold higher titers on 3T3 cells compared to th eir unamplified counterparts. These vectors were packaged in GP+envAm1 2 cells because vectors produced in this cell line transduced hematopo ietic cells more efficiently than other packaging cells tested. Bone m arrow mononuclear cells and purified CD34(+) cells were infected with virus supernatants four times in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) over 96 hours in culture. Cells were then plated in semisolid cultures and colony-forming unit-granulocyte /macrophage (CFU-GM) colonies were scored for vector presence by polym erase chain reaction (PCR). Transduction efficiency of CFU-GM colonies derived from CD34(+) cells was improved considerably using the amplif ied vectors in the GP+envAm12 packaging line. For A-LGSN, A-LG4, and u namplified LGSN, transduction efficiencies were 41, 42, and 25%, respe ctively. Therefore, multiple proviral copies resulting in higher titer improves retroviral transduction of human hematopoietic progenitor ce lls. Hematopoietic cells from Gaucher patients were transduced and pla ced into long-term bone marrow culture (LTBMC). Viral supernatant from the amplified producer lines transduced long-term culture initiating cells (LTCIC) efficiently (30 to 50%) using this clinically acceptable protocol. Both sustained mRNA expression and GC enzyme production are achieved in the long-term culture of LTCIC and lead to correction of the GC deficiency in their progeny cells.