FLUORESCENCE-BASED SELECTION OF RETROVIRALLY TRANSDUCED CELLS IN THE ABSENCE OF A MARKER GENE - DIRECT SELECTION OF TRANSDUCED TYPE-B NIEMANN-PICK DISEASE CELLS AND EVIDENCE FOR BYSTANDER CORRECTION

Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorde rs resulting from the deficient activity of acid sphingomyelinase (ASM ). Type A NPD is characterized by the absence of residual ASM activity , massive accumulation of sphingomyelin and cholesterol within lysosom es, and a rapid, neurodegenerative course that leads to death by 3 yea rs of age. In contrast, type B NPD patients have low, but detectable, levels of residual ASM activity and little or no neurologic disease. T hus, individuals with type B NPD may survive into late adolescence or adulthood and are considered excellent candidates for somatic cell gen e therapy. To facilitate the development of gene therapy for this diso rder, a novel procedure was devised to isolate metabolically corrected type B NPD cells in the absence of marker gene expression. Type B NPD cells were transduced with retroviral vectors expressing ASM, labeled with lissamine rhodamine sphingomyelin (LR-SPM), and subjected to pre parative fluorescence-activated cell sorting (FACS). Two non-overlappi ng cell populations were isolated, corresponding to enzymatically corr ected (i.e., low fluorescence) and noncorrected (i.e., high fluorescen ce) cells. Quantitative PCR analysis demonstrated that the enzymatical ly corrected cells were enriched for vector sequences. Moreover, the c orrected cells could be regrown and continued to express high levels o f ASM activity after numerous passages, consistent with the fact that they were stably transduced. Notably, coculture of FAGS-sorted, overex pressing cells with untreated type B NPD fibroblasts resulted in a hom ogeneous cell population with low fluorescence whose FAGS distribution overlapped that of the corrected cells. Computerized fluorescence mic roscopy confirmed that nearly all of these cocultured cells expressed ASM activity and could hydrolyze LR-SPM. Thus, this procedure provides a rapid, preparative method to isolate metabolically corrected type B NPD cells in the absence of marker gene expression. Because transduce d cells were able to cross-correct untreated cells, these results also suggested that ''bystander correction'' may occur following autologou s transplantation of retrovirally transduced cells into NPD patients, providing further rationale for the development of gene therapy for th is disorder.