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

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Abstract
Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders 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 lysosomes, and a rapid, neurodegenerative course that leads to death by 3 years of age. In contrast, type B NPD patients have low, but detectable, levels of residual ASM activity and little or no neurologic disease. Thus, individuals with type B NPD may survive into late adolescence or adulthood and are considered excellent candidates for somatic cell gene therapy. To facilitate the development of gene therapy for this disorder, 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 preparative fluorescence-activated cell sorting (FACS). Two non-overlapping cell populations were isolated, corresponding to enzymatically corrected (i.e., low fluorescence) and noncorrected (i.e., high fluorescence) cells. Quantitative PCR analysis demonstrated that the enzymatically corrected cells were enriched for vector sequences. Moreover, the corrected cells could be regrown and continued to express high levels of ASM activity after numerous passages, consistent with the fact that they were stably transduced. Notably, coculture of FACS-sorted, overexpressing cells with untreated type B NPD fibroblasts resulted in a homogeneous cell population with low fluorescence whose FACS distribution overlapped that of the corrected cells. Computerized fluorescence microscopy 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 transduced cells were able to cross-correct untreated cells, these results also suggested that “bystander correction” may occur following autologous transplantation of retrovirally transduced cells into NPD patients, providing further rationale for the development of gene therapy for this disorder. To facilitate the development of ex vivo gene therapy, it is advantageous to design efficient selection procedures for the isolation of genetically corrected patient cells prior to autologous transplantation. Current selection methods rely on the expression of cotransferred marker genes (e.g., neo). However, in almost all vector constructs, the marker and therapeutic genes are under independent transcriptional control and the selected cells must be subsequently analyzed for metabolic correction prior to transplantation. Moreover, the procedures that are commonly used to select for marker gene expression often require weeks of labor-intensive cell culture and employ potentially toxic compounds. Thus, in the current study, type B Niemann-Pick disease (NPD) was used as a model to develop a simple, fluorescence-based selection procedure that permits the direct isolation of metabolically corrected cells in the absence of marker gene expression. Previous studies using skin fibroblasts obtained from type A NPD patients had indicated the potential of this system using cells that had no detectable ASM activity (Dinur et al., 1992). In this manuscript, data is presented that demonstrates the usefulness of this technique for type B NPD patients with residual ASM activity. Importantly, preparative isolation of retrovirally transduced and nontransduced type B NPD cells was performed for the first time, permitting comprehensive cell, molecular, and biochemical analysis of the sorted cells and the first demonstration of “bystander” correction for this disorder. These techniques should facilitate the development of somatic cell gene therapy for NPD and provide the basis for the development of direct selection methods for other lysosomal storage disorders (e.g., Gaucher disease, metachromatic leukodystropy) in which similar fluorescent substrates are available.