Toward Gene Therapy for Niemann-Pick Disease (NPD): Separation of Retrovirally Corrected and Noncorrected NPD Fibroblasts Using a Novel Fluorescent Sphingomyelin

The neurologic (type A) and nonneurologic (type B) forms of Niemann-Pick disease (NPD) both result from deficiencies of acid sphingomyelinase (ASM) activity leading to the accumulation of sphingomyelin and other related lipids within lysosomes. Recently, the full-length cDNA and genomic sequences encoding ASM have been isolated and the nature of the molecular lesions causing NPD has been investigated. Although these developments have facilitated diagnosis for this debilitating disease, no effective treatment is currently available. Toward this latter goal, our laboratories recently reported the effectiveness of retroviral-mediated gene transfer for the in vitro correction of the cellular pathology in NPD fibroblasts (Suchi et al., 1992). In addition, novel selection procedures were developed to separate retrovirally corrected and noncorrected NPD fibroblasts based on the receptor-mediated delivery of a fluorescently (pyrene)-labeled sphingomyelin (P12-SPM) to the lysosomes of cells using liposomes coated with apolipoprotein E. In this study, we have used a different, fluorescent derivative of sphingomyelin (lissamine-rhodamine dodecanoyl sphingomyelin; LR12-SPM) to extend and improve this selection system. LR12-SPM offers a number of advantages over P12-SPM, including the facts that apolipoprotein E is not required for its efficient uptake and targeting to lysosomes and that the product of LR12-SPM degradation by ASM is efficiently transported out of cells. Thus, when analyzed in a fluorescence-activated cell sorter (FACS), there was complete separation (i.e., no overlap) of retrovirally corrected and noncorrected NPD cells after the administration of LR12-SPM. In addition, mixing experiments demonstrated that even when the corrected cells represented as few as 4% of the total cell population, they could be clearly detected and sterilely isolated using a FACS system. These studies should facilitate the development of gene therapy for NPD and provide a model system for the development of similar selection procedures for other lipid storage diseases.

Overview summary

The major cellular site of pathology in the nonneurologic forms of Niemann-Pick disease (i.e., type B) is the bone marrow-derived macrophage. Therefore, bone marrow-mediated gene therapy is a realistic approach for the treatment of this disease. As a first step toward this goal, retroviral vectors containing the acid sphingomyelinase cDNA were previously used to correct the metabolic defect in cultured fibroblasts from Niemann-Pick disease patients. We now report the development of a novel selection system that permits the rapid and efficient isolation of retrovirally corrected Niemann-Pick disease cells without the need for G418 selection.