Physician/Scientist

Diagnosis and Treatment

Treatment

Current Status

At present, there is no specific treatment available for NPD. Orthotopic liver transplantation in an infant with Type A disease and amniotic cell transplantation in several Type B NPD patients have been attempted with little or no success. BMT also has been accomplished in Type A NPD patients with no evidence of neurologic improvement. However, BMT in one Type B NPD patient was successful in reducing the spleen and liver volumes, the sphingomyelin content in the liver, the number of NPD cells in the marrow, and the radiologic infiltration of the lungs.

Recently, BMT studies also were carried out in the NPD mouse model (i.e., ASMKO mice; see "Animal Models"). An almost complete correction of the histologic and biochemical phenotype was achieved in the reticuloendothelial system organs of these animals, providing further evidence that bone marrow transplantation and hematopoietic stem cell-mediated gene therapy should be considered as therapeutic options for Type B NPD patients. In addition, the onset of ataxia was delayed by several months in the transplanted mice, leading to almost twofold increase in their life expectancy. This clinical result could be correlated with the recovery of the Purkinje cell layer in the transplanted animals. However, despite these positive neurologic results, the treated mice still developed ataxia (albeit at a later timepoint) and died prematurely.

These studies, together with those obtained from the Niemann-Pick disease mouse model (see "Animal Models"), suggest that BMT may have a significant, positive effect on the clinical course of severely affected Type B NPD patients if performed early in life. However, as is the case for many other genetic disorders, BMT is not a viable option for many Type B NPD patients because of the lack of histocompatibly-matched normal or heterozygous donors, and alternative therapeutic approaches must therefore be investigated. To date, lung transplantation has not been performed in any severely compromised Type B patient.

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Future Prospects

Enzyme Replacement Therapy
Since the seminal discoveries by Neufeld and others in the early 1970s that low levels of the appropriate normal enzymes could correct the metabolic defects in cultured fibroblasts from patients with lysosomal storage diseases, these disorders have been important models for the development of novel therapeutic strategies. Among these, enzyme replacement therapy has been actively pursued for more than two decades. Until recently, this therapeutic approach was severely handicapped by the inability to produce and purify large quantities of the normal human enzymes. This limitation has been overcome by the use of mammalian expression systems to produce large amounts of recombinant proteins. The first long-term clinical trials of enzyme replacement have been successfully undertaken in patients with Type 1 (nonneuronopathic) Gaucher disease, and clinical trials are underway for several other disorders. The success of enzyme therapy in Gaucher disease resulted, in part, from the modification of the enzyme's oligosaccharide chains (i.e., mannose-terminated ?-glucosidase) for targeting to macrophages. Since Type 1 Gaucher disease and Type B NPD both result from primary involvement of the monocyte-macrophage system, it is possible that infusion of a macrophage-targeted ASM glycoprotein also would prove therapeutic.

Thus, Type B NPD should be considered an excellent candidate for enzyme replacement therapy. The isolation of the human ASM cDNA has provided the capability to express stably high levels of recombinant human ASM in mammalian expression systems (see "Overexpression of Human ASM in Chinese Hamster Ovary Cells"), stably, as has been accomplished for other human recombinant human lysosomal enzymes. The clinical efficacy of recombinant human ASM is currently under evaluation in the NPD mouse model (see "Animal Models"), and if preclinical trials are successful, in the future the availability of this enzyme will permit the clinical evaluation of enzyme replacement to be undertaken in Type B NPD patients. For Type A NPD, enzyme replacement is unlikely to be successful since the injected enzyme is not expected to cross the blood brain barrier.

Somatic Cell Gene Therapy

The application of somatic cell gene transfer techniques to treat selected inherited metabolic diseases, including selected lysosomal storage diseases, is currently an area of intense investigation. To evaluate the potential of gene therapy for NPD, retroviral-mediated gene transfer has been used to introduce the full-length ASM cDNA into cultured fibroblasts from unrelated Type A NPD patients. The ASM activities in the nontransduced cells were less than 4 percent of the mean normal levels, and consequently, these cell lines had about threefold elevated levels of sphingomyelin. Three different retroviral vectors have been evaluated: pBC140, DCTK, and MFG. pBC140 and DCTK contain the neomycin resistance gene expressed from the viral long terminal repeat (LTR) and use heterologous promoters to express the ASM cDNA. In contrast, MFG has no selectable marker and expresses the ASM cDNA from the viral LTR. Following retroviral-mediated transduction of Type A NPD fibroblasts, the ASM activities were increased up to 23 times the endogenous activity of those found in normal fibroblasts. The MFG vector consistently expressed considerably higher levels of ASM activities than the pBC140 or DCTK vectors.

In NPD cells transduced with either vector, the sphingomyelin content was reduced to normal levels, indicating that the vector-encoded enzyme was properly targeted to lysosomes, where it was enzymatically active and able to degrade the accumulated substrate. In situ cell-loading studies were also performed to evaluate the effects of retroviral-mediated gene transfer on the chemical pathology of NPD fibroblasts. When a pyrene derivative of sphingomyelin was introduced into the lysosomes of cultured fibroblasts from a Type A NPD patient using ApoE-mediated endocytosis, only about 6 percent of the delivered substrate was degraded. In contrast, normal cells and NPD cells transduced by retroviral-mediated gene transfer were able to degrade about 80 percent of the endocytosed sphingomyelin. These results provided further evidence that retroviral-mediated gene transfer may be used to correct the metabolic defect in Type A NPD cells.

Cell-loading studies also were used to develop a selection system for discrimination of untransduced and retroviral-transduced NPD cells and those transduced by retroviral-mediated gene transfer. This selection scheme was based on the fluorescence emission of intact NPD cells, which, when loaded with pyrene- or lissamine rhodamine-labeled sphingomyelin, exceeded by three to five times those of normal or transduced cells. As a consequence, transduced cells could be efficiently sorted from nonexpressing cells by flow cytometry using a fluorescence-activated cell sorter (FACS).

This selection system was orginally developed using cultured skin fibroblasts from NPD patients. However, it has recently been adapted to hematopoietic stem cells using a different fluorescent compound, Bodipy sphingomyelin. (Erlich et al. 1998). Bodipy sphingomyelin was chosen for these studies for two reasons: the excitation/emission wavelengths of Bodipy are optimized for FACS detection, as compared to lissamine rhodamine or pyrene, and Bodipy selection of ASM expressing cells can be easily used in combination with commercially available stem cell markers, such as phycoerythrin conjugated antibodies against CD34 or c-Kit, since the emission wavelengths of Bodipy and phycoerythrin are non-overlapping.

Using bone marrow cells obtained from the NPD mouse model of NPD as an experimental system, this selection scheme has been used to isolate a population of transduced NPD cells that are highly enriched for hematopoietic stem cells (Erlich et al. 1998). In the absence of selection, these cells represent <1% of the total bone marrow cultures, but after selection they may represent up to 60-70% of the FACS-sorted cells. CFU-S assays also were carried out on the FACS-sorted cells. The results indicated that the number of vector-positive day 14 spleen colonies was ~80% in animals transplanted with the FACS-sorted cells, as compared to ~30% in non-sorted cells. Such FACS-selected, transduced stem cells have been transplanted into NPD mice for long-term evaluation of clinical efficacy of this gene therapy procedure.

Based on these results and the encouraging BMT results obtained in the NPD mice, hematopoietic stem cell-mediated gene therapy may be an effective treatment for Type B NPD. The availability of a method for the selection of gene corrected NPD stem cells also may be particularly advantageous for this disorder. Furthermore, it is now known that ASM is secreted at high levels by many cell types and is stable under physiologic conditions in the presence of zinc for long periods of time (see "Acid Sphingomyelinase"). This secreted enzyme can also be taken up readily by cells and is transported to lysosomes, facilitating in vivo cross-correction. However, many technical obstacles remain to be overcome before hematopoietic stem cell-mediated gene therapy can be accomplished successfully in human patients. This approach to therapy remains experimental and is not likely to be available to NPD families until its efficacy is proven further.

With regards to Type A NPD, the results of BMT in the NPD knock-out mice suggest that very limited improvements in the central nervous system involvement can be achieved by hematopoietic stem cell gene therapy by these procedures. Thus, new methods, such as the use of neurotropic expression vectors or direct injection of neuronal-targeted DNA complexes, must be developed. Moreover, because of the rapid progression of the neurologic disease in Type A NPD, these procedures must be accomplished in early infancy or during fetal development. Clearly, these latter studies will require extensive animal studies prior to the initiation of human clinical trials, and the murine models of NPD should be particularly valuable.

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