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Lysosomal storage diseases
Lysosomal storage results when any of several lysosomal enzymes is severely deficient and substrates for that enzyme accumulate. This accumulation causes distended lysosomes that increasingly occupy the cytoplasmic space and eventually compromise cell function. Thus, the course of storage disease is typically progressive and the outcome severe. More than 40 different storage diseases have been identified, and although each is rare, together they add up to a significant health problem (about one in every 3,000 to 5,000 births). Mucopolysaccharidosis (MPS) is a subclass of storage diseases in which glycosaminoglycans (GAGs) are the non-degraded storage material. MPS type seven (MPS VII) is caused by a deficiency in ß-glucuronidase (GUS), and although it is quite rare, it has been studied extensively as a model for lysosomal storage in general. Children with MPS VII may show little or no disease symptoms at birth but fail to continue developing normally. The result is a progressive deterioration including dwarfism, mental retardation, corneal clouding, loss of hearing, and multiple organ problems. Life expectancy is less than 20 years.

Figure 1. Brain section from an adult MPS VII mouse injected intrathecally with AAV-GUS as a newborn. GUS enzyme activity (red stain) is seen in multiple cells including neurons (especially Purkinje cells) and ependymal cells. This section was counterstained with methyl green.

To treat MPS VII in the mouse model, which closely mimics the characteristics of the human disease, we have used a viral vector to deliver the GUS gene to cells in the intact mouse. The vector is adeno-associated virus (AAV) in which the viral genes have been replaced with the coding sequence for GUS driven by a heterologous promoter (usually CMV). Intravenous administration of this vector to either newborn or adult MPS VII mice results in the production of GUS in multiple tissues, especially liver. Because transduced cells overproduce GUS, some of it is secreted and taken up by non-transduced cells. This cross-correction is responsible for elimination of storage material even in cells that have not been transduced. A single administration of vector provides a long-term source of GUS (at least 1.5 years and presumably for life). Treated mice show improved health and extended life span. In animals treated as newborns, abnormal skeletal development is improved but not completely corrected. We have been able to dramatically improve the efficiency of cross-correction by modifying the N- and C-terminal ends of the GUS polypeptide such that it is secreted at 10 times the normal rate. This increases the amount of circulating GUS that can be taken up by cells that are not targets for transduction by the AAV vector. The result is a broader distribution of GUS and more widespread elimination of storage vacuoles. However, intravenous administration of vector is not very effective in treating the brain, especially when treating adults. Neither the vector nor GUS enzyme is able to cross the blood/brain barrier. To circumvent the blood/brain barrier, we administer the vector by intrathecal (spinal) injection into the cerebrospinal fluid. This appears to provide sufficient GUS in the brain to provide widespread elimination of storage vacuoles. Combined intrathecal and intravenous treatment of MPS VII mice with the viral vector provides a dramatic, but still not quite complete, reversal of the disease phenotype. The recent development of more efficient vectors may make a complete "cure" possible.
MPS I is a related lysosomal storage disease with consequences similar to those of MPS VII. MPS I, which occurs at a higher frequency in the human population, is caused by deficient a-iduronidase (IDU). The best current treatment for most MPS I patients is enzyme replacement therapy; however, due to the blood/brain barrier, systemic infusion of IDU is not sufficient to treat the CNS aspects of this disease. Using an AAV vector to deliver the IDU gene, we used the intrathecal route to treat the brain in a mouse model of MPS I. Experiments showed that relatively low doses of vector resulted in widespread expression of IDU in the brain. This was accompanied by a reduction in storage vacuoles in the brain. Thus, the intrathecal route of vector administration again appears to successfully circumvent the blood/brain barrier. These results have positive implications for lysosomal storage diseases in particular and genetic diseases of the CNS in general.

Deficient Cholesterol Metabolism

Currently, our major effort has shifted to Smith-Lemli-Opitz syndrome (SLOS). This is a dysmorphology and mental retardation disorder caused by cholesterol deficiency due to inactivity of 7-dehydrocholesterol reductase (DHCR7). Biochemically, it is characterized by low concentrations of cholesterol in blood and tissues and abnormally high concentrations of dehydrocholesterol (DHC). There is currently no cure, although dietary cholesterol administration results in some clinical improvement. Among inherited errors of metabolism, SLOS is relatively frequent, and diagnostic methods developed by Dr. Cedric Shackleton at CHORI aid its early detection. SLOS is an excellent candidate for the development of gene therapy because: it is a single gene (enzyme) disorder; the desired biochemical consequences of therapy are predictable and readily measured; cross-correction through scavenging DHC should eliminate the need to treat all cells in a tissue; mouse models that mimic human SLOS open the door to in vivo experimentation; fetal diagnosis in humans via large scale, noninvasive screening is now feasible; and finally, there is significant medical and humanitarian need for new treatment of this devastating disease. In collaboration with Dr. Shackleton, we recently showed that gene therapy can have a positive impact on the synthesis of cholesterol in SLOS mice. Now it appears that gene therapy also improves the rate of weight gain in affected mice. As we have done for MPS I and VII, we are utilizing AAV vectors to deliver the therapeutic gene, in this case a human DHCR7 gene. Because some irreversible damage due to cholesterol deficiency probably occurs very early in fetal development, a complete cure for SLOS may not be possible. Nevertheless, we hope to be able to ameliorate symptoms of SLOS that are due to chronic cholesterol deficiency and ultimately to improve the health and wellbeing of children who suffer from this disease.

Fig. 2. Syndactyly, the fusion of two or more digits, is a minor, but common, symptom of SLOS, as seen in our mouse models (left) and in human patients (right).

Fig. 3. Two male littermates at three weeks of age. As in human patients, SLOS mice experience considerably diminished growth compared to non-mutant siblings.

Revised: Monday, May 20, 2013 5:14 PM


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