Genetic engineering of a therapeutic lysosomal enzyme to improve its delivery into the central nervous system

Mucopolysaccharidosis type VII (MPS VII) is a prototypical lysosomal storage disease, caused by inherited deficiency of beta-glucuronidase (GUSB). GUSB is an acid hydrolase involved in the degradation of glycosaminoglycans (GAGs). In MPS VII affected individuals, undegraded GAGs accumulate in lysosomes, resulting in progressive cell and organ dysfunction. One major manifestation of MPS VII and many other lysosomal storage diseases is mental retardation. However, treatment of CNS storage is restricted by the limited exogenous enzyme targeting efficiency of the existing delivery systems. One approach to increase GUSB uptake by the CNS is attachment of a moiety that binds to neurons with high affinity and specificity. A candidate is the atoxic tetanus toxin C fragment (TTC), which binds to polysialogangliosides that are abundant on neurons. For gene therapy, genetic fusions of GUSB and TTC are needed to provide a continuous source of the therapeutic protein for delivery.; Fusion constructs were made with different TTC fragments attached in frame to the 3′ end of the GUSB cDNA. These constructs were transfected into a MPS VII mouse fibroblast cell line. Qualitative and quantitative enzyme assays demonstrated production and secretion of functional GUSB proteins by transfected cells. ELISA and metabolic labeling experiments demonstrated fusion protein expression from one construct. The fusion protein had similar properties as the wild-type GUSB, however, unlike GUSB, it also exhibited a TTC-dependent neuronal uptake profile. In vivo peripheral cell inoculation experiments showed that the secreted fusion protein underwent retrograde transport into the central nervous system. The protein was also detected in second-order neurons.; This dissertation demonstrated successful production of a functional GUSB-TTC fusion protein from a genetic fusion construct. The fusion protein exhibited significantly increased neuronal affinity and specificity characteristic of native tetanus toxin. These findings illustrated an alternate CNS delivery pathway for macromolecules that do not normally penetrate the blood-brain barrier and those that have relatively low neuronal affinity. This strategy has potential not only in the treatment of lysosomal storage diseases but also other diseases involving the central nervous system.