Enzyme replacement therapy (ERT) is the primary mode of treatment for several lysosomal storage diseases. For each ERT, a chosen cell line is transfected with an expression construct bearing the coding DNA sequence for the human enzyme. The recombinant enzyme is delivered to the patient via intravenous infusions; or in one case, into the cerebrospinal fluid, to treat disease in the brain. Successful ERT depends on the recombinant protein binding to the mannose 6-phosphate receptor (MPR), which takes up the enzyme and traffics it to the lysosome. However, in some cases the recombinant enzyme is poorly phosphorylated and does not bind well to the MPR, limiting its therapeutic efficacy and/or requiring very high doses to be administered to overcome this limitation. Alpha-N-acetylglucosaminidase (NAGLU), the enzyme that is deficient in Sanfilippo B disease (mucopolysaccharidosis type IIIB), shows poor mannose 6-phosphate (Man-6-P) content when it is made recombinantly. As a result, recombinant NAGLU shows little to no binding to the MPR and does not readily enter cells. Here, we propose to use co-transfection with a novel, S1-S3 alpha-N-acetylglucosamine-1- phosphotransferase to increase the Man-6-P content of NAGLU. N-acetylglucosamine-1-phosphotransferase is naturally found in cells and phosphorylates mannose residues of N-glycosylated proteins to generate Man-6-P. The S1-S3 form of this phosphotransferase carries sequence modifications so that it does not require cleavage for activation, is highly expressed, and has good enzymatic activity toward several lysosomal enzymes. Initial studies suggest co-transfection with S1-S3 can increase the Man-6-P content of recombinant lysosomal enzymes in vitro, including NAGLU. As the symptoms of Sanfilippo B syndrome are largely localized to the brain, we propose to deliver recombinant NAGLU into cerebrospinal fluid. In the R61 phase, we propose in vitro experiments to optimize the Man-6-P content of recombinant NAGLU, characterize its uptake and lysosomal targeting as well as its other biochemical properties, and develop a process for producing and purifying the protein. In the R33 phase, we propose administration of recombinant NAGLU with enhanced Man- 6-P content into cerebrospinal fluid to evaluate its biodistribution and in vivo efficacy. The ability to enhance the Man-6-P content of recombinant NAGLU would solve a major barrier in the development of successful ERT for this devastating, untreatable disease.
Sanfilippo syndrome is a devastating, genetic disorder of childhood that is sometimes called ?childhood Alzheimer's.? We propose to develop a novel therapy that would replace the missing enzyme. The research could lead to a treatment for this fatal, neurodegenerative disease.
