# Recombinant alpha-N-acetylglucosaminidase with improved mannose 6-phosphorylation for treatment of Sanfilippo B syndrome

> **NIH NIH R33** · WASHINGTON UNIVERSITY · 2022 · $644,183

## Abstract

PROJECT SUMMARY/ABSTRACT
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.

## Key facts

- **NIH application ID:** 10520201
- **Project number:** 4R33NS111079-02
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** PATRICIA I DICKSON
- **Activity code:** R33 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $644,183
- **Award type:** 4N
- **Project period:** 2019-12-01 → 2024-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10520201

## Citation

> US National Institutes of Health, RePORTER application 10520201, Recombinant alpha-N-acetylglucosaminidase with improved mannose 6-phosphorylation for treatment of Sanfilippo B syndrome (4R33NS111079-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10520201. Licensed CC0.

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