Delivery of Enzyme Replacement Therapy Across the Blood-Brain Barrier for Hunter Syndrome

Background

Hunter syndrome (Mucopolysaccharidosis II, MPS II) is a rare inherited metabolic disorder caused by a deficiency of the iduronate-2-sulfatase (IDS) enzyme. This enzyme deficiency leads to the accumulation of glycosaminoglycans (such as heparan sulfate and dermatan sulfate) in cells and tissues, resulting in severe neurological and peripheral tissue pathologies. Although enzyme replacement therapy (ERT) has achieved some success in treating peripheral symptoms, traditional intravenous ERT is ineffective in alleviating central nervous system (CNS) symptoms due to the presence of the blood-brain barrier (BBB).

The blood-brain barrier, formed by tight junctions between brain microvascular endothelial cells, prevents most large molecules from entering the brain. Therefore, effectively delivering macromolecular drugs (such as enzymes) to the brain has become a key challenge in treating CNS disorders like Hunter syndrome. In recent years, researchers have developed various technologies based on receptor-mediated transcytosis (RMT) to enable the delivery of macromolecular drugs into the brain by targeting specific receptors.

Research Source

This study was led by Will J. Costain and colleagues from the National Research Council Canada and Oxyrane in Belgium. Published in 2025 in the journal Fluids and Barriers of the CNS, the study is titled In vivo brain delivery of BBB-enabled iduronate 2-sulfatase in rats. The research aimed to develop an enzyme replacement therapy capable of crossing the blood-brain barrier by fusing single-domain antibodies (sdAbs) with the IDS enzyme to treat CNS symptoms in Hunter syndrome.

Research Process and Results

1. Protein Design and Production

The research team first designed and produced various fusion proteins of the IDS enzyme with single-domain antibodies. These fusion proteins included IDS combined with single-domain antibodies targeting the insulin-like growth factor 1 receptor (IGF1R) (e.g., IGF1R3H5), human serum albumin (HSA), or anti-serum albumin single-domain antibodies (e.g., R28 and M79). These fusion proteins were expressed in the yeast strain Yarrowia lipolytica and purified through multiple chromatography steps.

Experimental Results:

• Using surface plasmon resonance (SPR), the team confirmed the high affinity of the IGF1R3H5 single-domain antibody for human IGF1R (Kd = 7.5 nM). Although the affinity of the fusion proteins decreased, they retained their ability to bind to IGF1R.
• Anti-serum albumin single-domain antibodies (e.g., R28 and M79) also exhibited high affinity for human and rat serum albumin.

2. In Vitro Blood-Brain Barrier Permeability Studies

The team evaluated the blood-brain barrier permeability of the fusion proteins using a rat brain endothelial cell model (SV-ARBEC). By measuring the apparent permeability coefficient (Papp), they found that the IDS enzyme fused with IGF1R3H5 (IGF1R3H5-IDS) exhibited significantly higher permeability than the IDS enzyme alone in the in vitro model.

Experimental Results:

• The Papp value of IGFR3H5-IDS was 250, significantly higher than that of the IDS enzyme (Papp = 9) and the negative control A20.1 (Papp = 5).
• IDS enzymes fused with HSA or anti-serum albumin single-domain antibodies (e.g., IGF1R3H5-IDS-HSA and IGF1R3H5-IDS-R28) also demonstrated high permeability.

3. In Vivo Pharmacokinetic Studies

The team conducted in vivo pharmacokinetic (PK) studies in a rat model to assess the distribution and clearance of different fusion proteins in serum and cerebrospinal fluid (CSF).

Experimental Results:

• The half-life (t1/2α) of the IDS enzyme and IGF1R3H5-IDS in serum was less than 10 minutes, indicating rapid clearance.
• IDS enzymes fused with HSA or anti-serum albumin single-domain antibodies (e.g., IGF1R3H5-IDS-HSA and IGF1R3H5-IDS-R28) exhibited significantly prolonged serum half-lives, with an 8-11 fold increase in AUC (area under the curve).
• In CSF, the AUC of IGF1R3H5-IDS-HSA and IGF1R3H5-IDS-R28 increased by 42-fold and 52-fold, respectively, indicating a significant increase in brain exposure to the IDS enzyme.

4. Brain Distribution Studies

The team further validated the distribution of fusion proteins in brain parenchyma and blood vessels through brain tissue fractionation experiments.

Experimental Results:

• The IDS enzyme was almost undetectable in brain parenchyma, while IGF1R3H5-IDS and IGF1R3H5-IDS-R28 showed significantly higher concentrations in parenchyma than in blood vessels, demonstrating effective delivery to brain parenchyma.

Conclusions and Significance

This study successfully developed an IDS enzyme replacement therapy capable of crossing the blood-brain barrier. By fusing single-domain antibodies with HSA, the serum half-life of the IDS enzyme was significantly extended, and its exposure in the brain was increased. This strategy not only provides new possibilities for treating CNS symptoms in Hunter syndrome but also offers an important technological platform for other enzyme replacement therapies requiring BBB penetration.

Research Highlights:

1. Innovative Delivery Strategy: By fusing single-domain antibodies with HSA, the IDS enzyme achieved BBB penetration and serum half-life extension.
2. Significant Brain Delivery Efficacy: IGF1R3H5-IDS-HSA and IGF1R3H5-IDS-R28 showed significantly increased concentrations in CSF and brain parenchyma, demonstrating their potential for CNS treatment.
3. Broad Application Prospects: This technological platform can be applied to other enzyme replacement therapies requiring BBB penetration, offering significant scientific and clinical value.

Additional Valuable Information

The research team also noted that although the affinity of the fusion proteins decreased, their in vivo functionality was not significantly affected. Additionally, the yeast expression system and purification process developed by the team provide feasibility for large-scale production.

This study offers new insights and methods for treating Hunter syndrome and other CNS disorders, holding significant scientific and clinical importance.