Characterization of an MPS I-H knock-in mouse that carries a nonsense mutation analogous to the human IDUA-W402X mutation

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Abstract

Here we report the characterization of a knock-in mouse model for the autosomal recessive disorder mucopolysaccharidosis type I-Hurler (MPS I-H), also known as Hurler syndrome. MPS I-H is the most severe form of α-l-iduronidase deficiency. α-l-iduronidase (encoded by the IDUA gene) is a lysosomal enzyme that participates in the degradation of dermatan sulfate and heparan sulfate. Using gene replacement methodology, a nucleotide change was introduced into the mouse Idua locus that resulted in a nonsense mutation at codon W392. The Idua-W392X mutation is analogous to the human IDUA-W402X mutation commonly found in MPS I-H patients. We found that the phenotype in homozygous Idua-W392X mice closely correlated with the human MPS I-H disease. Homozygous W392X mice showed no detectable α-l-iduronidase activity. We observed a defect in GAG degradation as evidenced by an increase in sulfated GAGs excreted in the urine and stored in multiple tissues. Histology and electron microscopy also revealed evidence of GAG storage in all tissues examined. Additional assessment revealed bone abnormalities and altered metabolism within the Idua-W392X mouse. This new mouse will provide an important tool to investigate therapeutic approaches for MPS I-H that cannot be addressed using current MPS I-H animal models.

Introduction

The mucopolysaccharidosis (MPS) diseases are a group of lysosomal storage disorders caused by a deficiency in one of the lysosomal enzymes that catalyzes the degradation of glycosaminoglycans (GAGs). Lysosomal accumulation of GAGs results in cellular dysfunctions, organ abnormalities, and metabolic defects through mechanisms that are not entirely understood [1], [2], [3], [4]. Currently, there are eleven known lysosomal enzyme deficiencies that lead to seven distinct MPS diseases. MPS I is caused by a deficiency of α-l-iduronidase (EC 3.2.1.76, encoded by the IDUA gene) that leads to the lysosomal accumulation of dermatan sulfate and heparan sulfate. Depending upon the amount of residual α-l-iduronidase activity, the severity of the MPS I phenotype can vary widely. Consequently, MPS I has been categorized as having three distinct phenotypic subtypes: MPS I-Hurler (MPS I-H), the severe form; MPS I-Scheie (MPS I-S), the mild form; and MPS I-Hurler/Scheie (MPS I-HS), an intermediate form. MPS I-H is a progressive disorder with multiple organ and tissue involvement that includes skeletal deformities, hearing loss, corneal clouding, heart failure and mental retardation. Patients usually die within their first decade as a result of obstructive airway disease, respiratory infection, or cardiac complications [2].

Two naturally occurring MPS I-H animal models have previously been characterized. The first was a feline model with a three-nucleotide deletion in the Idua gene that results in the loss of a single amino acid in the α-l-iduronidase protein [5], [6]. A canine model was later identified with a splice site mutation that causes retention of an intron in the Idua mRNA and leads to premature termination of α-l-iduronidase protein synthesis [7], [8], [9]. In addition, two MPS I-H mouse models have been generated using knock-out strategies that disrupt the Idua gene using an insertion cassette [10], [11]. The MPS I-H animal models have phenotypes that are generally consistent with the disease manifestations of MPS I-H patients, including deficiency of α-l-iduronidase activity [11], [12], accumulation of GAGs in most tissues [6], [10], [11], [13], [14], increased urine GAG excretion [11], [15], accumulation of GM2 and GM3 gangliosides in the brain [16], [17], abnormal facial appearance [16], [18], bone deformities [11], [19], neuropathology [20], [21], and cardiac manifestations [22], [23].

These existing animal models have proven to be valuable tools to investigate the pathogenesis of MPS I-H, and to evaluate several therapeutic strategies such as stem cell transplantation [12], enzyme replacement therapy [24], and gene therapy [15], [19]. However, the current animal models are not useful in evaluating the effectiveness of other promising therapeutic approaches. In particular, the lack of MPS I-H mouse models that carry mutations identified in MPS I-H patients limits many prospective investigations since some therapeutic approaches target a specific mutation or mutation type. In order to obtain an MPS I-H animal model that can be used to investigate a wider range of therapeutic approaches, we generated an Idua-W392X knock-in mouse model that carries a nonsense mutation corresponding to the IDUA-W402X mutation, the most common mutation found in MPS I-H patients. Here we report the characterization of the IDUA-W392X mouse. We evaluated the phenotype of Idua-W392X mice at three ages and found that mutant mice developed a quantifiable disease progression. We found evidence of biochemical, metabolic, and morphological abnormalities that correlate closely with the phenotype described for other MPS I-H animal models [10], [11], [14] as well with the human MPS I-H disease [2]. Thus, the Idua-W392X mouse will allow us to evaluate the efficacy of therapeutic approaches that previously have been limited by the availability of a suitable MPS I-H animal model.

Section snippets

Generation of the Idua-W392X knock-in mouse

The Idua-W392X targeting construct was made using a 129/Sv mouse genomic DNA fragment containing Idua exons 3–14 [11]. The W392X mutation (TGG  TAG) was introduced into exon 9 of the Idua gene by site-directed mutagenesis and was verified by sequencing. A viral thymidine kinase negative selectable marker was placed directly upstream of Idua exon 3, and a loxP-flanked neomycin-resistance positive selectable marker was cloned into the BstZ17I restriction site within the intron between exons 8 and

Replacement targeting of the Idua locus in murine ES cells

A replacement-targeting construct was generated using a 129/Sv mouse genomic DNA fragment containing Idua exons 3–14 (Fig. 1A) [11]. The W392X mutation (TGG  TAG), which corresponds to the W402X mutation found in MPS I-H patients, was introduced into exon 9 of the Idua gene. Thymidine kinase and neomycin-resistance genes were introduced into the targeting construct to provide a means of identifying ES cell clones that had undergone homologous recombination. After transfecting 129/Sv ES cells

Discussion

In this study, we report the generation and characterization of an Idua-W392X knock-in mouse model of MPS I-H. The Idua-W392X mutation is analogous to the IDUA-W402X mutation found in MPS I-H patients. We found that this mouse has a phenotype similar to other MPS I-H animal models and to human MPS I-H. The Idua-W392X mutation results in loss of α-l-iduronidase activity, leading to a significant increase in GAG levels in multiple tissues and an increase in urine GAG excretion. Abnormal tissue

Conclusions

The Idua-W392X mouse was found to have biochemical, metabolic, and morphologic defects that are consistent with the MPS I-H disease phenotype. These quantitative phenotypes are highly reproducible and relatively convenient to measure, thus serving as excellent markers to monitor therapeutic effects. Most importantly, the Idua-W392X mouse is suitable for evaluating therapeutic interventions that target premature stop mutations and NMD.

Acknowledgments

This study was supported by NIH Grant R01 NS057412. The authors also acknowledge the assistance of the UAB Transgenic Mouse Facility (NIH P30 CA13148, P30 AR048311, and P30 AR046031) and the UAB Small Animal Phenotyping Laboratory (NIH P30 DK56336, P30 AR046031, P30 NS057098, and P60 DK079626).

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