Reversal of neuroinflammation in novel GS model mice by single i.c.v. administration of CHO-derived rhCTSA precursor protein.

Galactosialidosis (GS) is a lysosomal cathepsin A (CTSA) deficiency. It associates with a simultaneous decrease of neuraminidase 1 (NEU1) activity and sialylglycan storage. Central nervous system (CNS) symptoms reduce the quality of life of juvenile/adult-type GS patients, but there is no effective therapy. Here, we established a novel GS model mouse carrying homozygotic Ctsa IVS6+1g→a mutation causing partial exon 6 skipping with concomitant deficiency of Ctsa/Neu1. The GS mice developed juvenile/adult GS-like symptoms, such as gargoyle-like face, edema, proctoprosia due to sialylglycan accumulation, and neurovisceral inflammation, including activated microglia/macrophage appearance and increase of inflammatory chemokines. We produced human CTSA precursor proteins (proCTSA), a homodimer carrying terminal mannose 6-phosphate (M6P)-type N-glycans. The CHO-derived proCTSA was taken up by GS patient-derived fibroblasts via M6P receptors and delivered to lysosomes. Catalytically active mature CTSA showed a shorter half-life due to intralysosomal proteolytic degradation. Following single i.c.v. administration, proCTSA was widely distributed, restored the Neu1 activity, and reduced the sialylglycans accumulated in brain regions. Moreover, proCTSA suppressed neuroinflammation associated with reduction of activated microglia/macrophage and up-regulated Mip1α. The results show therapeutic effects of intracerebrospinal enzyme replacement utilizing CHO-derived proCTSA and suggest suppression of CNS symptoms.

Use of modified U1 small nuclear RNA for rescue from exon 7 skipping caused by 5'-splice site mutation of human cathepsin A gene.

Cathepsin A (CTSA) is a multifunctional lysosomal enzyme, and its hereditary defect causes an autosomal recessive disorder called galactosialidosis. In a certain number of galactosialidosis patients, a base substitution from adenine to guanine is observed at the +3 position of the 7th intron (IVS7 +3a>g) of the CTSA gene. With this mutation, a splicing error occurs; and consequently mRNA lacking the 7th exon is produced. This skipping of exon 7 causes a frame shift of the transcripts, resulting in a non-functional CTSA protein and hence galactosialidosis. This mutation seems to make the interaction between the 5'-splice site of intron 7 of pre-mRNA and U1 small nuclear RNA (U1 snRNA) much weaker. In the present study, to produce properly spliced mRNA from the CTSA gene harboring this IVS7 +3a>g mutation, we examined the possible usefulness of modified U1 snRNA that could interact with the mutated 5'-splice site. Toward this goal, we first prepared a model system using a mutant CTSA mini gene plasmid for delivery into HeLa cells. Then, we examined the effectiveness of modified U1 snRNA on the formation of properly spliced mRNA from this mutant CTSA mini gene. As a result, we succeeded in obtaining improved formation of properly spliced CTSA mRNA. Our results suggest the usefulness of modified U1 snRNA for rescue from exon 7 skipping caused by the IVS7 +3a>g mutation of the CTSA gene.