Towards Splicing Therapy for Lysosomal Storage Disorders: Methylxanthines and Luteolin Ameliorate Splicing Defects in Aspartylglucosaminuria and Classic Late Infantile Neuronal Ceroid Lipofuscinosis.

Splicing defects caused by mutations in the consensus sequences at the borders of introns and exons are common in human diseases. Such defects frequently result in a complete loss of function of the protein in question. Therapy approaches based on antisense oligonucleotides for specific gene mutations have been developed in the past, but they are very expensive and require invasive, life-long administration. Thus, modulation of splicing by means of small molecules is of great interest for the therapy of genetic diseases resulting from splice-site mutations. Using minigene approaches and patient cells, we here show that methylxanthine derivatives and the food-derived flavonoid luteolin are able to enhance the correct splicing of the AGA mRNA with a splice-site mutation c.128-2A>G in aspartylglucosaminuria, and result in increased AGA enzyme activity in patient cells. Furthermore, we also show that one of the most common disease causing TPP1 gene variants in classic late infantile neuronal ceroid lipofuscinosis may also be amenable to splicing modulation using similar substances. Therefore, our data suggest that splice-modulation with small molecules may be a valid therapy option for lysosomal storage disorders.

Aspartylglucosaminuria: Clinical Presentation and Potential Therapies.

Aspartylglucosaminuria (AGU) is a recessively inherited neurodegenerative lysosomal storage disease characterized by progressive intellectual disability, skeletal abnormalities, connective tissue overgrowth, gait disturbance, and seizures followed by premature death. AGU is caused by pathogenic variants in the aspartylglucosaminidase (AGA) gene, leading to glycoasparagine accumulation and cellular dysfunction. Although more prevalent in the Finnish population, more than 30 AGA variants have been identified worldwide. Owing to its rarity, AGU may be largely underdiagnosed. Recognition of the following early clinical features may aid in AGU diagnosis: developmental delays, hyperactivity, early growth spurt, inguinal and abdominal hernias, clumsiness, characteristic facial features, recurring upper respiratory and ear infections, tonsillectomy, multiple sets of tympanostomy tube placement, and sleep problems. Although no curative therapies currently exist, early diagnosis may provide benefit through the provision of anticipatory guidance, management of expectations, early interventions, and prophylaxis; it will also be crucial for increased clinical benefits of future AGU disease-modifying therapies.

Pre-clinical Gene Therapy with AAV9/AGA in Aspartylglucosaminuria Mice Provides Evidence for Clinical Translation.

Aspartylglucosaminuria (AGU) is an autosomal recessive lysosomal storage disease caused by loss of the enzyme aspartylglucosaminidase (AGA), resulting in AGA substrate accumulation. AGU patients have a slow but progressive neurodegenerative disease course, for which there is no approved disease-modifying treatment. In this study, AAV9/AGA was administered to Aga-/- mice intravenously (i.v.) or intrathecally (i.t.), at a range of doses, either before or after disease pathology begins. At either treatment age, AAV9/AGA administration led to (1) dose dependently increased and sustained AGA activity in body fluids and tissues; (2) rapid, sustained, and dose-dependent elimination of AGA substrate in body fluids; (3) significantly rescued locomotor activity; (4) dose-dependent preservation of Purkinje neurons in the cerebellum; and (5) significantly reduced gliosis in the brain. Treated mice had no abnormal neurological phenotype and maintained body weight throughout the whole experiment to 18 months old. In summary, these results demonstrate that treatment of Aga-/- mice with AAV9/AGA is effective and safe, providing strong evidence that AAV9/AGA gene therapy should be considered for human translation. Further, we provide a direct comparison of the efficacy of an i.v. versus i.t. approach using AAV9, which should greatly inform the development of similar treatments for other related lysosomal storage diseases.

Early initiation of enzyme replacement therapy improves metabolic correction in the brain tissue of aspartylglycosaminuria mice.

Aspartylglycosaminuria (AGU) is a lysosomal storage disease caused by deficient activity of glycosylasparaginase (AGA), and characterized by motor and mental retardation. Enzyme replacement therapy (ERT) in adult AGU mice with AGA removes the accumulating substance aspartylglucosamine from and reverses pathology in many somatic tissues, but has only limited efficacy in the brain tissue of the animals. In the current work, ERT of AGU mice was initiated at the age of 1 week with three different dosage schedules of recombinant glycosylasparaginase. The animals received either 3.4 U of AGA/kg every second day for 2 weeks (Group 1), 1.7 U/kg every second day for 9 days followed by an enzyme injection once a week for 4 weeks (Group 2) or 17 U/kg at the age of 7 and 9 days (Group 3). In the Group 1 and Group 3 mice, ERT reduced the amount of aspartylglucosamine by 34 and 41% in the brain tissue, respectively. No therapeutic effect was observed in the brain tissue of Group 2 mice. As in the case of adult AGU mice, the AGA therapy was much more effective in the somatic tissues than in the brain tissue of the newborn AGU mice. The combined evidence demonstrates that a high dose ERT with AGA in newborn AGU mice is up to twofold more effective in reducing the amount of the accumulated storage material from the brain tissue than ERT in adult AGU animals, indicating the importance of early detection and treatment of the disease.