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.

The T99K variant of glycosylasparaginase shows a new structural mechanism of the genetic disease aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is an inherited disease caused by mutations in a lysosomal amidase called aspartylglucosaminidase (AGA) or glycosylasparaginase (GA). This disorder results in an accumulation of glycoasparagines in the lysosomes of virtually all cell types, with severe clinical symptoms affecting the central nervous system, skeletal abnormalities, and connective tissue lesions. GA is synthesized as a single-chain precursor that requires an intramolecular autoprocessing to form a mature amidase. Previously, we showed that a Canadian AGU mutation disrupts this obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterization of a model enzyme corresponding to a new American AGU allele, the T99K variant. Unlike other variants with known 3D structures, this T99K model enzyme still has autoprocessing capacity to generate a mature form. However, its amidase activity to digest glycoasparagines remains low, consistent with its association with AGU. We have determined a 1.5-Å-resolution structure of this new AGU model enzyme and built an enzyme-substrate complex to provide a structural basis to analyze the negative effects of the T99K point mutation on KM and kcat of the amidase. It appears that a "molecular clamp" capable of fixing local disorders at the dimer interface might be able to rescue the deficiency of this new AGU variant.

Biochemical and structural insights into an allelic variant causing the lysosomal storage disorder - aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by defects of the hydrolase glycosylasparaginase (GA). Previously, we showed that a Canadian AGU mutation disrupts an obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterizations of a model enzyme corresponding to a Finnish AGU allele, the T234I variant. Unlike the Canadian counterpart, the Finnish variant is capable of a slow autoprocessing to generate detectible hydrolyzation activity of the natural substrate of GA. We have determined a 1.6 Å-resolution structure of the Finnish AGU model and built an enzyme-substrate complex to provide a structural basis for analyzing the negative effects of the point mutation on KM and kcat of the mature enzyme. ENZYME: Glycosylasparaginase or aspartylglucosaminidase, EC3.5.1.26.

Crystal structure of a mutant glycosylasparaginase shedding light on aspartylglycosaminuria-causing mechanism as well as on hydrolysis of non-chitobiose substrate.

Glycosylasparaginase (GA) is an amidase that cleaves Asn-linked glycoproteins in lysosomes. Deficiency of this enzyme causes accumulation of glycoasparagines in lysosomes of cells, resulting in a genetic condition called aspartylglycosaminuria (AGU). To better understand the mechanism of a disease-causing mutation with a single residue change from a glycine to an aspartic acid, we generated a model mutant enzyme at the corresponding position (named G172D mutant). Here we report a 1.8Å resolution crystal structure of mature G172D mutant and analyzed the reason behind its low hydrolase activity. Comparison of mature G172D and wildtype GA models reveals that the presence of Asp 172 near the catalytic site affects substrate catabolism in mature G172D, making it less efficient in substrate processing. Also recent studies suggest that GA is capable of processing substrates that lack a chitobiose (Glycan, N-acetylchiobios, NAcGlc) moiety, by its exo-hydrolase activity. The mechanism for this type of catalysis is not yet clear. l-Aspartic acid ß-hydroxamate (ß-AHA) is a non-chitobiose substrate that is known to interact with GA. To study the underlying mechanism of non-chitobiose substrate processing, we built a GA-ß-AHA complex structure by comparing to a previously published G172D mutant precursor in complex with a ß-AHA molecule. A hydrolysis mechanism of ß-AHA by GA is proposed based on this complex model.

Aspartylglycosaminuria: a review.

Aspartylglucosaminuria (AGU), a recessively inherited lysosomal storage disease, is the most common disorder of glycoprotein degradation with a high prevalence in the Finnish population. It is a lifelong condition affecting on the patient's appearance, cognition, adaptive skills, physical growth, personality, body structure, and health. An infantile growth spurt and development of macrocephalia associated to hernias and respiratory infections are the key signs to an early identification of AGU. Progressive intellectual and physical disability is the main symptom leading to death usually before the age of 50 years.The disease is caused by the deficient activity of the lysosomal enzyme glycosylasparaginase (aspartylglucosaminidase, AGA), which leads to a disorder in the degradation of glycoasparagines - aspartylglucosamine or other glycoconjugates with an aspartylglucosamine moiety at their reducing end - and accumulation of these undegraded glycoasparagines in tissues and body fluids. A single nucleotide change in the AGA gene resulting in a cysteine to serine substitution (C163S) in the AGA enzyme protein causes the deficiency of the glycosylasparaginase activity in the Finnish population. Homozygosity for the single nucleotide change causing the C163S mutation is responsible for 98% of the AGU cases in Finland simplifying the carrier detection and prenatal diagnosis of the disorder in the Finnish population. A mouse strain, which completely lacks the Aga activity has been generated through targeted disruption of the Aga gene in embryonic stem cells. These Aga-deficient mice share most of the clinical, histopathologic and biochemical characteristics of human AGU disease. Treatment of AGU mice with recombinant AGA resulted in rapid correction of the pathophysiologic characteristics of AGU in non-neuronal tissues of the animals. The accumulation of aspartylglucosamine was reduced by up to 40% in the brain tissue of the animals depending on the age of the animals and the therapeutic protocol. Enzyme replacement trials on human AGU patients have not been reported so far. Allogenic stem cell transplantation has not proved effective in curing AGU.

Identification of Small Molecule Compounds for Pharmacological Chaperone Therapy of Aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder that is caused by genetic deficiency of the enzyme aspartylglucosaminidase (AGA) which is involved in glycoprotein degradation. AGU is a progressive disorder that results in severe mental retardation in early adulthood. No curative therapy is currently available for AGU. We have here characterized the consequences of a novel AGU mutation that results in Thr122Lys exchange in AGA, and compared this mutant form to one carrying the worldwide most common AGU mutation, AGU-Fin. We show that T122K mutated AGA is expressed in normal amounts and localized in lysosomes, but exhibits low AGA activity due to impaired processing of the precursor molecule into subunits. Coexpression of T122K with wildtype AGA results in processing of the precursor into subunits, implicating that the mutation causes a local misfolding that prevents the precursor from becoming processed. Similar data were obtained for the AGU-Fin mutant polypeptide. We have here also identified small chemical compounds that function as chemical or pharmacological chaperones for the mutant AGA. Treatment of patient fibroblasts with these compounds results in increased AGA activity and processing, implicating that these substances may be suitable for chaperone mediated therapy for AGU.

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.

Structural basis of aspartylglucosaminuria.

To elucidate the basis of aspartylglucosaminuria (AGU) from the viewpoint of enzyme structure, we constructed structural models of mutant aspartylglucosaminidase (AGA) proteins using molecular modeling software, TINKER. We classified the amino acid substitutions responsible for AGU and divided them into three groups based on the biochemical phenotype. Then, we examined the structural changes in the AGA protein for each group by calculating the solvent-accessible surface area (ASA), the number of atoms affected, and the root-mean-square deviation (RMSD). Our results revealed that the structural changes in group 1, which exhibits folding/transport defects and a complete deficiency of AGA activity, were generally large and located in the core region of the enzyme molecule. In group 2, exhibiting the mature AGA protein but no AGA activity, the functionally important region of the enzyme molecule was seriously affected. In group 3 exhibiting residual AGA activity, the structural changes in AGA were small and localized near the surface of the enzyme molecule. Coloring of affected atoms based on the distances between the wild-type and mutant ones revealed the characteristic structural changes in the AGA protein geographically and semi-quantitatively. Structural investigation provides us with a deeper insight into the basis of AGU.