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

Abstract

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.

Keywords: AAV; AGA; AGU; CNS; adeno-associated virus; aspartylglucosaminidase; aspartylglucosaminuria; central nervous system; gene therapy; lysosomal storage disease.

Graphical abstract

Figure 1

AAV9/AGA Vector Construct to Express Human AGA and Study Plan of Mouse Experiment (A) Schematic diagram of the AAV9/AGA gene transfer cassette comprising a mutant AAV2 inverted terminal repeat (ITR) with the D element deleted (Δ ITR), the CBh promoter (CMV enhancer, chicken beta actin promoter, synthetic intron), codon-optimized human AGA DNA coding sequence (hAGAopt), the bovine growth hormone polyadenylation (BGHpA) signal, and WT AAV2 ITR. (B) Study plan, duration, and readouts. Various doses of AAV9/AGA vector were administered either intravenously (i.v.) or intrathecally (i.t.) to the mice at 2 months old (pre-symptomatic) or 6 months old (early-symptomatic). Study readouts at each time point after dose administration or at specified age are listed from top to bottom.

Figure 2

AAV9/AGA Gene Therapy (GT) Dose Dependently Increases AGA Activity in Serum, Tissues, and Cerebrospinal Fluid (CSF) to a Supraphysiological Level (A–D) Various doses of AAV9/AGA vector were administered either i.t. or i.v. to Aga^−/− mice at 6 months old (A and C) or 2 months old (B and D). AGA activity was assayed in serum sampled at 1, 4, 24, 48 (A and B), and 64 (B) weeks following AAV9/AGA administration. AGA activity was assayed in tissue lysates from heart, liver, brain, and cervical spinal cord (c. cord) and in CSF collected at necropsy (C and D) when the mice reached 18 months old. All data are presented as mean ± SEM. ∗ depicts significant difference (p < 0.05) by ordinary one-way ANOVA followed by Dunnett’s multiple-comparisons test compared to the untreated Aga^−/− control. n = 12∼39 in (A), n = 15∼66 in (B), n = 3–6 in (C), and n = 4–8 in (D).

Figure 3

AAV9/AGA GT Rapidly and Sustainedly Reduces AGA Substrate Accumulation in Serum, Urine, and CSF (A–F) Various doses of AAV9/AGA vector were administered either i.t. or i.v. to Aga^−/− mice at 6 months old (A, C, and E) or 2 months old (B, D, and F). AGA substrate was assayed in serum (A and B) and urine (C and D) sampled at 1, 4, 24, 48 (A–D), and 64 (B and D) weeks following AAV9/AGA GT. AGA substrate was assayed in CSF (E and F) collected at necropsy when the mice reached 18 months old. All data are presented as mean ± SEM. ∗ depicts significant difference (p < 0.05) by ordinary one-way ANOVA followed by Dunnett’s multiple-comparisons test compared to the untreated Aga^−/− control. n = 4–12 in (A), n = 4–10 in (B), n = 4–16 in (C), n = 3–10 in (D), n = 2–14 in (E), and n = 4–14 in (F).

Figure 4

AAV9/AGA GT Rescues Abnormal Behavior of Aga^−/− Mice during the First 5 Min of Open-Field Tests (A–F) Various doses of AAV9/AGA vector were administered either i.t. or i.v. to Aga^−/− mice at 6 months old (A–C) or 2 months old (D–F). At 14 months old, the mice were allowed to survey an open-field apparatus. The distance traveled in the first 5 min (distance traveled, A and D), time spent highly moving around (mobility, B and E), and time spent still (immobility, C and F) were recorded and quantified by an automated Noldus video tracking system. All data are presented as mean ± SEM. ∗ depicts significant difference (p < 0.05) by ordinary one-way ANOVA followed by Dunnett’s multiple-comparisons test compared to the untreated Aga^−/− control. n = 7–21 in (A)–(C), and n = 11–37 in (D)–(F).

Figure 5

AAV9/AGA GT Significantly Preserves Purkinje Cells in the Cerebellum in Aga^−/− Mice (A–C) Various doses of AAV9/AGA vector were administered either i.t. or i.v. to Aga^−/− mice treated at 6 months old (A and B) or 2 months old (C). At 18 months old, mouse brain was harvested for hematoxylin and eosin (H&E) staining. Arrows in (A) indicate Purkinje cells, and scale bars in (A) represent 200 μm. All data in (B) and (C) are presented as mean ± SEM. ∗ depicts significant difference (p < 0.05) by ordinary one-way ANOVA followed by Dunnett’s multiple-comparisons test compared to the untreated Aga^−/− control. n = 6 in (B), and n = 3–6 in (C).

Figure 6

AAV9/AGA GT Significantly Reduces Gliosis in Aga^−/− Mice (A–C) Various doses of AAV9/AGA vector were administered either i.t. or i.v. to Aga^−/− mice at 6 months old (A and B) or 2 months old (C). At 18 months old, mouse brain was harvested for glial fibrillary acidic protein (GFAP) staining. Scale bars in (A) represent 500 μm. All data in (B) and (C) are presented as mean ± SEM. Asterisk (∗) depicts significant difference (p < 0.05) by ordinary one-way ANOVA followed by Dunnett’s multiple-comparisons test compared to the untreated Aga^−/− control. n = 6–7 in (B), and n = 7 in (C).