A single intravenous rAAV injection as late as P20 achieves efficacious and sustained CNS Gene therapy in Canavan mice

Abstract

Canavan's disease (CD) is a fatal pediatric leukodystrophy caused by mutations in aspartoacylase (AspA) gene. Currently, there is no effective treatment for CD; however, gene therapy is an attractive approach to ameliorate the disease. Here, we studied progressive neuropathology and gene therapy in short-lived (≤ 1 month) AspA(-/-) mice, a bona-fide animal model for the severest form of CD. Single intravenous (IV) injections of several primate-derived recombinant adeno-associated viruses (rAAVs) as late as postnatal day 20 (P20) completely rescued their early lethality and alleviated the major disease symptoms, extending survival in P0-injected rAAV9 and rAAVrh8 groups to as long as 2 years thus far. We successfully used microRNA (miRNA)-mediated post-transcriptional detargeting for the first time to restrict therapeutic rAAV expression in the central nervous system (CNS) and minimize potentially deleterious effects of transgene overexpression in peripheral tissues. rAAV treatment globally improved CNS myelination, although some abnormalities persisted in the content and distribution of myelin-specific and -enriched lipids. We demonstrate that systemically delivered and CNS-restricted rAAVs can serve as efficacious and sustained gene therapeutics in a model of a severe neurodegenerative disorder even when administered as late as P20.

Figure 1

Progressive neuropathology and retinopathy in postnatal CD mice. (a) Representative images of hematoxyline and eosin (H&E) stained brain sections (n = 4). Bars: 4.8 mm. (b) Semiquantitative analyses of neuropathology normalized to WT animals using Image J. Ob, olfactory bulb; Cx, cortex; Hp, hippocampus; St, striatum; Th, thalamus; Ce, cerebellum; BS, brain stem. (c) Scotopic ERGs of overnight dark-adapted WT and CD mice (n = 4 each) to test the response of rods recorded as waveforms (μV) to flickers of light in increasing intensity called steps. CD, Canavan's disease; ERG, electroretinography; WT, wild-type.

Figure 2

Enzymatic and metabolic correction of AspA deficiency by systemically delivered rAAV-mediated gene therapy in CD mice. (a) Western blot of brain homogenates from WT (P90), Het (P90), and CD mice treated with PBS (P27) or rAAVhAspA (P90). Loading control: GAPDH. (b) Aspartoacylase activity in brain homogenates of the study groups. (c) Persistent vector genomes (by quantitative PCR) and aspartoacylase expression (by western blot) in P90 CD/rAAV9hAspA and CD/rAAV9hAspAmiRBS. (d) Representative images of avidin-biotin-complex (ABC) stained brain sections. (e) Immunofluorescence images of brain sections from P90 WT and CD/rAAV9 for AspA (green) and neuronal marker, NeuN (red) and colocalization (yellow). Bars: 10 μm. (f) Representative MRS spectra of brain NAA levels in WT and CD/PBS mice at P26 (n = 3 each) showing NAA peaks at 2.018 ppm. (g) Quantification of in vivo brain metabolite levels from the proton spectra as ratio to total creatine. Ins: inositol, tCho: total choline, tNAA: total NAA, tCr: total creatine, Glx: glutamate + glutamine. (h) Mass spectrometry of urine from P27 WT, Het and CD/PBS or CD/rAAV9 mice. WT, wild-type; Het, heterozygote; CD/PBS, CD/rAAV9, CD mice treated with PBS or rAAV9 respectively. CD, Canavan's disease; MRS, magnetic resonance spectroscopy; NAA, N-acetyl aspartic acid; NS, not significant; PBS, phosphate-buffered saline; rAAV, recombinant adeno-associated virus.

Figure 3

Intravenous (IV) gene therapy using rAAV9 mitigates neuropathology, reduces water accumulation in the brain and improves visual acuity. (a) Representative images from hematoxylin and eosin (H&E) stained paraffin sections (n = 4 for each group). (b) Quantification of neuropathology by ImageJ. Ob, olfactory bulb; Cx, cortex; Hp, hippocampus; St, striatum; Th, thalamus; Ce, cerebellum; BS, brain stem; CSP cervical spinal cord; TSP, thoracic spinal cord; LSP, lumbar spinal cord. (c) Representative T2-weighted magnetic resonance imaging images showing water accumulation (white) in P25, CD/PBS; P60, P365 GC; and WT animals. (d) Quantification of MRS data for metabolites in brains of mice injected with rAAV9 or rAAV9BS at P0 at age P90. (e) Scotopic ERG of WT, CD/PBS and CD/rAAV9 at P25 and P365, respectively, to test visual acuity. WT, wild-type; CD/PBS, CD/rAAV9, CD mice treated with PBS or rAAV9 respectively. CD, Canavan's disease; ERG, electroretinography; MRS, magnetic resonance spectroscopy; NS, not significant; PBS, phosphate-buffered saline; rAAV, recombinant adeno-associated virus.

Figure 4

Intravenous (IV) gene therapy using novel rAAVs improves growth profile, rescues early lethality and corrects motor function defects of CD mice. (a) Weight of animals at 2-week intervals plotted as a function of time. Early growth rate was assessed by weights at 2-day intervals for the first month (inset). (b) Kaplan–Meier survival curves for groups treated with different vectors at P0 (“closed circles” = WT, “closed squares” = CD/PBS, “closed triangles” = CD/rAAV9, “closed up-side down triangles” = CD/rAAV9mirBS, “closed diamonds” = CD/rAAVrh.8, and “closed hexagon” = CD/rAAVrh.10, respectively). (c) Negative geotaxis test at intervals of 1 day from P7 (day 8) to P17 (day 16). Motor functions of study groups were tested at P27, P90, and P180 based on their performance on the (d) balance beam, (e) inverted screen and rotarod moving at (f) fixed speed (3 rpm) and (g) accelerated speed (4–40 rpm in 5 minutes). The same tests were performed on (h) P90 CD/rAAV9 and CD/rAAV9mirBS. WT, wild-type; CD/PBS, CD/rAAV9, CD/rAAV9mirBS, CD/rAAVrh.8, and CD/rAAVrh.10: CD mice treated with PBS, rAAV9hAspA, rAAV9hAspA-mirBS, rAAVrh.8hAspA, and rAAVrh.10hAspA at P0, respectively. CD, Canavan's disease; NS, not significant; PBS, phosphate-buffered saline; rAAV, recombinant adeno-associated virus.

Figure 5

Intravenous (IV) gene therapy with rAAVs as late as P20 improves growth profile, rescues early lethality and corrects motor function defects of CD mice. (a) Kaplan–Meier survival curves for all groups treated with different vectors at P0. (b) Weight of animals at 2-week intervals plotted as a function of time. (c) Early growth rate assessed by weights at 2-day intervals for the first month. (d) Negative geotaxis test at intervals of 1 day from P7 (day 8) to P17 (day 16). Motor functions of study groups were tested at P90 based on their performance on the (e) balance beam, (f) inverted screen and rotarod moving at (g) fixed speed (3 rpm) and (h) accelerated speed (4–40 rpm in 5 minutes). WT, wild-type; CD/PBS, CD/P0, CD/P6, CD/P13, and CD/P20: CD mice treated with PBS and rAAV9hAspA at P0, P6, P13 and P20 respectively. CD, Canavan's disease; NS, not significant; PBS, phosphate-buffered saline; rAAV, recombinant adeno-associated virus.

Figure 6

Intravenous (IV) gene therapy using rAAV9 improves myelin synthesis and partially corrects lipid profile of myelin in CD mice. (a) Electron microscopy of the motor cortex of P27 age-matched mice (n = 3 for each cohort). Bar: 2 μm. Inset: higher magnification. Black arrows: unmyelinated axons; white arrows: myelin sheath. (b) G ratios of myelinated axons calculated from toluidine blue stained sections of the cortex using ImageJ. (c) High-performance thin-layer chromatography of gangliosides in cerebrum, cerebellum, and medulla of different mouse groups (n = 3 for each group). Lower panel: quantification of the plate. (d) Quantification of the high-performance thin-layer chromatography plate. Canavan's disease; GC, gene-corrected mice; NS, not significant; PBS, phosphate-buffered saline; rAAV, recombinant adeno-associated virus.