Functional correction of neurological and somatic disorders at later stages of disease in MPS IIIA mice by systemic scAAV9-hSGSH gene delivery

Haiyan Fu, Marcela P Cataldi, Tierra A Ware, Kimberly Zaraspe, Aaron S Meadows, Darren A Murrey, Douglas M McCarty, Haiyan Fu, Marcela P Cataldi, Tierra A Ware, Kimberly Zaraspe, Aaron S Meadows, Darren A Murrey, Douglas M McCarty

Abstract

The reversibility of neuropathic lysosomal storage diseases, including MPS IIIA, is a major goal in therapeutic development, due to typically late diagnoses and a large population of untreated patients. We used self-complementary adeno-associated virus (scAAV) serotype 9 vector expressing human N-sulfoglucosamine sulfohydrolase (SGSH) to test the efficacy of treatment at later stages of the disease. We treated MPS IIIA mice at 1, 2, 3, 6, and 9 months of age with an intravenous injection of scAAV9-U1a-hSGSH vector, leading to restoration of SGSH activity and reduction of glycosaminoglycans (GAG) throughout the central nervous system (CNS) and somatic tissues at a dose of 5E12 vg/kg. Treatment up to 3 months age improved learning ability in the Morris water maze at 7.5 months, and lifespan was normalized. In mice treated at 6 months age, behavioral performance was impaired at 7.5 months, but did not decline further when retested at 12 months, and lifespan was increased, but not normalized. Treatment at 9 months did not increase life-span, though the GAG storage pathology in the CNS was improved. The study suggests that there is potential for gene therapy intervention in MPS IIIA at intermediate stages of the disease, and extends the clinical relevance of our systemic scAAV9-hSGSH gene delivery approach.

Figures

Figure 1
Figure 1
Expression of N-sulfoglucosamine sulfohydrolase (SGSH) enzyme and correction of CNS and somatic pathology in MPS IIIA mice at 10 days postinjection. MPS IIIA mice (n = 2) were injected intravenously with 5E12 vg/kg of scAAV9-U1a-hSGSH vector at 2 months of age. Tissues were assayed at 10 days postinjection for (a) SGSH enzyme activity expressed as units/mg protein (1 unit = 1 nmol 4MU/17 hours). (b) hSGSH immunofluorescence. Red fluorescent hSGSH-positive cells indicated by yellow arrows. (c) lysosomal storage of GAGs by toluidine blue staining. Black arrows: neurons; green arrows: microglia. (d) Glial fibrillary acidic protein (GFAP). Green fluorescent GFAP-positive cells indicated by red arrows. (e) LAMP1 immunofluorescence. The indicated tissues show LAMP1 staining in red. Yellow arrows indicate myenteric neurons. BS, brain stem; cc, corpus callosum; CTX, cerebral cortex; Hrt, heart; Int, intestine; Liv, liver; TH, thalamus. Blue fluorescence: 4’,6-diamidino-2-phenylindole (DAPI)-positive nuclei. Scale bar: 50 μm.
Figure 2
Figure 2
Tissue N-sulfoglucosamine sulfohydrolase (SGSH) enzyme activity in vector-treated MPS IIIA mice. (a) MPS IIIA mice were treated at 1 month age with 1E12 or 5E12 vg/kg scAAV9-U1a-hSGSH vector. Tissues were analyzed for SGSH activity at either 8 months age or at the humane endpoint for mice in the longevity study. (b) MPS IIIA mice were treated with 5E12 vg/kg at the indicated ages and assayed at either 8 months age or at the humane endpoint as indicated in each graph (mice treated at 9 months age were assayed only at the humane endpoint). SGSH activity is expressed as units/mg protein (1 unit = nmol 4MU released/17 hours). Data presented are means ± SD. Background residual SGSH activity values in tissues from untreated MPS IIIA mice were <3% of wt levels, and were subtracted from the values for AAV9-treated mice to show vector-expressed enzyme activity. The indicated values in (a) (^) were not significantly different from wt using repeated measures analysis of variance with Bonferroni-Holm correction for group comparisons. All other values were statistically different from wt. Hrt, heart; Int, intestine; Kid, kidney; Liv, liver; Lg, lung; Mus, skeletal muscle; Spl, spleen.
Figure 3
Figure 3
Persistence of tissue N-sulfoglucosamine sulfohydrolase (SGSH) expression. MPS IIIA mice were treated by an IV injection of 5E12 vg/kg scAAV9-U1a-hSGSH vector at different ages (1 to 9 months), and tissues were assayed for hSGSH by immunofluorescence at either age 8 months or the endpoint for mice in the longevity study (8 to 24 months). In each image, treatment mo/termination mo is indicated. Red fluorescence: rSGSH-positive cells and signals; blue fluorescence: DAPI-positive nuclei. (a) Liver. (b) Brain (CTX, cortex): blue arrows: SGSH-positive cells; green arrows: autofluorescent signals. (c) Intestine: SM: submucosa; ME: muscularis externa; red arrows: peritoneal surface of intestine; white arrows: SGSH positive neurons of myenteric plexus and submucsal plexus. Scale bar: 50 um.
Figure 4
Figure 4
Reduction of stored GAGs in vector-treated MPS IIIA mice. MPS IIIA mice were treated at 1 month age with 1E12 or 5E12 vg/kg scAAV9-U1a-hSGSH vector (a), or at ages 1–9 months with 5E12 vg/kg vector (b). Tissues were analyzed for GAG content at either 8 months age or at the humane endpoint for mice in the longevity study. Data presented are means ± SD. Hrt, heart; Int, intestin; Kid, kidney; Liv, liver; Mus, skeletal muscle; NT, nontreated MPS IIIA mice; Spl, spleene; WT, wild-type mice. The following symbols indicate statistical results using repeated measures analysis of variance with Bonferroni-Holm correction for group comparisons: *difference versus wt; #difference versus IIIA; ^no statistical difference versus wt; @no statistical difference versus IIIA.
Figure 5
Figure 5
Reduction in lysosomal marker, LAMP1, in vector-treated MPS IIIA mice. MPS IIIA mice were treated with 5E12 vg/kg scAAV9-U1a-hSGSH vector at the indicated ages (1–9 months). Tissues were assayed for LAMP1 by immunofluorescence at either 8 months or endpoint for mice in the longevity study. In each image, treatment mo/termination mo is indicated. Red fluorescence: LAMP1-positive cell and signals; blue fluorescence: DAPI-positive nuclei. IIIA-NT: tissues from nontreated MPS IIIA mice; IIIA-AAV9; tissues from vector treated MPS IIIB mice. (a) Brain tissues: CTX: cortex; BS: brainstem; TH: thalamus; and CP: choroid plexus. (b) Somatic tissues: Liv: liver; Hrt: heart; Mu: skeletal muscle; Int: intestine; SM, submucosa; ME, muscularis externa; yellow arrows, neurons of the myenteric plexus. Scale bar: 50 μm.
Figure 6
Figure 6
Correction of astrocytosis in vector-treated MPS IIIA mice. MPS IIIA mice were treated at 1 month age with an intravenous injection of 5E12 vg/kg scAAV9-U1a-hSGSH vector and tissues were assayed for glial fibrillary acidic protein (GFAP) (astrocytic marker) by immunofluorescence at age 8 months. Green fluorescence: GFAP-positive cells and signals; blue fluorescence: DAPI-positive nuclei. IIIA-NT: tissues from nontreated MPS IIIA mice; AAV9: tissues from vector-treated MPS IIIA mice. (a) CNS tissues: HP: hippocampus; BS: brain stem; CTX: cortex; and SC: spinal cord. G: Gray matter; W: white matter. (b) Intestine: SM: submucosa; ME: muscularis externa; pink arrows: neurons of myenteric plexus; white arrows: neurons in submucosal plexus. (c) Eye (retina): GFAP-positive cell processes associated with retina. Yellow arrows: outer nuclear layer; red arrows: inner nuclear layers. Scale bar: 50 μm.
Figure 7
Figure 7
Improvement in behavior performance and survival in vector-treated MPS IIIA mice. MPS IIIA mice treated at 1 month age with 1E12 or 5E12 vg/kg scAAV9-U1a-hSGSH vector (a) or at different ages with 5E12 vg/kg vector (b), were evaluated for behavior performance in the Morris water maze at age 7–7.5 months. Data presented are latency to find a hidden platform (left chart) and swimming speed (right chart) over 4 days of testing. WT: wild-type mice (n = 76); IIIA: nontreated MPS IIIA mice (n = 34). (a) 5E12: n = 8; 1E12: n = 10. (b) Comparison of MPS IIIA mice treated at ages 1 (n = 8), 2 (n = 12), 3 (n = 12), and 6 (n = 10) months. (c) MPS IIIA mice treated at 6 months age, wild type, and untreated MPS IIIA mice, were retested at age 12 months.The following symbols indicate results of pairwise T-tests without correction for group comparisons: *P < 0.05 versus wt; #P < 0.05 versus IIIA; ^P > 0.05 versus wt; @P > 0.05 versus IIIA. (d) Kaplan-Meier graphs comparing longevity of wild-type and untreated MPS IIIA mice compared to mice treated with 1E12 or 5E12 vg/kg (left) or treated at different ages (1, 2, 3, 6, and 9 months) with 5E12 vg/kg (right).
Figure 8
Figure 8
Persistence of scAAV9-U1a- N-sulfoglucosamine sulfohydrolase vector genomes in MPS IIIA mice treated at different ages. At ages 4 months, 8 months, and the humane endpoint for mice in the longevity study, total DNA was extracted from the indicated tissues and assayed by quantitative polymerase chain reaction for vector and cellular genome copy numbers. Data are expressed as vector genome per diploid genome (VG/DG) from individual animals.

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