Mannitol-facilitated CNS entry of rAAV2 vector significantly delayed the neurological disease progression in MPS IIIB mice

Abstract

The presence of the blood-brain barrier (BBB) presents the most critical challenge in therapeutic development for mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease with severe neurological manifestation, because of alpha-N-acetylglucosaminidase (NaGlu) deficiency. Earlier, we showed a global central nervous system (CNS) transduction in mice by mannitol-facilitated entry of intravenous (IV)-delivered recombinant adeno-associated viral serotype 2 (rAAV2) vector. In this study, we optimized the approach and showed that the maximal transduction in the CNS occurred when the rAAV2 vector was IV injected at 8 min after mannitol administration, and was approximately 10-fold more efficient than IV delivery of the vector at 5 or 10 min after mannitol infusion. Using this optimal (8 min) regimen, a single IV infusion of rAAV2-CMV-hNaGlu vector is therapeutically beneficial for treating the CNS disease of MPS IIIB in adult mice, with significantly extended survival, improved behavioral performance, and reduction of brain lysosomal storage pathology. The therapeutic benefit correlated with maximal delivery to the CNS, but not peripheral tissues. This milestone data shows the first effective gene delivery across the BBB to treat CNS disease. The critical timing of vector delivery and mannitol infusion highlights the important contribution of this pretreatment to successful intervention, and the long history of safe use of mannitol in patients bodes well for its application in CNS gene therapy.

Fig. 1. Mannitol-facilitated AAV-CNS entry, diffuse global CNS transduction and somatic transduction in mice

scAAV2-CMV-GFP viral vector (4×10¹¹ viral particles) was injected into 6–8-week-old wt mice through tail vein, 8 minutes after an IV infusion of mannitol or without mannitol pretreatment. Vibratom tissue sections (50μm) were obtained at 4 weeks pi and probed for GFP expression by immunofluorescence using a polyclonal antibody against GFP and a secondary antibody conjugated with AlexaFluo⁵⁶⁸. A: Transgene expression in the CNS. a–h: samples of mice injected with AAV2 vector following mannitol pretreatment. a. olfactory; b. cerebral cortex; c. striatum; d. thalamus; e. hippocampus; f. brain stem; g. cerebellum. G: granule layer, M: molecular layer, blue arrows: Purkinje cells; h. spinal cord, G: grey matter, W: white matter; i. cerebral cortex of mice injected with AAV2 vector without mannitol pretreatment. GFP was stained in red. Blue arrows: neurons; Yellow arrows: glial cells; Yellow arrowhead: processes in white matter. Images: 10×. B: Somatic transduction. i. non-treated liver of non-treated mouse, ii. AAV2-treated live, red fluorescent cells are GFP-positive hepatocytes; iii. AAV2-treated heart (myocardium), yellow arrowhead: GFP-positive cardiac myocytes: iv. non-treated kidney (medulla), v. AAV2-treated kidney, yellow arrowhead: GFP-positive cuboidal epithelial cells; vi. AAV2-treated intestine, yellow arrowhead: GFP-positive enteric plexus neurons. Blue arrow: muscularis externa. i–ii: 10×; iii–vi: 20×.

Fig. 1. Mannitol-facilitated AAV-CNS entry, diffuse global CNS transduction and somatic transduction in mice

scAAV2-CMV-GFP viral vector (4×10¹¹ viral particles) was injected into 6–8-week-old wt mice through tail vein, 8 minutes after an IV infusion of mannitol or without mannitol pretreatment. Vibratom tissue sections (50μm) were obtained at 4 weeks pi and probed for GFP expression by immunofluorescence using a polyclonal antibody against GFP and a secondary antibody conjugated with AlexaFluo⁵⁶⁸. A: Transgene expression in the CNS. a–h: samples of mice injected with AAV2 vector following mannitol pretreatment. a. olfactory; b. cerebral cortex; c. striatum; d. thalamus; e. hippocampus; f. brain stem; g. cerebellum. G: granule layer, M: molecular layer, blue arrows: Purkinje cells; h. spinal cord, G: grey matter, W: white matter; i. cerebral cortex of mice injected with AAV2 vector without mannitol pretreatment. GFP was stained in red. Blue arrows: neurons; Yellow arrows: glial cells; Yellow arrowhead: processes in white matter. Images: 10×. B: Somatic transduction. i. non-treated liver of non-treated mouse, ii. AAV2-treated live, red fluorescent cells are GFP-positive hepatocytes; iii. AAV2-treated heart (myocardium), yellow arrowhead: GFP-positive cardiac myocytes: iv. non-treated kidney (medulla), v. AAV2-treated kidney, yellow arrowhead: GFP-positive cuboidal epithelial cells; vi. AAV2-treated intestine, yellow arrowhead: GFP-positive enteric plexus neurons. Blue arrow: muscularis externa. i–ii: 10×; iii–vi: 20×.

Fig. 2. Significant behavioral improvement and extended survival in MPS IIIB mice treated with an IV infusion of rAAV2 vector

MPS IIIB mice (4–6-wk-old) were treated with an IV injection of rAAV2-CMV-hNaGlu, at 8 or 10 minutes after an IV infusion of mannitol. The mice were tested for behavioral performance at 5–5.5 months of age, and were observed for longevity. a. Latency to find a hidden platform in water maze. b. Swimming ability. c. Latency to fall from an accelerating rotarod. +/+: wt (n=20); −/−: MPS IIIB (n=24); −/− +IV: MPS IIIB mice given an IV vector injection at 8 minutes after mannitol pretreatment (n=14); d. Survival. The lifespan of MPS IIIB mice were significantly prolonged when treated with an IV injection of rAAV2 vector at 8 minutes (P<0.01) and 10 minutes (P<0.05) after mannitol pretreatment (n=16–20/group). IV-8’ and IV-10’: MPS IIIB mice given an IV vector injection at 8 or 10 minutes after mannitol pretreatment; *: P<0.05 (vs. −/− +IV); #: P<0.05 (vs. +/+); @: P>0.05 (vs. +/+).

Fig. 3. Expression of recombinant NaGlu and decrease in lysosomal storage pathology in MPS IIIB mouse brain. A

Immunofluorescence staining for rNaGlu expression in the brain of MPS IIIB mouse (17-month-old) treated with an IV injection of rAAV-hNaGlu vector at 8 minutes after mannitol pretreatment, using a polyclonal antibody against hNaGlu and a secondary antibody labeled with AlexaFluo⁵⁶⁸ (a–e). a. brain stem, b. hypothalamus, c. thalamus, d. Dorsal 3^rd ventricle and periventricular thalamic area, e. choroid plexus in lateral ventricle, f. thalamus of non-treated MPS IIIB mouse. Red arrows: NaGlu-positive neurons/glia and process. Yellow arrowheads: NaGlu-positive ependymal cells (d) and choroid plexus cells (e). D3V: dorsal 3^rd ventricle. Nuclei are labeled blue. Green fluorescence was used to separate autofluorecence from specific fluorescence signals (red). Scale bars: 10μm. B. NaGlu activity in brain (endpoint, n>8/group). C. Histopathology staining with toluidine blue. +/+: wt; −/−: non-treated; −/−+AAV: AAV-treated. CB: cerebella; TH: Thalamus. M: molecular layer; G: granule layer, red arrows: Purkinje cells; red arrowhead: enlarged lysosomes. Scale bar: 20μm. D. GAG contents in the brain of rAAV-treated MPS IIIB mice (3 months pi, n>8/group): expressed as μg/mg tissue (wet). *: P<0.05 (vs. −/−), #: P<0.05 (vs. +/+).

Fig. 3. Expression of recombinant NaGlu and decrease in lysosomal storage pathology in MPS IIIB mouse brain. A

Immunofluorescence staining for rNaGlu expression in the brain of MPS IIIB mouse (17-month-old) treated with an IV injection of rAAV-hNaGlu vector at 8 minutes after mannitol pretreatment, using a polyclonal antibody against hNaGlu and a secondary antibody labeled with AlexaFluo⁵⁶⁸ (a–e). a. brain stem, b. hypothalamus, c. thalamus, d. Dorsal 3^rd ventricle and periventricular thalamic area, e. choroid plexus in lateral ventricle, f. thalamus of non-treated MPS IIIB mouse. Red arrows: NaGlu-positive neurons/glia and process. Yellow arrowheads: NaGlu-positive ependymal cells (d) and choroid plexus cells (e). D3V: dorsal 3^rd ventricle. Nuclei are labeled blue. Green fluorescence was used to separate autofluorecence from specific fluorescence signals (red). Scale bars: 10μm. B. NaGlu activity in brain (endpoint, n>8/group). C. Histopathology staining with toluidine blue. +/+: wt; −/−: non-treated; −/−+AAV: AAV-treated. CB: cerebella; TH: Thalamus. M: molecular layer; G: granule layer, red arrows: Purkinje cells; red arrowhead: enlarged lysosomes. Scale bar: 20μm. D. GAG contents in the brain of rAAV-treated MPS IIIB mice (3 months pi, n>8/group): expressed as μg/mg tissue (wet). *: P<0.05 (vs. −/−), #: P<0.05 (vs. +/+).

Fig. 3. Expression of recombinant NaGlu and decrease in lysosomal storage pathology in MPS IIIB mouse brain. A

Immunofluorescence staining for rNaGlu expression in the brain of MPS IIIB mouse (17-month-old) treated with an IV injection of rAAV-hNaGlu vector at 8 minutes after mannitol pretreatment, using a polyclonal antibody against hNaGlu and a secondary antibody labeled with AlexaFluo⁵⁶⁸ (a–e). a. brain stem, b. hypothalamus, c. thalamus, d. Dorsal 3^rd ventricle and periventricular thalamic area, e. choroid plexus in lateral ventricle, f. thalamus of non-treated MPS IIIB mouse. Red arrows: NaGlu-positive neurons/glia and process. Yellow arrowheads: NaGlu-positive ependymal cells (d) and choroid plexus cells (e). D3V: dorsal 3^rd ventricle. Nuclei are labeled blue. Green fluorescence was used to separate autofluorecence from specific fluorescence signals (red). Scale bars: 10μm. B. NaGlu activity in brain (endpoint, n>8/group). C. Histopathology staining with toluidine blue. +/+: wt; −/−: non-treated; −/−+AAV: AAV-treated. CB: cerebella; TH: Thalamus. M: molecular layer; G: granule layer, red arrows: Purkinje cells; red arrowhead: enlarged lysosomes. Scale bar: 20μm. D. GAG contents in the brain of rAAV-treated MPS IIIB mice (3 months pi, n>8/group): expressed as μg/mg tissue (wet). *: P<0.05 (vs. −/−), #: P<0.05 (vs. +/+).

Fig. 3. Expression of recombinant NaGlu and decrease in lysosomal storage pathology in MPS IIIB mouse brain. A

Immunofluorescence staining for rNaGlu expression in the brain of MPS IIIB mouse (17-month-old) treated with an IV injection of rAAV-hNaGlu vector at 8 minutes after mannitol pretreatment, using a polyclonal antibody against hNaGlu and a secondary antibody labeled with AlexaFluo⁵⁶⁸ (a–e). a. brain stem, b. hypothalamus, c. thalamus, d. Dorsal 3^rd ventricle and periventricular thalamic area, e. choroid plexus in lateral ventricle, f. thalamus of non-treated MPS IIIB mouse. Red arrows: NaGlu-positive neurons/glia and process. Yellow arrowheads: NaGlu-positive ependymal cells (d) and choroid plexus cells (e). D3V: dorsal 3^rd ventricle. Nuclei are labeled blue. Green fluorescence was used to separate autofluorecence from specific fluorescence signals (red). Scale bars: 10μm. B. NaGlu activity in brain (endpoint, n>8/group). C. Histopathology staining with toluidine blue. +/+: wt; −/−: non-treated; −/−+AAV: AAV-treated. CB: cerebella; TH: Thalamus. M: molecular layer; G: granule layer, red arrows: Purkinje cells; red arrowhead: enlarged lysosomes. Scale bar: 20μm. D. GAG contents in the brain of rAAV-treated MPS IIIB mice (3 months pi, n>8/group): expressed as μg/mg tissue (wet). *: P<0.05 (vs. −/−), #: P<0.05 (vs. +/+).

Fig.4. Expression of hNaGlu and correction of lysosomal storage in somatic tissues

Somatic tissues (endpoint, n>8/group) were assayed for NaGlu expression (a, b) and correction of lysosomal storage (c, d). a. NaGlu activity is expressed as unite (U)/mg protein. 1U = 1nmol 4MU released/h at 37°C. +/+: wt mice; −/−:/ non-treated MPS IIIB mice; −/− +AAV: AAV-treated MPS IIIB mice; −/− +AAV-8’ and −/− +AAV-10’: MPS IIIB mice IV injected with AAV vector at 8 or 10 min after mannitol infusion. L: liver, K: Kidney; S: spleen; H: heart; Lg: lung; I: intestine; M: skeletal muscle. b. Immunofluorescence staining for hNaGlu on cryostat liver sections of AAV-treated (−/− +AAV, 17-month-old) and non-treated (−/−) MPS IIIB mice. Scale bar: 50μm. c. GAG content in somatic tissues. d. Ultrastructural correction of lysosomal storage in liver of AAV-treated MPS IIIB mice. Arrow: a Kupffer cell. Arrow heads: enlarged lysosomes. e. Urine GAG content (3 months pi, n>4/group). GAG content was expressed as μg/mg tissue (wet) or μg/ml (urine). $: P<0.05 (vs. −/−). @: P<0.01 (vs. −/−); *: P<0.05 (vs. +/+); #: P<0.01 (vs. +/+); ˆ: P>0.05 (vs. −/−).

Fig.4. Expression of hNaGlu and correction of lysosomal storage in somatic tissues

Somatic tissues (endpoint, n>8/group) were assayed for NaGlu expression (a, b) and correction of lysosomal storage (c, d). a. NaGlu activity is expressed as unite (U)/mg protein. 1U = 1nmol 4MU released/h at 37°C. +/+: wt mice; −/−:/ non-treated MPS IIIB mice; −/− +AAV: AAV-treated MPS IIIB mice; −/− +AAV-8’ and −/− +AAV-10’: MPS IIIB mice IV injected with AAV vector at 8 or 10 min after mannitol infusion. L: liver, K: Kidney; S: spleen; H: heart; Lg: lung; I: intestine; M: skeletal muscle. b. Immunofluorescence staining for hNaGlu on cryostat liver sections of AAV-treated (−/− +AAV, 17-month-old) and non-treated (−/−) MPS IIIB mice. Scale bar: 50μm. c. GAG content in somatic tissues. d. Ultrastructural correction of lysosomal storage in liver of AAV-treated MPS IIIB mice. Arrow: a Kupffer cell. Arrow heads: enlarged lysosomes. e. Urine GAG content (3 months pi, n>4/group). GAG content was expressed as μg/mg tissue (wet) or μg/ml (urine). $: P<0.05 (vs. −/−). @: P<0.01 (vs. −/−); *: P<0.05 (vs. +/+); #: P<0.01 (vs. +/+); ˆ: P>0.05 (vs. −/−).

Fig.4. Expression of hNaGlu and correction of lysosomal storage in somatic tissues

Somatic tissues (endpoint, n>8/group) were assayed for NaGlu expression (a, b) and correction of lysosomal storage (c, d). a. NaGlu activity is expressed as unite (U)/mg protein. 1U = 1nmol 4MU released/h at 37°C. +/+: wt mice; −/−:/ non-treated MPS IIIB mice; −/− +AAV: AAV-treated MPS IIIB mice; −/− +AAV-8’ and −/− +AAV-10’: MPS IIIB mice IV injected with AAV vector at 8 or 10 min after mannitol infusion. L: liver, K: Kidney; S: spleen; H: heart; Lg: lung; I: intestine; M: skeletal muscle. b. Immunofluorescence staining for hNaGlu on cryostat liver sections of AAV-treated (−/− +AAV, 17-month-old) and non-treated (−/−) MPS IIIB mice. Scale bar: 50μm. c. GAG content in somatic tissues. d. Ultrastructural correction of lysosomal storage in liver of AAV-treated MPS IIIB mice. Arrow: a Kupffer cell. Arrow heads: enlarged lysosomes. e. Urine GAG content (3 months pi, n>4/group). GAG content was expressed as μg/mg tissue (wet) or μg/ml (urine). $: P<0.05 (vs. −/−). @: P<0.01 (vs. −/−); *: P<0.05 (vs. +/+); #: P<0.01 (vs. +/+); ˆ: P>0.05 (vs. −/−).

Fig.4. Expression of hNaGlu and correction of lysosomal storage in somatic tissues

Somatic tissues (endpoint, n>8/group) were assayed for NaGlu expression (a, b) and correction of lysosomal storage (c, d). a. NaGlu activity is expressed as unite (U)/mg protein. 1U = 1nmol 4MU released/h at 37°C. +/+: wt mice; −/−:/ non-treated MPS IIIB mice; −/− +AAV: AAV-treated MPS IIIB mice; −/− +AAV-8’ and −/− +AAV-10’: MPS IIIB mice IV injected with AAV vector at 8 or 10 min after mannitol infusion. L: liver, K: Kidney; S: spleen; H: heart; Lg: lung; I: intestine; M: skeletal muscle. b. Immunofluorescence staining for hNaGlu on cryostat liver sections of AAV-treated (−/− +AAV, 17-month-old) and non-treated (−/−) MPS IIIB mice. Scale bar: 50μm. c. GAG content in somatic tissues. d. Ultrastructural correction of lysosomal storage in liver of AAV-treated MPS IIIB mice. Arrow: a Kupffer cell. Arrow heads: enlarged lysosomes. e. Urine GAG content (3 months pi, n>4/group). GAG content was expressed as μg/mg tissue (wet) or μg/ml (urine). $: P<0.05 (vs. −/−). @: P<0.01 (vs. −/−); *: P<0.05 (vs. +/+); #: P<0.01 (vs. +/+); ˆ: P>0.05 (vs. −/−).

Fig.4. Expression of hNaGlu and correction of lysosomal storage in somatic tissues

Somatic tissues (endpoint, n>8/group) were assayed for NaGlu expression (a, b) and correction of lysosomal storage (c, d). a. NaGlu activity is expressed as unite (U)/mg protein. 1U = 1nmol 4MU released/h at 37°C. +/+: wt mice; −/−:/ non-treated MPS IIIB mice; −/− +AAV: AAV-treated MPS IIIB mice; −/− +AAV-8’ and −/− +AAV-10’: MPS IIIB mice IV injected with AAV vector at 8 or 10 min after mannitol infusion. L: liver, K: Kidney; S: spleen; H: heart; Lg: lung; I: intestine; M: skeletal muscle. b. Immunofluorescence staining for hNaGlu on cryostat liver sections of AAV-treated (−/− +AAV, 17-month-old) and non-treated (−/−) MPS IIIB mice. Scale bar: 50μm. c. GAG content in somatic tissues. d. Ultrastructural correction of lysosomal storage in liver of AAV-treated MPS IIIB mice. Arrow: a Kupffer cell. Arrow heads: enlarged lysosomes. e. Urine GAG content (3 months pi, n>4/group). GAG content was expressed as μg/mg tissue (wet) or μg/ml (urine). $: P<0.05 (vs. −/−). @: P<0.01 (vs. −/−); *: P<0.05 (vs. +/+); #: P<0.01 (vs. +/+); ˆ: P>0.05 (vs. −/−).