Global CNS correction in a large brain model of human alpha-mannosidosis by intravascular gene therapy

Abstract

Intravascular injection of certain adeno-associated virus vector serotypes can cross the blood-brain barrier to deliver a gene into the CNS. However, gene distribution has been much more limited within the brains of large animals compared to rodents, rendering this approach suboptimal for treatment of the global brain lesions present in most human neurogenetic diseases. The most commonly used serotype in animal and human studies is 9, which also has the property of being transported via axonal pathways to distal neurons. A small number of other serotypes share this property, three of which were tested intravenously in mice compared to 9. Serotype hu.11 transduced fewer cells in the brain than 9, rh8 was similar to 9, but hu.32 mediated substantially greater transduction than the others throughout the mouse brain. To evaluate the potential for therapeutic application of the hu.32 serotype in a gyrencephalic brain of larger mammals, a hu.32 vector expressing the green fluorescent protein reporter gene was evaluated in the cat. Transduction was widely distributed in the cat brain, including in the cerebral cortex, an important target since mental retardation is an important component of many of the human neurogenetic diseases. The therapeutic potential of a hu.32 serotype vector was evaluated in the cat homologue of the human lysosomal storage disease alpha-mannosidosis, which has globally distributed lysosomal storage lesions in the brain. Treated alpha-mannosidosis cats had reduced severity of neurological signs and extended life spans compared to untreated cats. The extent of therapy was dose dependent and intra-arterial injection was more effective than intravenous delivery. Pre-mortem, non-invasive magnetic resonance spectroscopy and diffusion tensor imaging detected differences between the low and high doses, and showed normalization of grey and white matter imaging parameters at the higher dose. The imaging analysis was corroborated by post-mortem histological analysis, which showed reversal of histopathology throughout the brain with the high dose, intra-arterial treatment. The hu.32 serotype would appear to provide a significant advantage for effective treatment of the gyrencephalic brain by systemic adeno-associated virus delivery in human neurological diseases with widespread brain lesions.

Keywords: adeno-associated virus; alpha-mannosidosis; global correction; gyrencephalic brain; systemic delivery.

Figure 1

Comparison of mouse brain transduction following intravenous delivery of transportable AAV vectors. (A) Intravenous injection of 2.9 × 10¹² vg total (1.4 × 10¹⁴ vg/kg) of AAV9, hu.11, rh.8 and hu.32 expressing eGFP in adult mice resulted in GFP expression throughout the brain 4 weeks post-injection (n = 3 mice for each group). Scale bar = 500 μm. (B) The amount of transduction was quantified by counting the number of GFP-positive objects throughout the brain in sections at distances from Bregma as shown. Horizontal lines represent means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Intracarotid injection of AAVhu.32 in cats results in widespread transduction in the brain. Three 6-week-old cats were injected with 6 × 10¹³ vg/kg of AAVhu.32-eGFP into the carotid artery. Vector transduction was analysed by immunohistochemistry for eGFP at 12 weeks of age. (A) Locations of the six brain sections analysed in each cat. The numbers indicate the position of the coronal sections shown in B. (C) Negative control brain section with no primary antibody. (D) Image from spinal cord; right panel is magnification of the square area in the left panel. Scale bars = 500 μm (B); 600 μm (D left panel); 60 μm (D right panel).

Figure 3

Neuronal transduction in the cat brain by carotid injection of AAVhu.32. The neuronal phenotype of the transduced cells in the brain was verified by dual immunofluorescent staining with antibodies against GFP and the neuron marker NeuN in the striatum, cortex and hippocampus. Images in the right-hand columns of GFP and merge are magnifications of the boxed area in the adjacent images on the left. Scale bars = 100 μm (left column); 50 μm (right column).

Figure 4

Clinical improvement in AMD cats following intracarotid injection of AAVhu.32. AMD cats were treated at 4–6 weeks of age with a single intracarotid injection of AAVhu.32 expressing fMANB at 2.9 × 10¹³ vg/kg (low dose, n = 3) or 4.8-5.0 × 10¹³ vg/kg (high dose, n = 3). Serum and CSF samples were collected before and after injection and assayed for MANB enzymatic activity in individual cats. (A) Serum MANB activity in the high dose group was elevated above untreated cats from 1 to 2 months and the low dose group beginning from 3 to 4 months post-injection and they remained elevated until the end point. (B) CSF MANB activity was elevated in the high dose treated animals by 1–2 months post-injection and remained elevated above untreated cats until the end point but declined over time. (C) All of the treated AMD cats (n = 6) lived longer than any of the untreated cats (n = 18) (P < 0.0001) and the high-dose group lived significantly longer than the low-dose group. (D) Monthly neurological examinations showed high dose treated cats had a delayed onset of whole-body tremor but not the onset of ataxia. At 18 weeks of age, both the whole-body tremor and truncal ataxia were less severe for both treatment groups compared to untreated cats. Values at each time point represent means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 5

Non-invasive MRS imaging show AAV dose-dependent correction in AMD cats. AMD cats were treated at 4–6 weeks of age with a single intra-carotid injection of AAVhu.32 expressing fMANB at 2.9 × 10¹³ vg/kg (low dose, n = 3) or 4.8 × 10¹³ vg/kg (high dose, n = 3). (A) ¹H-MRS at 3 T from thalamus shows correction by AAVhu.32-fMANB at the high dose. Large arrows point to the major peak of mannose-rich oligosaccharides (MS); small arrows to the minor peak (Magnitsky et al., 2010). (B) Representative haematoxylin and eosin stained brain sections of normal, untreated, and high and low dose of AAVhu.32-fMANB-treated cats. Storage lesions present in the cerebral cortex of untreated animals are reduced in AAVhu.32-fMANB treated animals. While the low dose treatment produced partial storage reduction, the high dose treatment resulted in complete correction of the storage lesions. Age at analysis: untreated 19 weeks (end of life), normal 38 weeks, low dose 37 weeks and high dose 56 weeks.

Figure 6

MANB enzymatic activity in treated AMD cat brain and peripheral organs. (A) MANB enzymatic activity in treated and untreated brains (n = 3 for high dose, n = 3 for low dose, n = 4 for untreated) was measured in coronal sections taken at intervals along the rostral-caudal axis (as shown in Fig. 2A). The mean activity was expressed as a percentage of normal. The transverse slices along the rostral-caudal axis show the wide spatial distribution within the brains of animals treated with high dose AAVhu.32-fMANB. (B) A significant increase in MANB enzymatic activity was measured in the liver, kidney, heart and spleen of high dose treated AMD cats. Horizontal lines represent means + SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Correction of lysosomal storage lesions in brain tissue of treated AMD cats. Representative haematoxylin and eosin stained brain sections of normal, untreated, and high and low dose AAVhu.32-fMANB-treated cats. Only partial storage reduction was seen in the low dose treated cats, while the high dose treatment group had complete correction of lysosomal storage vacuoles throughout the brain. Scale bar = 60 μm.