Intravitreal gene therapy reduces lysosomal storage in specific areas of the CNS in mucopolysaccharidosis VII mice

Abstract

The mucopolysaccharidoses (MPSs) are lysosomal storage diseases resulting from impaired catabolism of sulfated glycosaminoglycans. MPS VII mice lack lysosomal beta-glucuronidase (GUSB) activity, leading to the accumulation of partially degraded chondroitin, dermatan, and heparan sulfates in most tissues. Consequently, these mice develop most of the symptoms exhibited by human MPS VII patients, including progressive visual and cognitive deficits. To investigate the effects of reducing lysosomal storage in nervous tissues, we injected recombinant adeno-associated virus encoding GUSB directly into the vitreous humor of young adult mice. Interestingly, GUSB activity was subsequently detected in the brains of the recipients. At 8-12 weeks after treatment, increased GUSB activity and reduced lysosomal distension were found in regions of the thalamus and tectum that received inputs from the injected eye. Lysosomal storage was also reduced in adjacent nonvisual regions, including the hippocampus, as well as in the visual cortex. The findings suggest that both diffusion and trans-synaptic transfer contribute to the dissemination of enzyme activity within the CNS. Intravitreal injection may thus provide a means of delivering certain therapeutic gene products to specific areas within the CNS.

Fig. 1.

GUSB activity is present in the brain along the visual pathway. A, An oblique coronal section through the brain of an MPS VII mouse that received a unilateral intravitreal injection of AAVβGEnh. GUSB activity (red) is present in the optic nerve (ON) from the mouse's left (injected) eye but not in the ON from the untreated right eye. B, An adjacent section stained with cresyl violet shows the cellular architecture. C, A parallel section through the optic chiasm (OC) and tract (OT), lateral geniculate nucleus (LGN), and superior colliculus (SC) of the same brain shows prominent GUSB activity on the side contralateral to the injected eye, as well as a lower level of activity in the ipsilateral LGN. This pattern of enzyme activity was seen in all brains from MPS VII mice that received unilateral intravitreal AAVβGEnh.D, A section adjacent to that in C, stained with cresyl violet. E, A coronal section through the brain of an MPS VII mouse that received bilateral intravitreal AAVβGEnh shows a symmetrical pattern of high-level enzyme activity.F, A parallel section from the same brain asE shows high-level activity in both SCs. L, Mouse's left; R, mouse's right. Sections stained for GUSB activity are counterstained with methyl green.

Fig. 2.

The distribution of GUSB activity throughout the brain is seen in a three-dimensional reconstruction from another 16-week-old MPS VII mouse treated unilaterally with intravitreal AAVβGEnh at 4 weeks of age. A, Lateral view of the reconstructed image shows surface contours of the cerebral hemisphere, cerebellum, and brainstem, from anterior (ant.) to posterior (post.).B, Rendering the image transparent shows the pattern of GUSB activity along the visual pathway. C, Frontal view, showing the surface contours of the reconstruction. The right (blue) and left (red) optic nerves (ON) can be seen entering the brain. Right and left indicate the right and left sides of the mouse.D, In the transparent image, GUSB activity is most prominent in the optic nerve and tract, lateral geniculate nucleus (LGN), and superior colliculus (SC) on the right side, contralateral to the injected eye. Lower levels of activity can also be seen in the ipsilateral optic pathway.

Fig. 3.

Intravitreal AAVβGEnh decreases lysosomal storage in hippocampus and visual cortex as well as LGN. Pairs of toluidine blue-stained, 0.5-μm-thick sections from the CA1 and CA4 regions of the hippocampus, visual cortex (VC), nonvisual cortex (NVC), and lateral geniculate nucleus (LGN) are from two 16-week-old MPS VII mice. The sections indicated by “−” are from the mouse that received no treatment. The sections indicated by “+” are from a littermate that received unilateral intravitreal AAVβGEnh at 4 weeks of age. Black arrows indicate distended vacuoles in neurons; white arrows indicate storage in microglia and other nonneuronal cells. The approximate sites of the respective histologic samples are indicated on the GUSB-stained cryostat section.

Fig. 4.

PCR reveals AAVβGEnh DNA in the injected eyes of treated mice, but not in brain samples. A, DNA from the right optic nerve (lane 1), left (injected) eye (lane 2), left optic nerve (lane 3), optic chiasm (lane 4), right LGN (lane 5), posterior LGN + anterior SC (lane 6), and SC (lane 7) of a 14-week-old MPS VII mouse that received an intravitreal injection at 6 weeks of age. Primers amplified a fragment of 240 bp from the human GUSB cDNA sequence encoded by AAVβGEnh as well as a 454 bp fragment from the endogenous mouse GUSB gene. B, Murine fibroblasts (3521 cells) containing one retroviral hGUSB cDNA insert per cell (lane 1) were titered with untransduced 3521 cells (lane 7). Cell mixtures contained one copy of hGUSB per 100 haploid genomes (lane 2), one per 1 × 10³ (lane 3), one per 2 × 10³ (lane 4), one per 10⁴ (lane 5), and one per 2 × 10⁴ (lane 6). Fragment sizes are given in base pairs.