7T MRI Predicts Amelioration of Neurodegeneration in the Brain after AAV Gene Therapy

Abstract

GM1 gangliosidosis (GM1) is a fatal neurodegenerative lysosomal storage disease that occurs most commonly in young children, with no effective treatment available. Long-term follow-up of GM1 cats treated by bilateral thalamic and deep cerebellar nuclei (DCN) injection of adeno-associated virus (AAV)-mediated gene therapy has increased lifespan to 8 years of age, compared with an untreated lifespan of ~8 months. Due to risks associated with cerebellar injection in humans, the lateral ventricle was tested as a replacement route to deliver an AAVrh8 vector expressing feline β-galactosidase (β-gal), the defective enzyme in GM1. Treatment via the thalamus and lateral ventricle corrected storage, myelination, astrogliosis, and neuronal morphology in areas where β-gal was effectively delivered. Oligodendrocyte number increased, but only in areas where myelination was corrected. Reduced AAV and β-gal distribution were noted in the cerebellum with subsequent increases in storage, demyelination, astrogliosis, and neuronal degeneration. These postmortem findings were correlated with endpoint MRI and magnetic resonance spectroscopy (MRS). Compared with the moderate dose with which most cats were treated, a higher AAV dose produced superior survival, currently 6.5 years. Thus, MRI and MRS can predict therapeutic efficacy of AAV gene therapy and non-invasively monitor cellular events within the GM1 brain.

Keywords: AAV; GM1; MRI; adeno-associated virus; biomarkers; gangliosidosis; gene therapy; lysosomal storage disease; spectroscopy.

Figure 1

Clinical Disease Progression and Survival of GM1+AAV-Treated Cats (A) Clinical rating scores representing neurologic disease in normal cats (black circles), a representative untreated GM1 cat (closed gray triangles), a GM1 cat treated with AAV at a high dose (closed gray circles), and GM1 cats treated with AAV at a low dose (open symbols). (B) Kaplan-Meier survival curve. GM1 cat treated with AAV at a high dose (black line), GM1+AAV low-dose cats (dark gray line), and untreated GM1 cats (light gray line).

Figure 2

7T MRI of GM1 Cats after Gene Therapy (A) In T2-weighted images from normal cats, white matter is represented by dark gray regions and gray matter by light gray regions. CSF is represented by white pixels. In the GM1 cat, there is an overall isointensity or lack of difference between the gray and white matter in the cerebral cortex and cerebellum. At 8 months in the AAV-treated cats (low dose), the brain is largely normalized except for the temporal lobe (white box) and parts of the cerebellum. (B–E) Intensities on MRI were calculated in an individual slice at the level of the striatum (B), thalamus (C), occipital cortex (D), and cerebellum (E) in normal cats, untreated GM1 cats, and low-dose GM1+AAV cats at 8 months and at the humane endpoint. The intensity associated with increased lipid content (dark gray) was increased in GM1 untreated cats because of ganglioside storage in cell bodies of the gray matter. After treatment, gray and white matter intensities are normalized. *p < 0.05, **p < 0.01 from age-matched normal cats; ^†p < 0.05, ^Ŧp < 0.01 from untreated GM1 cats at the humane endpoint. Error bars represent standard deviation.

Figure 3

MRS of the Cerebellum after AAV Gene Therapy Brain metabolites were measured in untreated GM1 cats at early disease stage (4 months) and humane endpoint (~8 months), and in age-matched normal controls (yellow line). Also measured were GM1+AAV-treated cats at 8 months or long term. The following metabolites were included: myoinositol (Ins, a marker of gliosis), N-acetylaspartate (NAA, a marker of neuroaxonal health), glycerophosphocholine and phosphocholine (GPC+PCh, an indication of demyelination), NAA+N-acetyl aspartyl glutamate (NAAG, also indicating neuroaxonal health), creatine and phosphocreatine (Cr+PCr, markers of metabolism), and glutamate and glutamine (Glu+Gln, markers of the glutaminergic neurotransmitter recycling system). (A) Partially or fully corrected metabolites of GM1 cats treated by gene therapy included Ins, GPC+PCh, and Cr+PCr. *p < 0.05, **p < 0.01 versus age-matched normal; ^†p < 0.05, ^Ŧp < 0.01 from untreated GM1 cats at the humane endpoint. (B) Metabolites were plotted against clinical status (clinical rating scores) at the time of MRS to evaluate correlation with clinical disease (R²). Error bars represent standard deviation.

Figure 4

Biodistribution of β-Gal in the Brain and Spinal Cord of GM1+AAV (Low-Dose) Cats (A) Solid white dots indicate thalamic injection sites, whereas the open white dot represents the lateral ventricular injection site. Cat brain was divided at necropsy into 6-mm blocks, shown by white lines, and blocks were labeled A–I. The right hemisphere was used for β-gal staining and activity assays. (B) Blue histochemical staining represents β-gal activity throughout the brain. Below each stained section is shown quantitative β-gal activity expressed as fold of normal levels. (C) Spinal cord blocks are denoted by letters J–P and correspond to the coronal sections stained for β-gal activity (blue). Shown is a representative example of a GM1 cat treated with the low dose and followed to the humane endpoint (8-1701), as well as control sections from untreated normal and GM1 cats.

Figure 5

Storage Material in GM1 Cats Treated with the Low Dose of Gene Therapy Periodic acid-Schiff stain (converted to grayscale) is shown to illustrate the storage pattern in the striatum/parietal cortex, caudal thalamus/temporal cortex, occipital cortex/midbrain, and cerebellum/brainstem. In the normal cat, white matter is darker than gray matter, and this is inverted in the GM1 cat because of high storage levels in neuronal cell bodies. Gene therapy normalized storage levels except in discrete areas of the parietal cortex, temporal lobe, and cerebellum.

Figure 6

Astrocyte (GFAP) Staining after AAV Gene Therapy (Low Dose) (A) Representative GFAP staining of the parietal cortex in normal, GM1, or GM1+AAV (low-dose) cats at 8 months and at the humane endpoint. (B) Quantification of GFAP staining in the striatum, thalamus, parietal cortex, temporal lobe, occipital cortex, and cerebellum. (C) Correlation of cerebellar GFAP staining with myoinositol (Ins) concentrations from MRS. (D) Correlation of GFAP staining in the cerebellum with the clinical rating score of cats. *p < 0.05, **p < 0.01 from age-matched normal controls; ^†p < 0.05, ^Ŧp < 0.01 from GM1 cats at the humane endpoint. Error bars represent standard deviation.

Figure 7

Myelin (Luxol Fast Blue) Staining and the Effect of AAV Gene Therapy (A) Representative Luxol fast blue stains from normal, GM1, and GM1+AAV (low dose) cats at 8 months and at the humane endpoint (scale bars, 10 μm). (B) Myelin density in the internal capsule, thalamus, parietal cortex, temporal lobe, occipital cortex, and cerebellum. (C) Transmission electronic microscopy (TEM) of the white matter of the temporal lobe of normal (left), untreated GM1 (middle), and GM1+AAV at the humane endpoint (right). White arrows show the myelin sheath. *p < 0.05 or **p < 0.01 from age-matched normal; ^†p < 0.05 or ^Ŧp < 0.01 from GM1 cats at endpoint; ^§p < 0.05 from 8-month time point. Error bars represent standard deviation.

Figure 8

Oligodendrocyte (Olig2) Alterations in GM1 Cats after AAV-Mediated Gene Therapy (A) Representative photomicrograph of oligodendrocytes in the white matter of normal, GM1, and GM1+AAV-treated cats in the parietal cortex (scale bars, 10 μm). (B) Quantification of oligodendrocyte density in the gray and white matter of the striatum, thalamus, parietal cortex, temporal lobe, occipital cortex, and cerebellum. (C) Transmission electron microscopy (TEM) of oligodendrocytes in the temporal lobe of the normal (left), GM1 (middle), and GM1+AAV cat at the humane endpoint (right). *p < 0.05, **p < 0.01 from age-matched normal controls; ^†p < 0.05 from GM1 cats at the humane endpoint. Error bars represent standard deviation.