Combination small molecule PPT1 mimetic and CNS-directed gene therapy as a treatment for infantile neuronal ceroid lipofuscinosis

Abstract

Infantile neuronal ceroid lipofuscinosis (INCL) is a profoundly neurodegenerative disease of children caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). There is currently no effective therapy for this invariably fatal disease. To date, preclinical experiments using single treatments have resulted in incremental clinical improvements. Therefore, we determined the efficacy of CNS-directed AAV2/5-mediated gene therapy alone and in combination with the systemic delivery of the lysosomotropic PPT1 mimetic phosphocysteamine. Since CNS-directed gene therapy provides relatively high levels of PPT1 activity to specific regions of the brain, we hypothesized that phosphocysteamine would complement that activity in regions expressing subtherapeutic levels of the enzyme. Results indicate that CNS-directed gene therapy alone provided the greatest improvements in biochemical and histological measures as well as motor function and life span. Phosphocysteamine alone resulted in only minor improvements in motor function and no increase in lifespan. Interestingly, phosphocysteamine did not increase the biochemical and histological response when combined with AAV2/5-mediated gene therapy, but it did result in an additional improvement in motor function. These data suggest that a CNS-directed gene therapy approach provides significant clinical benefit, and the addition of the small molecule PPT1 mimetic can further increase that response.

Fig. 1a, b

PPT1 activity and secondary lysosomal enzyme elevations in the brain at 7 months of age. a PPT1 activity in the treated and untreated brains. Ppt1-/- mice treated with either AAV only or AAV+ phosphocysteamine resulted in a twofold increase in PPT1 enzyme activity compared to WT brains. Untreated Ppt1-/- brains or those treated with only phosphocysteamine had undetectable levels of PPT1 activity in the brain. b Secondary lysosomal enzyme elevations in the treated and untreated brains. To ascertain whether secondary elevations in lysosomal enzyme activity were decreased in response to therapy, we performed β-glucoronidase activities on the brains from treated and untreated mice. There was a six fold increase in β-glucoronidase activity in the brains of untreated Ppt1-/- mice compared to normal. A similar increase in β-glucoronidase activity was observed in the phosphocysteamine only group. Following either AAV only or AAV+phosphocysteamine treatment, there was a decrease in secondary enzyme elevation, returning β-glucoronidase activity to WT levels. (*p<0.05, ***p<0.001)

Fig. 2a-d

Histological improvement in the cortex following therapy. a Representative micrographs of the S1BF cortex at 7 months. Nissl- stained sections reveal the pronounced thinning of the primary somato- sensory cortex (S1BF) of Ppt1-/- mice compared to WT controls. Mutant mice treated with phosphocysteamine display a similar level of cortical thinning as the Ppt1-/- mice. Treatment with either AAV only or AAV+phophocysteamine appeared to prevent the thinning of S1BF. b Cortical thickness measurements at 7 months. Both the Ppt1-/- group and the phosphocysteamine-treated mutant mice displayed a significant thinning of the S1BF compared to WT mice. Following treatment, the AAV only and AAV+phosphocysteamine treatment groups both exhibited a significant increase in cortical thickness when compared to untreated Ppt1-/- mice. c Overall brain atrophy in the treated and untreated mice at 7 months and terminal time points. Ppt1-/- mice display a significant reduction in brain mass at 7 months of age in comparison to WT mice. A similar reduction in brain mass was seen in phosphocysteamine-treated mutant mice. Notably, the AAVonly and AAV+phosphocysteamine–treated mutant mice displayed a significant increase in brain mass at 7 months of age. However, this therapeutic benefit was lost at a terminal time point in both treated groups. d Auto- fluorescent storage material accumulation in 7 month S1BF cortex of treated and untreated mice. Compared to WT mice, untreated Ppt1-/- mice and phosphocysteamine only groups display a significant accumulation of autofluorescent storage material within the S1BF cortex. Following treatment, there is a significant reduction in autofluorescent material in the AAV only and AAV+phosphocysteamine groups. (*p< 0.05, **p<0.01, n.s. 0 no significant difference)

Fig. 3a-d

Decreased neuroinflammation following therapeutic intervention. a Astrocyte activation in the S1BF cortex at 7 months. Untreated Ppt1-/-mice and phosphocysteamine-treated mice display pronounced GFAP staining in the S1BF cortex, indicative of astrocyte activation in this brain region. This was reduced following AAV only or AAV+phosphocysteamine treatment. b Quantification of GFAP immunoreactivity in the S1BF cortex at 7 months. Thresholding image analysis reveals a significant increase in GFAP immunoreacitvity in the S1BF of both untreated Ppt1-/- and phosphocysteamine-treated mice compared to WT controls. Treatment with either AAV only or AAV+ phosphocysteamine resulted in a significant decrease in GFAP immunoreactivity within the S1BF. Notably, AAV+phosphocysteamine–treated mice still displayed significant elevations in GFAP staining compared to AAV only–treated mutant mice. c Microglial activation in the S1BF cortex at 7 months. Staining for CD68 revealed a pronounced increase in staining in the S1BF cortex of Ppt1-/- mice compared to WTcontrols. A similar pattern of CD68 immunoreactivity was apparent in the phosphocysteamine-treated mice. Conversely, the S1BF of Ppt1-/- mice treated with either AAVonly or AAV+phosphocysteamine appeared to have fewer, less intensely stained CD68+ cells than were seen in untreated mutants. d Quantification of CD68 immunoreactivity in the S1BF cortex at 7 months. Both the Ppt1-/- and phosphocysteamine-treated mice displayed significantly more CD68 staining in the S1BF compared to WT controls. Relative to untreated Ppt1-/- mice, treatment with either AAV only or AAV+phosphocysteamine resulted in a significant reduction in the level of CD68 immunoreactivity in the S1BF. (*p<0.05, **p<0.01, ***p<0.001)

Fig. 4

Autofluorescent storage material accumulation, astrocytosis, and microglia activation in treated and untreated Ppt1-/- mice. Fluorescent micrographs of autofluorescent storage, GFAP+astrocytes, and CD68+ microglia in the S1BF cortex of WT, Ppt1-/- , phosphocysteamine-treated, AAV-treated, and AAV+phosphocysteamine–treated Ppt1-/- mice. No autofluorescent accumulation or glial activation is present in the WT mice at 7 months of age. Conversely, there are high levels of autofluorescent accumulation in the S1BF cortex of untreated Ppt1-/- mice, which coincides with both astrocyte (GFAP) and microglial (CD68) upregulation. In the inset, note that there is more autofluorescent accumulation within CD68+ cells than GFAP+cells, although the majority of storage material appears to be localized to another cell type, most likely the neurons. The storage material in phosphocysteamine-treated mice appears less than the untreated Ppt1-/- mice, while treatment with phosphocysteamine does not appear to affect GFAP or CD68 levels in the S1BF. Conversely, treatment with either AAVonly or AAV+phosphocysteamine resulted in decreased autofluorescence, GFAP+immunostaining, and CD68+ immunostaining. Scale bar 0100 μm

Fig. 5a, b

Functional improvement on the rocking rotorod paradigm. a Motor improvement observed following therapeutic intervention. At 5 months of age, all treated and untreated mice performed similarly on the rocking rotorod paradigm. At 6 months, the performance of the phosphocysteamine only and untreated Ppt1-/- mice began to deteriorate and was significantly worse compared to WTcontrols (p<0.0001). Phosphocysteamine-treated mutant mice performed better at 6 months of age compared to untreated Ppt1-/- mice (ε). Remarkably, the performance of the AAV only or AAV+phosphocysteamine treated–mice was comparable to W T. The motor function of both phosphocysteamine-treated and untreated Ppt1-/- mice deteriorated dramatically by 7 months of age. At 7 months, AAV+phosphocysteamine–treated mice not only performed significantly better than AAV only–treated mice (p<0.001), but the motor performance of the combination group was indistinguishable from WT (ϕ). At 8 months, motor function in AAV only and AAV+phosphocysteamine–treated mice began to decline, but the performance of the AAV+phophocysteamine treatment group was still significantly improved compared to AAV only–treated mutant mice (γ; p<0.05). Motor performance continued to decline in both the AAVonly and AAV+ phophocysteamine–treated mice until they were unable to accomplish this task at 12 months of age. b Scatter plot of rocking rotorod data for 7-month-old mice. Each point represents an individual trial for each mouse tested within each treatment group. In the WT group, all mice stayed on the rod for 60 s. In comparison, the phosphocysteamine and untreated Ppt1-/- mice performed poorly, and each individual trial clustered around 10 s. Of particular interest is the comparison between the AAV only and AAV+phosphocysteamine groups. The variability within the AAV only treatment group was far greater than the AAV+phosphocysteamine group, suggesting that combination therapy was more effective than AAV treatment alone

Fig. 6

Improvement in lifespan following AAV-mediated gene therapy with or without phosphocysteamine. The median lifespan of the untreated Ppt1-/-mice was 36.5 weeks compared to 34 weeks in the phosphocysteamine-treated mutant mice. There was a significant improvement in lifespan in mutant mice treated with either AAV only or AAV+phosphocysteamine. Treatment with AAV only increased their median lifespan to 54 weeks while AAV+ phosphocysteamine increased median lifespan to 59 weeks, although this difference was not significant