Wendy Yang

Delivery of AAV-IGF-1 to the CNS extends survival in ALS mice through modification of aberrant glial cell activity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. Recent work in rodent models of ALS has shown that insulin-like growth factor-1 (IGF-1) slows disease progression when delivered at disease onset. However, IGF-1s mechanism of action along the neuromuscular axis remains unclear. In this study, symptomatic ALS mice received IGF-1 through stereotaxic injection of an IGF-1-expressing viral vector to the deep cerebellar nuclei (DCN), a region of the cerebellum with extensive brain stem and spinal cord connections. We found that delivery of IGF-1 to the central nervous system (CNS) reduced ALS neuropathology, improved muscle strength, and significantly extended life span in ALS mice. To explore the mechanism of action of IGF-1, we used a newly developed in vitro model of ALS. We demonstrate that IGF-1 is potently neuroprotective and attenuates glial cell-mediated release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO). Our results show that delivering IGF-1 to the CNS is sufficient to delay disease progression in a mouse model of familial ALS and demonstrate for the first time that IGF-1 attenuates the pathological activity of non-neuronal cells that contribute to disease progression. Our findings highlight an innovative approach for delivering IGF-1 to the CNS.

Intracranial Delivery of CLN2 Reduces Brain Pathology in a Mouse Model of Classical Late Infantile Neuronal Ceroid Lipofuscinosis

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a lysosomal storage disorder caused by mutations in CLN2, which encodes lysosomal tripeptidyl peptidase I (TPP1). Lack of TPP1 results in accumulation of autofluorescent storage material and curvilinear bodies in cells throughout the CNS, leading to progressive neurodegeneration and death typically in childhood. In this study, we injected adeno-associated virus (AAV) vectors containing the human CLN2 cDNA into the brains of CLN2−/− mice to determine therapeutic efficacy. AAV2CUhCLN2 or AAV5CUhCLN2 were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age, and mice were then killed at 13 weeks after injection. Mice treated with AAV2CUhCLN2 and AAV5CUhCLN2 contained TPP1 activity at each injection tract that was equivalent to 0.5- and 2-fold that of CLN2+/+ control mice, respectively. Lysosome-associated membrane protein 1 immunostaining and confocal microscopy showed intracellular targeting of TPP1 to the lysosomal compartment. Compared with control animals, there was a marked reduction of autofluorescent storage in the AAV2CUhCLN2 and AAV5CUhCLN2 injected brain regions, as well as adjacent regions, including the striatum and hippocampus. Analysis by electron microscopy confirmed a significant decrease in pathological curvilinear bodies in cells. This study demonstrates that AAV-mediated TPP1 enzyme replacement corrects the hallmark cellular pathologies of cLINCL in the mouse model and raises the possibility of using AAV gene therapy to treat cLINCL patients.

Intracerebral Transplantation of Adult Mouse Neural Progenitor Cells into the Niemann-Pick-A Mouse Leads to a Marked Decrease in Lysosomal Storage Pathology

Niemann-Pick disease is caused by a genetic deficiency in acid sphingomyelinase (ASM) leading to the intracellular accumulation of sphingomyelin and cholesterol in lysosomes. In the present study, we evaluated the effects of direct intracerebral transplantation of neural progenitor cells (NPCs) on the brain storage pathology in the ASM knock-out (ASMKO) mouse model of Type A Niemann-Pick disease. NPCs derived from adult mouse brain were genetically modified to express human ASM (hASM) and were transplanted into multiple regions of the ASMKO mouse brain. Transplanted NPCs survived, migrated, and showed region-specific differentiation in the host brain up to 10 weeks after transplantation (the longest time point examined). In vitro, gene-modified NPCs expressed up to 10 times more and released five times more ASM activity into the culture media compared with nontransduced NPCs. In vivo, transplanted cells expressed hASM at levels that were barely detectable by immunostaining but were sufficient for uptake and cross-correction of host cells, leading to reversal of distended lysosomal pathology and regional clearance of sphingomyelin and cholesterol storage. Within the host brain, the area of correction closely overlapped with the distribution of the hASM-modified NPCs. No correction of pathology occurred in brain regions that received transplants of nontransduced NPCs. These results indicate that the presence of transduced NPCs releasing low levels of hASM within the ASMKO mouse brain is necessary and sufficient to reverse lysosomal storage pathology. Potentially, NPCs may serve as a useful gene transfer vehicle for the treatment of CNS pathology in other lysosomal storage diseases and neurodegenerative disorders.

Intracerebroventricular infusion of acid sphingomyelinase corrects CNS manifestations in a mouse model of Niemann-Pick A disease.

Niemann-Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we showed that the storage pathology in the ASM knockout (ASMKO) mouse brain could be corrected by intracerebral injections of cell, gene and protein based therapies. However, except for instances where distal areas were targeted with viral vectors, correction of lysosomal storage pathology was typically limited to a region within a few millimeters from the injection site. As NPA is a global neurometabolic disease, the development of delivery strategies that maximize the distribution of the enzyme throughout the CNS is likely necessary to arrest or delay progression of the disease. To address this challenge, we evaluated the effectiveness of intracerebroventricular (ICV) delivery of recombinant human ASM into ASMKO mice. Our findings showed that ICV delivery of the enzyme led to widespread distribution of the hydrolase throughout the CNS. Moreover, a significant reduction in lysosomal accumulation of sphingomyelin was observed throughout the brain and also within the spinal cord and viscera. Importantly, we demonstrated that repeated ICV infusions of ASM were effective at improving the disease phenotype in the ASMKO mouse as indicated by a partial alleviation of the motor abnormalities. These findings support the continued exploration of ICV delivery of recombinant lysosomal enzymes as a therapeutic modality for LSDs such as NPA that manifests substrate accumulation within the CNS.

Relationship between neuropathology and disease progression in the SOD1G93A ALS mouse

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. However, recent reports suggest an active role of non-neuronal cells in the pathogenesis of the disease. Here, we examined quantitatively the temporal development of neuropathologic features in the brain and spinal cord of a mouse model of ALS (SOD1(G93A)). Four phases of the disease were studied in both male and female SOD1(G93A) mice: presymptomatic (PRE-SYM), symptomatic (SYM), endstage (ES) and moribund (MB). Compared to their control littermates, SOD1(G93A) mice showed an increase in astrogliosis in the motor cortex, spinal cord and motor trigeminal nucleus in the SYM phase that worsened progressively in ES and MB animals. Associated with this increase in astrogliosis was a concomitant increase in motor neuron cell death in the spinal cord and motor trigeminal nucleus in both ES and MB mice, as well as in the ventrolateral thalamus in MB animals. In contrast, microglial activation was significantly increased in all the same regions but only when the mice were in the MB phase. These results suggest that astrogliosis preceded or occurred concurrently with neuronal degeneration whereas prominent microgliosis was evident later (MB stage), after significant motor neuron degeneration had occurred. Hence, our findings support a role for astrocytes in modulating the progression of non-cell autonomous degeneration of motor neurons, with microglia playing a role in clearing degenerating neurons.

Injection of mouse and human neural stem cells into neonatal Niemann-Pick A model mice.

Cloned mouse C17.2 neural stem cells (NSCs) or human NSCs were injected into five CNS sites in very large numbers (100,000 cells/site, or a total of 500,000 cells) into 18 neonatal mice homozygous for a targeted deletion (knockout) of the acid sphingomyelinase (ASM) gene (called ASMKO mice), a faithful model of human Niemann-Pick type A (NP-A) disease, and into 10 wild-type mice, all on the C57BL/6J background. Injected mice were not immunosuppressed, and all survived to adulthood. Non-injected ASMKO controls had developed widespread neuronal and glial vacuolation and lysosomal accumulation of sphingomyelin and cholesterol when examined histologically at 16 weeks of age. Unlike children with NP-A disease, the ASMKO mice also lose cerebellar Purkinje neurons progressively, are ataxic, and show parallel progressive declines in rotorod performance. At 16 weeks NSC-injected mice showed a dramatic decrease in neuronal and glial vacuolation (by standard histological staining) and in cholesterol accumulation (by filipin fluorescence staining) throughout the cerebral neocortex, hippocampal formation, striatum and cerebellum, with lesser but clear improvement throughout the brainstem. Improvement was modestly but consistently better in human HFT13-injected than in mouse C17.2-injected ASMKO mice. Improvement in the ASMKO brains was more widespread than the distribution of NSCs, an indication that ASM must have been secreted and diffused at therapeutic concentrations beyond the territory occupied by NSCs. However, though Purkinje cell rescue has been achieved with NSCs in some other disease models, loss of Purkinje neurons and decline in rotorod performance were still present in injected ASMKO mice.

Intraparenchymal injections of acid sphingomyelinase results in regional correction of lysosomal storage pathology in the Niemann-Pick A mouse

Niemann-Pick A disease (NPD-A) is caused by a deficiency of acid sphingomyelinase (ASM) leading to the intracellular accumulation of sphingomyelin and cholesterol in lysosomes. We evaluated the effects of direct intraparenchymal brain injections of purified recombinant human ASM (hASM) at correcting the storage pathology in a mouse model of NPD-A (ASMKO). Different doses (0.1 ng to 10 mug of hASM) were injected into the right hemisphere of the hippocampus and thalamus of 12- to 14-week-old ASMKO mice. Immunohistochemical analysis after 1 week indicated that animals treated with greater than 1 mug hASM/site showed detectable levels of enzyme around the injected regions. However, localized clearance of sphingomyelin and cholesterol storage were observed in animals administered lower doses of enzyme, starting at 100 ng hASM/site. Areas of correction were also noted at distal sites such as in the contralateral hemispheres. Indications of storage re-accumulation were seen after 2 weeks post-injection. Injections of hASM did not cause any significant cell infiltration, astrogliosis, or microglial activation. These results indicate that intraparenchymal injection of hASM is associated with minimal toxicity and can lead to regional reductions in storage pathology in the ASMKO mouse.

427. AAV2- and AAV5-Mediated CNS Delivery of Human CLN2 Reduces Lysosomal Storage in a Mouse Model of Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a neurometabolic disorder caused by mutations in CLN2, which encodes lysosomal tripeptidyl peptidase (TPP-1). Lack of TPP-1 results in intracellular accumulation of autofluorescent storage material within the CNS, neurodegeneration and death typically in childhood. In this study, we injected clinical-grade AAV vectors containing the human CLN2 cDNA into the LINCL mouse brain to determine therapeutic efficacy. A total of 3.6 |[times]| 109 genome copies of AAV2CUhCLN2 (n=5) or AAV5CUhCLN2 (n=6) were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age. At 13 weeks post-injection (19 weeks of age), mice were sacrificed and brains hemispheres were separated from each other. The left hemisphere was analyzed for TPP-1 enzyme activity, and the right hemisphere was processed for immunohistochemistry and detection of autoflourescent storage material. Brains treated with AAV2CUhCLN2 contained TPP-1 activity at each of three injection tracts equivalent to that of levels in the brains of heterozygote which are phenotypically normal. In contrast, AAV5CUhCLN2-injected brains had approximately two fold wild-type levels of enzyme activity within these tracts. Immunohistochemistry and confocal microscopy of the right hemispheres showed the presence of TPP-1 protein in the lysosomes and neuronal processes of cells at the injection sites. Compared to control animals (AAV2CUNull, sham, uninjected), there was a marked reduction of autofluorescent storage material with AAV2CUCLN2 and AAV5CUCLN2 in the injected regions which include motor cortex, thalamus, cerebellum, as well as other regions such as the striatum and the hippocampus. There was no statistical difference in the amount of autofluorescent reduction mediated by the two serotype vectors despite the higher levels of enzyme activity with AAV5CUCLN2. Behavioral testing on the rotorod showed improvement of cerebellar motor function at 8 and 10 weeks post-injection with both viral vectors. This study demonstrates that the LINCL brain is responsive to TPP-1 replacement with different AAV serotype vectors, and that heterozygote levels of enzyme activity is sufficient to reverse lysosomal pathology and partially restore behavioral function.