Comparative Analysis of Acid Sphingomyelinase Distribution in the CNS of Rats and Mice Following Intracerebroventricular Delivery

Niemann-Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we reported that biochemical and functional abnormalities observed in ASM knockout (ASMKO) mice could be partially alleviated by intracerebroventricular (ICV) infusion of hASM. We now show that this route of delivery also results in widespread enzyme distribution throughout the rat brain and spinal cord. However, enzyme diffusion into CNS parenchyma did not occur in a linear dose-dependent fashion. Moreover, although the levels of hASM detected in the rat CNS were determined to be within the range shown to be therapeutic in ASMKO mice, the absolute amounts represented less than 1% of the total dose administered. Finally, our results also showed that similar levels of enzyme distribution are achieved across rodent species when the dose is normalized to CNS weight as opposed to whole body weight. Collectively, these data suggest that the efficacy observed following ICV delivery of hASM in ASMKO mice could be scaled to CNS of the rat

Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington's disease.

Huntington's disease (HD) is caused by a toxic gain-of-function associated with the expression of the mutant huntingtin (htt) protein. Therefore, the use of RNA interference to inhibit Htt expression could represent a disease-modifying therapy. The potent

hASM levels in (A) rat brain, (B) spinal cord and (C) serum following a 6 h ICV infusion of hASM at different doses (0.1, 1.0 and 10 mg/kg) or aCSF.

<p>Significant (p<.01) levels of hASM were detected in all regions of the brain (i.e., cortex, preoptic region, striatum, hippocampus, thalamus, hypothalamus, midbrain, cerebellum and brainstem) in rats infused with 1 and 10 mg/kg of the enzyme. Rats infused with the 0.1 mg/kg dose displayed significant hASM levels in the cortex, preoptic region, striatum and hippocampus. No significant differences in hASM levels between regions were observed regardless of the dose tested. In each region of the spinal cord, significant (p<.01) levels of hASM were also detected in rats infused at either 1 or 10 mg/kg vs. rats infused with aCSF (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016313#pone-0016313-g002" target="_blank">Figure 2b</a>). Detected hASM levels in rats infused with 0.1 mg/kg were not significantly above background. Significant (p<.01) levels of hASM were detected in serum for all hASM infused rats regardless of the dose of enzyme used.</p

Percentage of the total administered dose of hASM detected in the (A) brain, (B) spinal cord and (C) serum of rats following 6 h ICV infusion.

<p>Animals were infused with 0.1, 1.0 or 10 mg/kg of hASM or aCSF. For both the brain and spinal cord, less than 0.2% of the total dose could be detected in each region. Cumulatively, the level of hASM detected in the brain and spinal cord was less than 1% of the total dose administered. No significant differences were observed between regions or different dose groups. Significant (p<.01) levels of hASM were detected in serum for each dose.</p

CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by a deficiency of survival motor neuron (SMN) due to mutations in the SMN1 gene. In this study, an adeno-associated virus (AAV) vector expressing human SMN (AAV8-hSMN) was injected at birth into the CNS of mice modeling SMA. Western blot analysis showed that these injections resulted in widespread expression of SMN throughout the spinal cord, and this translated into robust improvement in skeletal muscle physiology, including increased myofiber size and improved neuromuscular junction architecture. Treated mice also displayed substantial improvements on behavioral tests of muscle strength, coordination, and locomotion, indicating that the neuromuscular junction was functional. Treatment with AAV8-hSMN increased the median life span of mice with SMA-like disease to 50 days compared with 15 days for untreated controls. Moreover, injecting mice with SMA-like disease with a human SMN–expressing self-complementary AAV vector — a vector that leads to earlier onset of gene expression compared with standard AAV vectors — led to improved efficacy of gene therapy, including a substantial extension in median survival to 157 days. These data indicate that CNS-directed, AAV-mediated SMN augmentation is highly efficacious in addressing both neuronal and muscular pathologies in a severe mouse model of SMA

(A) Distribution of hASM in the rat CNS following a 6 h ICV infusion of hASM or aCSF (S1 = most rostral brain section and S5 = most caudal brain section, S6 = cervical spinal cord).

<p>Positive hASM staining was observed throughout the brain and spinal cord of rats administered with the enzyme. Staining intensity was maximal in regions proximal to ventricular compartments and the sub-arachnoid space. (B) Higher magnification (4× for brain; 20× for spinal cord) of hASM staining in regions between ventricular compartments and the tissue proximal to the sub-arachnoid space (area enclosed within the white box of sections S2 and S6). Shown are images from the brain and cervical spinal cord.</p

Merits of Combination Cortical, Subcortical, and Cerebellar Injections for the Treatment of Niemann-Pick Disease Type A

Niemann-Pick disease Type A (NPA) is a neuronopathic lysosomal storage disease (LSD) caused by the loss of acid sphingomyelinase (ASM). The goals of the current study are to ascertain the levels of human ASM that are efficacious in ASM knockout (ASMKO) mice, and determine whether these levels can be attained in non-human primates (NHPs) using a multiple parenchymal injection strategy. Intracranial injections of different doses of AAV1-hASM in ASMKO mice demonstrated that only a small amount of enzyme (&lt;0.5 mg hASM/g tissue) was sufficient to increase survival, and that increasing the amount of hASM did not enhance this survival benefit until a new threshold level of &gt;10 mg hASM/g tissue was reached. In monkeys, injection of 12 tracts of AAV1-hASM resulted in efficacious levels of enzyme in broad regions of the brain that was aided, in part, by axonal transport of adeno-associated virus (AAV) and movement through the perivascular space. This study demonstrates that a combination cortical, subcortical, and cerebellar injection protocol could provide therapeutic levels of hASM to regions of the NHP brain that are highly affected in NPA patients. The information from this study might help design new AAV-mediated enzyme replacement protocols for NPA and other neuronopathic LSDs in future clinical trials