Rescue of a Mouse Model of Spinal Muscular Atrophy With Respiratory Distress Type 1 by AAV9-IGHMBP2 Is Dose Dependent.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive disease occurring during childhood. The gene responsible for disease development is a ubiquitously expressed protein, IGHMBP2. Mutations in IGHMBP2 result in the loss of α-motor neurons leading to muscle atrophy in the distal limbs accompanied by respiratory complications. Although genetically and clinically distinct, proximal SMA is also caused by the loss of a ubiquitously expressed gene (SMN). Significant preclinical success has been achieved in proximal SMA using viral-based gene replacement strategies. We leveraged the technologies employed in SMA to demonstrate gene replacement efficacy in an SMARD1 animal model. Intracerebroventricular (ICV) injection of single-stranded AAV9 expressing the full-length cDNA of IGHMBP2 in a low dose led to a significant level of rescue in treated SMARD1 animals. Consistent with drastically increased survival, weight gain, and strength, the rescued animals demonstrated a significant improvement in muscle, NMJ, motor neurons, and axonal pathology. In addition, increased levels of IGHMBP2 in lumbar motor neurons verified the efficacy of the virus to transduce the target tissues. Our results indicate that AAV9-based gene replacement is a viable strategy for SMARD1, although dosing effects and potential negative impacts of high dose and ICV injection should be thoroughly investigated.

Enzyme replacement therapy attenuates disease progression in a canine model of late-infantile neuronal ceroid lipofuscinosis (CLN2 disease).

Using a canine model of classical late-infantile neuronal ceroid lipofuscinosis (CLN2 disease), a study was conducted to evaluate the potential pharmacological activity of recombinant human tripeptidyl peptidase-1 (rhTPP1) enzyme replacement therapy administered directly to the cerebrospinal fluid (CSF). CLN2 disease is a hereditary neurodegenerative disorder resulting from mutations in CLN2, which encodes the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Infants with mutations in both CLN2 alleles develop normally but in the late-infantile/early-childhood period undergo progressive neurological decline accompanied by pronounced brain atrophy. The disorder, a form of Batten disease, is uniformly fatal, with clinical signs starting between 2 and 4 years of age and death usually occurring by the early teenage years. Dachshunds homozygous for a null mutation in the canine ortholog of CLN2 (TPP1) exhibit a similar disorder that progresses to end stage at 10.5-11 months of age. Administration of rhTPP1 via infusion into the CSF every other week, starting at approximately 2.5 months of age, resulted in dose-dependent significant delays in disease progression, as measured by delayed onset of neurologic deficits, improved performance on a cognitive function test, reduced brain atrophy, and increased life span. Based on these findings, a clinical study evaluating the potential therapeutic value of rhTPP1 administration into the CSF of children with CLN2 disease has been initiated.

Hypoxia Attenuates Purinergic P2X Receptor-Induced Inflammatory Gene Expression in Brainstem Microglia.

Hypoxia and increased extracellular nucleotides are frequently coincident in the brainstem. Extracellular nucleotides are potent modulators of microglial inflammatory gene expression via P2X purinergic receptor activation. Although hypoxia is also known to modulate inflammatory gene expression, little is known about how hypoxia or P2X receptor activation alone affect inflammatory molecule production in brainstem microglia, nor how hypoxia and P2X receptor signaling interact when they occur together. In this study, we investigated the ability of a brief episode of hypoxia (2hrs) in the presence and absence of the non-selective P2X receptor agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) to promote inflammatory gene expression in brainstem microglia in adult rats. We evaluated inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) mRNA levels in immunomagnetically-isolated brainstem microglia. Whereas iNOS and IL-6 gene expression increased with hypoxia and BzATP alone, TNFα expression was unaffected. Surprisingly, BzATP-induced inflammatory effects are lost after hypoxia, suggesting that hypoxia impairs pro-inflammatory P2X receptor signaling. We also evaluated the expression of key P2X receptors activated by BzATP, namely P2X1, P2X4 and P2X7 receptors. Whereas hypoxia did not alter their expression, BzATP upregulated P2X4 and P2X7 mRNAs; these effects were ablated in hypoxia. Although both P2X4 and P2X7 receptor expression correlated with increased microglial iNOS and IL-6 levels in microglia from normoxic rats, in hypoxia, P2X7 only correlated with IL-6, and P2X4 correlated only with iNOS. In addition, correlations between P2X7 and P2X4 were lost following hypoxia, suggesting that P2X4 and P2X7 receptor signaling differs in normoxia and hypoxia. Together, these data suggest that hypoxia suppresses P2X receptor-induced inflammatory gene expression, indicating a potentially immunosuppressive role of extracellular nucleotides in brainstem microglia following exposure to hypoxia.