Widespread and efficient transduction of spinal cord and brain following neonatal AAV injection and potential disease modifying effect in ALS mice.

The architecture of the spinal cord makes efficient delivery of recombinant adeno-associated virus (rAAV) vectors throughout the neuraxis challenging. We describe a paradigm in which small amounts of virus delivered intraspinally to newborn mice result in robust rAAV-mediated transgene expression in the spinal cord. We compared the efficacy of rAAV2/1, 2/5, 2/8, and 2/9 encoding EGFP delivered to the hindlimb muscle (IM), cisterna magna (ICM), or lumbar spinal cord (IS) of neonatal pups. IS injection of all four capsids resulted in robust transduction of the spinal cord with rAAV2/5, 2/8, and 2/9 vectors appearing to be transported to brain. ICM injection resulted in widespread expression of EGFP in the brain, and upper spinal cord. IM injection resulted in robust muscle expression, with only rAAV2/8 and 2/9 transducing spinal motor and sensory neurons. As proof of concept, we use the IS paradigm to express murine Interleukin (IL)-10 in the spinal cord of the SOD1-G93A transgenic mouse model of amyotrophic lateral sclerosis. We show that expression of IL-10 in the spinal axis of SOD1-G93A mice altered the immune milieu and significantly prolonged survival. These data establish an efficient paradigm for somatic transgene delivery of therapeutic biologics to the spinal cord of mice.

Substantially elevating the levels of αB-crystallin in spinal motor neurons of mutant SOD1 mice does not significantly delay paralysis or attenuate mutant protein aggregation.

There has been great interest in enhancing endogenous protein maintenance pathways such as the heat-shock chaperone response, as it is postulated that enhancing clearance of misfolded proteins could have beneficial disease modifying effects in amyotrophic lateral sclerosis and other neurodegenerative disorders. In cultured cell models of mutant SOD1 aggregation, co-expression of αB-crystallin (αB-crys) has been shown to inhibit the formation of detergent-insoluble forms of mutant protein. Here, we describe the generation of a new line of transgenic mice that express αB-crys at > 6-fold the normal level in spinal cord, with robust increases in immunoreactivity throughout the spinal cord grey matter and, specifically, in spinal motor neurons. Surprisingly, spinal cords of mice expressing αB-crys alone contained 20% more motor neurons per section than littermate controls. Raising αB-crys by these levels in mice transgenic for either G93A or L126Z mutant SOD1 had no effect on the age at which paralysis developed. In the G93A mice, which showed the most robust degree of motor neuron loss, the number of these cells declined by the same proportion as in mice expressing the mutant SOD1 alone. In paralyzed bigenic mice, the levels of detergent-insoluble, misfolded, mutant SOD1 were similar to those of mice expressing mutant SOD1 alone. These findings indicate that raising the levels of αB-crys in spinal motor neurons by 6-fold does not produce the therapeutic effects predicted by cell culture models of mutant SOD1 aggregation. Enhancing the protein chaperone function may present a therapeutic approach to amyotrophic lateral sclerosis caused by mutations in SOD1, and other neurodegenerative disorders characterized by cytosolic protein aggregation. Previous studies in cell models suggested that the chaperone known as αB-crystallin (αB-crys) can prevent mutant SOD1 aggregation. We report that transgenic expression of αB-crys at > 6-fold the normal level in spinal cords of mice expressing mutant SOD1 produces no therapeutic benefit.

Analysis of proteolytic processes and enzymatic activities in the generation of huntingtin n-terminal fragments in an HEK293 cell model.

BACKGROUND: N-terminal fragments of mutant huntingtin (htt) that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1), form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD). These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments. RESULTS: Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like), were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1. CONCLUSIONS: Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.