BACKGROUND
spinal muscular atrophy  is the leading genetic cause of infantile death.
the clinical categorization of sma is based upon disease severity and age of onset  <cit> .
sma results from the lack of a functional copy of the survival motor neuron  <dig>  gene  <cit> .
there is a nearly identical copy gene, smn <dig>  found only in humans  <cit> .
smn <dig> does not fully compensate for the loss of smn <dig>  due to an alternative splicing event induced by a non-polymorphic c to t transition within exon  <dig>  <cit> .
alternative pre-mrna splicing, therefore, results in the majority of smn <dig> transcripts excluding exon  <dig> , encoding a less stable protein product compared to full-length smn  <cit> .
loss of full-length smn protein leads to the death of α-motor neurons, however, it is unclear why motor neurons are particularly susceptible to low levels of this ubiquitously expressed protein.
however, two hypotheses suggest that axonal rna transport and/or snrnp biogenesis underlie the sma-associated activity  <cit> .
since the lack of smn exon  <dig> leads to sma development, this sequence is clearly important.
there is a cytoplasmic localization signal within smn exon  <dig>  that can transport smn exons 1- <dig> and other heterologous nuclear proteins to the cytoplasm  <cit> .
however, heterologous sequences can mediate proper smn localization in several cellular contexts in vitro.
in addition, heterologous sequences can also partially restore functionality in cell-based assays, such as protein stability, snrnp assembly and neurite extension assays  <cit> .
for these reasons, aminoglycosides have been examined in sma for their ability to induce translational "read-through."
these compounds are presumed to inhibit the recognition of the endogenous smnΔ <dig> translational stop codon, located four amino acids into exon  <dig>  "read-through" of the stop codon would thereby allow the incorporation of a c-terminal extension of an additional five amino acids  <cit> .
treating type i primary sma patient fibroblasts  or sma ips-derived neuronal cultures with aminoglycosides was shown to increase smn protein  <cit> .
when delivered via intra-peritoneal injection, treatment with the aminoglycoside g <dig> in a sma mouse model increased gross motor function at select time points  <cit> .
additionally, direct injection of tc <dig>  a novel aminoglycoside, into the central nervous system extends life span approximately 30% and elevates smn protein in the brain and spinal cord  <cit> .
to begin to examine a less invasive means of delivery, we have examined alternative routes of delivery for tc <dig>  here we demonstrate that subcutaneous administration of tc <dig>  while not extending survival, results in improved molecular and cellular aspects of the sma phenotype, as well as benefits in gross motor function in a sma mouse model.
RESULTS
subcutaneous dosing of sma mice with tc <dig> significantly increases gross motor function, but not lifespan
based on previous in vivo examinations of aminoglycosides and stop codon readthrough  <cit> , we administered subcutaneous doses of tc <dig>  <cit>  at  <dig> mg/kg daily from post-natal day   <dig> to  <dig> to the well-characterized Δ <dig> sma model   <cit> .
tc <dig> treated mice  lived approximately 16% longer  than vehicle treated mice , although this difference did not reach statistical significance .
the aminoglycosides appeared to prevent the death at a younger age, resulting in larger numbers of mice dying later .
however, the longest life spans were similar in the vehicle and tc007-treated mice.
moreover not all aminoglycosides that increase smn levels confer the same survival effect.
for example, administration of neo <dig>   <cit> , a compound containing the same chemical backbone as tc <dig>  significantly elevated smn levels in vitro , but only marginally affected lifespan .
total body weight was unaffected in tc <dig> and neo <dig> treated animals compared to vehicle treated sma animals .
since smn read-through therapies are proposed to function in part through a stabilization of smnΔ <dig> protein, various tissues were examined for an increase in steady-state levels of smn protein.
therefore brain, spinal cord and muscle of p10-treated mice were isolated.
under these experimental conditions, we could not detect a significant increase of smn , which may also explain the modest extension in lifespan.
tc007-treated mice, however, did exhibit increased gross motor function.
a previously quantifiable measure of gross motor function in these sma mice is determining the time it takes for a pup to right itself from a prone position, or time-to-right  <cit> .
not only did tc007-treated pups right faster, but they were also able to right significantly more often than their untreated counterparts at mid- to end-stage of disease .
when the gastrocnemius of p <dig> mice were examined, the fibers were significantly larger in the tc007-treated group , showing an anatomical correlation with our observed increase in overall gross motor function.
to determine if with the increase in fiber size, an increased survival of motor neurons was also observed, ventral horn cells  were examined of p <dig> tc007-treated and vehicle-treated mice.
while there was no increase in vhc cell body size, there was a slight trend towards an increased number of vhcs in treated tissues , although statistical significance was not achieved.
this time point was chosen for histological examination due to its correlation with the most prominent display of an increase in gross motor function by the tc007-treated group.
the increased fiber size along with the increase vhc numbers does provide a cellular corroboration to the improved gross motor phenotype seen upon subcutaneous administration of tc <dig>  however, more pronounced improvements were likely not observed if tc <dig> failed to cross the blood-brain-barrier or if the compound exhibits a short half life.
as no medicinal chemistry has been performed on tc <dig> to improve its drug-like qualities, it is not surprising that at this early stage the compound lacks properties typically associated with drugs for cns disorders.
to date, properties such as blood-brain-barrier permeability and pharmacokinetics have not been examined for tc <dig> but will be important to analyze in the event that tc <dig> or similar scaffolds move forward toward clinical application.
while it is possible that tc <dig> is functioning as a general neuroprotectant, it is more likely that smn levels were the basis for the cellular and gross motor activity increases.
unfortunately, the in vivo examination of this compound did not correlate with the in vitro activity when administered under these experimental conditions  <cit> , however, direct administration of tc <dig> into the cns by intraventricular injection did significantly elevate smn levels and extend life by approximately 30%  <cit> .
clearly, moving this compound forward and making it more amenable for central nervous system diseases will require an in depth analysis of its pharmacokinetics and the development of medicinal chemistry beyond these proof-of-concept experiments.
smnΔ <dig> read-through benefits sma mouse models
translation stop codon read-through occurs at a relatively low frequency in the presence of aminoglycosides and this is likely true for the smnΔ <dig> exon  <dig> stop codon based upon its specific stop codon and the proximal sequences flanking the stop signal  <cit> .
aminoglycoside treatment likely generates a low level of smn read-through protein that enters into an smn complexes with the existing full-length protein.
this theory is supported by previously published data, demonstrating a small increase in smn-fl protein after treatment, without increasing smn2-transcription or altering exon  <dig> splicing  <cit> .
in vitro studies have indicated that this possible hetero-oligomerization leads to an increase in the functionality of the smn protein  <cit> .
while it is still possible that read-through smn is not as active in sma-specific activities, experiments such as the development of a read-through sma mouse would be an important step towards determining the extent of smn read-through functionality and whether read-through based therapeutic strategies hold promise for sma.
CONCLUSIONS
in conclusion, similar benefits have been reported here for tc <dig> regarding a smnΔ <dig> read-through therapy compared to previously published work using a fda-approved aminoglycosides, g <dig>  <cit> .
while tc <dig> treatment does not result in a statistically significant extension in median lifespan, there were significant increases in gross motor function and muscle integrity at mid-stage of disease .
extension in life span was demonstrated when tc <dig> was administered directly to the central nervous system via intracerebral ventricular injection, suggesting that the importance in delivery strategies as well as likely target tissues for sma therapeutics  <cit> .
taken together, these results demonstrate the proof-of-concept utility of read-through compounds in sma cell and animal models.

 spinal muscular atrophy is the leading genetic cause of infantile death. the clinical categorization of sma is based upon disease severity and age of onset. sma results from the lack of a functional copy of the survival motor neuron gene. there is a nearly identical copy gene, smn found only in humans. smn does not fully compensate for the loss of smn due to an alternative splicing event induced by a non-polymorphic c to t transition within exon. alternative pre-mrna splicing, therefore, results in the majority of smn transcripts excluding exon, encoding a less stable protein product compared to full-length smn. loss of full-length smn protein leads to the death of α-motor neurons, however, it is unclear why motor neurons are particularly susceptible to low levels of this ubiquitously expressed protein. however, two hypotheses suggest that axonal rna transport and/or snrnp biogenesis underlie the sma-associated activity. since the lack of smn exon leads to sma development, this sequence is clearly important. there is a cytoplasmic localization signal within smn exon that can transport smn exons 1- and other heterologous nuclear proteins to the cytoplasm. however, heterologous sequences can mediate proper smn localization in several cellular contexts in vitro. in addition, heterologous sequences can also partially restore functionality in cell-based assays, such as protein stability, snrnp assembly and neurite extension assays. for these reasons, aminoglycosides have been examined in sma for their ability to induce translational "read-through." these compounds are presumed to inhibit the recognition of the endogenous smnΔ translational stop codon, located four amino acids into exon "read-through" of the stop codon would thereby allow the incorporation of a c-terminal extension of an additional five amino acids. treating type i primary sma patient fibroblasts or sma ips-derived neuronal cultures with aminoglycosides was shown to increase smn protein. when delivered via intra-peritoneal injection, treatment with the aminoglycoside g in a sma mouse model increased gross motor function at select time points. additionally, direct injection of tc a novel aminoglycoside, into the central nervous system extends life span approximately 30% and elevates smn protein in the brain and spinal cord. to begin to examine a less invasive means of delivery, we have examined alternative routes of delivery for tc here we demonstrate that subcutaneous administration of tc while not extending survival, results in improved molecular and cellular aspects of the sma phenotype, as well as benefits in gross motor function in a sma mouse model.  subcutaneous dosing of sma mice with tc significantly increases gross motor function, but not lifespan based on previous in vivo examinations of aminoglycosides and stop codon readthrough, we administered subcutaneous doses of tc at mg/kg daily from post-natal day to to the well-characterized Δ sma model. tc treated mice lived approximately 16% longer than vehicle treated mice, although this difference did not reach statistical significance. the aminoglycosides appeared to prevent the death at a younger age, resulting in larger numbers of mice dying later. however, the longest life spans were similar in the vehicle and tc007-treated mice. moreover not all aminoglycosides that increase smn levels confer the same survival effect. for example, administration of neo, a compound containing the same chemical backbone as tc significantly elevated smn levels in vitro, but only marginally affected lifespan. total body weight was unaffected in tc and neo treated animals compared to vehicle treated sma animals. since smn read-through therapies are proposed to function in part through a stabilization of smnΔ protein, various tissues were examined for an increase in steady-state levels of smn protein. therefore brain, spinal cord and muscle of p10-treated mice were isolated. under these experimental conditions, we could not detect a significant increase of smn, which may also explain the modest extension in lifespan. tc007-treated mice, however, did exhibit increased gross motor function. a previously quantifiable measure of gross motor function in these sma mice is determining the time it takes for a pup to right itself from a prone position, or time-to-right. not only did tc007-treated pups right faster, but they were also able to right significantly more often than their untreated counterparts at mid- to end-stage of disease. when the gastrocnemius of p mice were examined, the fibers were significantly larger in the tc007-treated group, showing an anatomical correlation with our observed increase in overall gross motor function. to determine if with the increase in fiber size, an increased survival of motor neurons was also observed, ventral horn cells were examined of p tc007-treated and vehicle-treated mice. while there was no increase in vhc cell body size, there was a slight trend towards an increased number of vhcs in treated tissues, although statistical significance was not achieved. this time point was chosen for histological examination due to its correlation with the most prominent display of an increase in gross motor function by the tc007-treated group. the increased fiber size along with the increase vhc numbers does provide a cellular corroboration to the improved gross motor phenotype seen upon subcutaneous administration of tc however, more pronounced improvements were likely not observed if tc failed to cross the blood-brain-barrier or if the compound exhibits a short half life. as no medicinal chemistry has been performed on tc to improve its drug-like qualities, it is not surprising that at this early stage the compound lacks properties typically associated with drugs for cns disorders. to date, properties such as blood-brain-barrier permeability and pharmacokinetics have not been examined for tc but will be important to analyze in the event that tc or similar scaffolds move forward toward clinical application. while it is possible that tc is functioning as a general neuroprotectant, it is more likely that smn levels were the basis for the cellular and gross motor activity increases. unfortunately, the in vivo examination of this compound did not correlate with the in vitro activity when administered under these experimental conditions, however, direct administration of tc into the cns by intraventricular injection did significantly elevate smn levels and extend life by approximately 30%. clearly, moving this compound forward and making it more amenable for central nervous system diseases will require an in depth analysis of its pharmacokinetics and the development of medicinal chemistry beyond these proof-of-concept experiments. smnΔ read-through benefits sma mouse models translation stop codon read-through occurs at a relatively low frequency in the presence of aminoglycosides and this is likely true for the smnΔ exon stop codon based upon its specific stop codon and the proximal sequences flanking the stop signal. aminoglycoside treatment likely generates a low level of smn read-through protein that enters into an smn complexes with the existing full-length protein. this theory is supported by previously published data, demonstrating a small increase in smn-fl protein after treatment, without increasing smn2-transcription or altering exon splicing. in vitro studies have indicated that this possible hetero-oligomerization leads to an increase in the functionality of the smn protein. while it is still possible that read-through smn is not as active in sma-specific activities, experiments such as the development of a read-through sma mouse would be an important step towards determining the extent of smn read-through functionality and whether read-through based therapeutic strategies hold promise for sma.  in conclusion, similar benefits have been reported here for tc regarding a smnΔ read-through therapy compared to previously published work using a fda-approved aminoglycosides, g. while tc treatment does not result in a statistically significant extension in median lifespan, there were significant increases in gross motor function and muscle integrity at mid-stage of disease. extension in life span was demonstrated when tc was administered directly to the central nervous system via intracerebral ventricular injection, suggesting that the importance in delivery strategies as well as likely target tissues for sma therapeutics. taken together, these results demonstrate the proof-of-concept utility of read-through compounds in sma cell and animal models. 
here we demonstrate that subcutaneous-administration of a read-through inducing compound to an intermediate sma model had beneficial effects on muscle fiber size and gross motor function. spinal muscular atrophy is the leading genetic cause of infantile death. there is a nearly identical copy gene, smn but it is unable to rescue from disease due to an alternative splicing event that excises a necessary exon from the majority of smn2-derived transcripts. delivery of the read-through inducing compound tc reduces the disease-associated phenotype in sma mice, however, does not significantly extend survival.
 spinal muscular atrophy is the leading genetic cause of infantile death. it is caused by the loss of functional survival motor neuron. there is a nearly identical copy gene, smn but it is unable to rescue from disease due to an alternative splicing event that excises a necessary exon from the majority of smn2-derived transcripts. while smnΔ protein has severely reduced functionality, the exon sequences may not be specifically required for all activities. therefore, aminoglycoside antibiotics previously shown to suppress stop codon recognition and promote translation read-through have been examined to increase the length of the smnΔ c-terminus.  here we demonstrate that subcutaneous-administration of a read-through inducing compound to an intermediate sma model had beneficial effects on muscle fiber size and gross motor function.  delivery of the read-through inducing compound tc reduces the disease-associated phenotype in sma mice, however, does not significantly extend survival. 