Leah Schmelzer

Proximal spinal muscular atrophy (SMA) is a debilitating neurological disease marked by isolated lower motor neuron death and subsequent atrophy of skeletal muscle. Historically, SMA pathology was thought to be limited to lower motor neurons and the skeletal muscles they control, yet there are several reports describing the coincidence of cardiovascular abnormalities in SMA patients. As new therapies for SMA emerge, it is necessary to determine whether these non-neuromuscular systems need to be targeted. Therefore, we have characterized left ventricular (LV) function of SMA mice (SMN2+/+; SMNΔ7+/+; Smn-/-) and compared it with that of their unaffected littermates at 7 and 14 days of age. Anatomical and physiological measurements made by electrocardiogram and echocardiography show that affected mouse pups have a dramatic decrease in cardiac function. At 14 days of age, SMA mice have bradycardia and develop a marked dilated cardiomyopathy with a concomitant decrease in contractility. Signs of decreased cardiac function are also apparent as early as 7 days of age in SMA animals. Delivery of a survival motor neuron-1 transgene using a self-complementary adeno-associated virus serotype 9 abolished the symptom of bradycardia and significantly decreased the severity of the heart defect. We conclude that severe SMA animals have compromised cardiac function resulting at least partially from early bradycardia, which is likely attributable to aberrant autonomic signaling. Further cardiographic studies of human SMA patients are needed to clarify the clinical relevance of these findings from this SMA mouse.

Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (ALS) resulting in progressive motor neuron death through one or more acquired toxicities. Involvement of wild-type SOD1 has been linked to sporadic ALS, as misfolded SOD1 has been reported in affected tissues of sporadic patients and toxicity of astrocytes derived from sporadic ALS patients to motor neurons has been reported to be reduced by lowering the synthesis of SOD1. We now report slowed disease onset and progression in two mouse models following therapeutic delivery using a single peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding an shRNA to reduce the synthesis of ALS-causing human SOD1 mutants. Delivery to young mice that develop aggressive, fatal paralysis extended survival by delaying both disease onset and slowing progression. In a later-onset model, AAV9 delivery after onset markedly slowed disease progression and significantly extended survival. Moreover, AAV9 delivered intrathecally to nonhuman primates is demonstrated to yield robust SOD1 suppression in motor neurons and glia throughout the spinal cord and therefore, setting the stage for AAV9-mediated therapy in human clinical trials. Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (ALS) resulting in progressive motor neuron death through one or more acquired toxicities. Involvement of wild-type SOD1 has been linked to sporadic ALS, as misfolded SOD1 has been reported in affected tissues of sporadic patients and toxicity of astrocytes derived from sporadic ALS patients to motor neurons has been reported to be reduced by lowering the synthesis of SOD1. We now report slowed disease onset and progression in two mouse models following therapeutic delivery using a single peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding an shRNA to reduce the synthesis of ALS-causing human SOD1 mutants. Delivery to young mice that develop aggressive, fatal paralysis extended survival by delaying both disease onset and slowing progression. In a later-onset model, AAV9 delivery after onset markedly slowed disease progression and significantly extended survival. Moreover, AAV9 delivered intrathecally to nonhuman primates is demonstrated to yield robust SOD1 suppression in motor neurons and glia throughout the spinal cord and therefore, setting the stage for AAV9-mediated therapy in human clinical trials. Amyotrophic lateral sclerosis (ALS) is an adult-onset, rapidly progressive, and fatal neurodegenerative disease, characterized by selective degeneration of both upper and lower motor neurons. ALS is the most prominent motor neuron disease, responsible for one in every 2,000 deaths. Most of the cases have no clear genetic linkage and are referred to as sporadic but in 10% of the instances, disease is familial with dominant inheritance. Twenty percent of the familial cases are caused by mutations in superoxide dismutase 1 (SOD1), with over 140 distinct mutations identified to date.1Da Cruz S Cleveland DW Understanding the role of TDP-43 and FUS/TLS in ALS and beyond.Curr Opin Neurobiol. 2011; 21: 904-919Crossref PubMed Scopus (257) Google Scholar,2Rosen DR Siddique T Patterson D Figlewicz DA Sapp P Hentati A et al.Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis.Nature. 1993; 362: 59-62Crossref PubMed Scopus (5508) Google Scholar Many efforts to identify how mutations alter the function of SOD1 have produced a consensus view that SOD1 mutants acquire one or more toxicities, whose nature still remains controversial.3Ilieva H Polymenidou M Cleveland DW Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond.J Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (788) Google Scholar However, there is clear evidence that a proportion of mutant SOD1 is misfolded and subsequently aggregates.4Chattopadhyay M Valentine JS Aggregation of copper-zinc superoxide dismutase in familial and sporadic ALS.Antioxid Redox Signal. 2009; 11: 1603-1614Crossref PubMed Scopus (125) Google Scholar,5Prudencio M Hart PJ Borchelt DR Andersen PM Variation in aggregation propensities among ALS-associated variants of SOD1: correlation to human disease.Hum Mol Genet. 2009; 18: 3217-3226Crossref PubMed Scopus (181) Google Scholar SOD1 aggregates are, in fact, one of the histological hallmarks of SOD1-related ALS cases.4Chattopadhyay M Valentine JS Aggregation of copper-zinc superoxide dismutase in familial and sporadic ALS.Antioxid Redox Signal. 2009; 11: 1603-1614Crossref PubMed Scopus (125) Google Scholar In the past 20 years, multiple animal models expressing mutant forms of human SOD1 have been generated. These models recapitulate the hallmarks of ALS, developing age-dependent motor axon degeneration and accompanying muscle denervation, glial inflammation, and subsequent motor neuron loss. Selective gene excision experiments have determined that mutant SOD1 expression within motor neurons themselves contributes to disease onset and early disease progression,6Boillée S Yamanaka K Lobsiger CS Copeland NG Jenkins NA Kassiotis G et al.Onset and progression in inherited ALS determined by motor neurons and microglia.Science. 2006; 312: 1389-1392Crossref PubMed Scopus (1277) Google Scholar as does mutant synthesis in NG2+ cells7Kang SH Li Y Fukaya M Lorenzini I Cleveland DW Ostrow LW et al.Degeneration and impaired regeneration of gray matter oligodendrocytes in amyotrophic lateral sclerosis.Nat Neurosci. 2013; 16: 571-579Crossref PubMed Scopus (390) Google Scholar that are precursors to oligodendrocytes. However, mutant SOD1 protein expression in microglia and astrocytes significantly drives rapid disease progression,6Boillée S Yamanaka K Lobsiger CS Copeland NG Jenkins NA Kassiotis G et al.Onset and progression in inherited ALS determined by motor neurons and microglia.Science. 2006; 312: 1389-1392Crossref PubMed Scopus (1277) Google Scholar,8Yamanaka K Chun SJ Boillee S Fujimori-Tonou N Yamashita H Gutmann DH et al.Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis.Nat Neurosci. 2008; 11: 251-253Crossref PubMed Scopus (874) Google Scholar findings which have led to the conclusion that ALS pathophysiology is noncell autonomous.3Ilieva H Polymenidou M Cleveland DW Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond.J Cell Biol. 2009; 187: 761-772Crossref PubMed Scopus (788) Google Scholar Furthermore, astrocytes have been found to be toxic to motor neurons in multiple in vitro models where mutant forms of human SOD1 were overexpressed.9Di Giorgio FP Boulting GL Bobrowicz S Eggan KC Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation.Cell Stem Cell. 2008; 3: 637-648Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar,10Di Giorgio FP Carrasco MA Siao MC Maniatis T Eggan K Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model.Nat Neurosci. 2007; 10: 608-614Crossref PubMed Scopus (638) Google Scholar,11Marchetto MC Muotri AR Mu Y Smith AM Cezar GG Gage FH Non-cell-autonomous effect of human SOD1 G37R astrocytes on motor neurons derived from human embryonic stem cells.Cell Stem Cell. 2008; 3: 649-657Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar A recent study by our group derived astrocytes from postmortem spinal cords of ALS patients with or without SOD1 mutations. In all cases, astrocytes from sporadic ALS patients were as toxic to motor neurons as astrocytes carrying genetic mutations in SOD1, but neither sporadic nor familial ALS glia were toxic to GABAergic neurons.12Haidet-Phillips AM Hester ME Miranda CJ Meyer K Braun L Frakes A et al.Astrocytes from familial and sporadic ALS patients are toxic to motor neurons.Nat Biotechnol. 2011; 29: 824-828Crossref PubMed Scopus (571) Google Scholar Even more strikingly, of SOD1 in astrocytes derived from both sporadic and familial ALS patients toxicity motor neurons. with the that misfolded SOD1 are found in the spinal cords of familial as as sporadic ALS DA G Y P et and mutant SOD1 an and a in Neurosci. PubMed Scopus Google M of and is associated with of SOD1 in amyotrophic lateral PubMed Scopus Google K Andersen PM D M et SOD1 in sporadic ALS PubMed Scopus Google Scholar has evidence for a role of wild-type SOD1 in sporadic the that animal models have for the in motor neuron for the of therapeutic has been S M ALS from the past and a 2008; PubMed Scopus Google Scholar as no with a reported survival in mutant mice has been in clinical with sporadic ALS We that in all but one the to human been reported to mutant SOD1 mouse survival and to a survival by delaying disease onset with no in slowing disease progression. one to which to the human extended survival of mutant mice and by slowing disease ME P in familial amyotrophic lateral sclerosis to of superoxide Biol. Google Scholar that human slowing of disease the mice have the of and the of of in human have been are that disease progression in have that adeno-associated virus 9 (AAV9) the and neurons and astrocytes in the and spinal cord A AAV9 neurons and Biotechnol. 2009; PubMed Scopus Google S T L AM et of AAV9 delivery to motor 2009; Full Text Full Text PDF PubMed Scopus Google Scholar We that of AAV9 be to SOD1 shRNA to disease progression in models of We have now in two mouse models using a peripheral injection of SOD1 Furthermore, delivery of SOD1 shRNA and protein nonhuman primates all of the spinal cord and significantly reduce the of SOD1. We the of AAV9 in the mouse that fatal were 1 or be referred to as and with from the were and spinal cords were for expression in astrocytes with in and of and spinal gray matter astrocytes by for glial protein to our report of in wild-type A AAV9 neurons and Biotechnol. 2009; PubMed Scopus Google Scholar neurons were a prominent all of the spinal cords of with significantly lower to motor neurons in have reported that astrocytes in wild-type spinal cords with for A AAV9 neurons and Biotechnol. 2009; PubMed Scopus Google Scholar of mutant SOD1 astrocytes synthesis of by the AAV9 disease mice were and with and to stage of the spinal cords from and a of astrocytes as were found after AAV9 injection These are with the survival of astrocytes for the of disease in mice and that gene expression is Furthermore, and and early in the develop disease in M Y et SOD1 mutants to motor neuron Neurosci. 2008; 11: PubMed Scopus Google Scholar affected AAV9 mice were with and stage of the spinal cords that the in to with astrocytes as the all cells in gray the human SOD1 shRNA that a of with the mouse for the human to in the shRNA an expression to of the Human cells were with were and SOD1 were by reduced SOD1 protein by produced the most a AAV9 that a gene whose expression identify cells to as that the shRNA of human SOD1, mice were with or were and the spinal cords were and by for both human and SOD1 and spinal cords and in mutant SOD1 SOD1 in to human SOD1 be mice of both were or and to of and mice a in to mice no and in motor by as as in and of were and of both and from both no in the of and all the were the of by a to group no in the with the We that both of AAV9 and shRNA expression were and the of AAV9-mediated SOD1 of mice with a single injection of and or after that but motor be the two of disease by from muscle significantly by a of P in the but affected by of the and and with the muscle and motor function survival significantly extended by AAV9 injection all survival that of mice P P and P disease as the from onset to stage that the group significantly of slowed disease with the P with slowed disease progression in the two P and P lower of cells that a of cells be to disease progression in the with an that but mouse SOD1, to mutant SOD1 in spinal cords of and Human SOD1 in spinal cord with survival and lower of mutant SOD1 with the SOD1 expression within motor neurons by and reduced with the neurons that been to Moreover, of spinal cords in but no in in AAV9-mediated mutant SOD1 disease a of S Yamanaka K Lobsiger CS Copeland NG Jenkins NA Kassiotis G et al.Onset and progression in inherited ALS determined by motor neurons and microglia.Science. 2006; 312: 1389-1392Crossref PubMed Scopus (1277) Google Scholar were with after disease onset onset of in P A of and were as no and therefore, the were as and are in were for and and after disease onset significantly extended survival by over SOD1 P disease by the from to 10% significantly slowed SOD1 P A slowing of disease to SOD1 P disease following therapy to after disease onset in mice P with the slowed disease AAV9 therapy to in survival the in that expression to the mutant K Chun SJ Boillee S Fujimori-Tonou N Yamashita H Gutmann DH et al.Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis.Nat Neurosci. 2008; 11: 251-253Crossref PubMed Scopus (874) Google Scholar AAV9 of astrocytes after peripheral delivery and the of S Yamanaka K Lobsiger CS Copeland NG Jenkins NA Kassiotis G et al.Onset and progression in inherited ALS determined by motor neurons and microglia.Science. 2006; 312: 1389-1392Crossref PubMed Scopus (1277) Google Scholar whose synthesis of mutant SOD1 disease progression. of of in with or expression within motor neurons and astrocytes of both and SOD1 expression in of spinal cord from mice an in protein in with SOD1 be using AAV9 in the nonhuman spinal AAV9 intrathecally through over delivery to the of virus and to from of SOD1 in peripheral of from from and the that the shRNA a single to shRNA expression a which to of that the SOD1 reduced by in cells with the cells virus with through the of the and one with from the were spinal cords were for the of expression and SOD1 expression in and cells throughout the gray and matter of the spinal the to the of injection of of spinal cord in SOD1 protein to from motor neurons as as from glia in the by using for SOD1 to a in the motor neuron and a in the as with the from a animal the of throughout the spinal cord and found to be within all neuron a to in with the more of motor to in the and in the In to the of SOD1 with for SOD1 on from and cord robust SOD1 all spinal cord from a in the a in the and an in the with the proportion of cells in one is by and as a therapy for ALS, a survival DH for amyotrophic lateral sclerosis neuron disease 3: PubMed Google Scholar the of familial cases caused by in SOD1, therapy by synthesis of SOD1 have been the of multiple therapeutic and were in Yamanaka K G et therapy for neurodegenerative 2006; PubMed Scopus Google Scholar and mouse T S G et of SOD1 through disease onset and progression in a mouse of 11: PubMed Scopus Google MA et mutant SOD1 using and survival in an ALS model.Nat 11: PubMed Scopus Google Yamanaka K Gage FH et in amyotrophic lateral PubMed Scopus Google Scholar that develop fatal paralysis from human disease onset produced SOD1 and a slowing of disease Yamanaka K G et therapy for neurodegenerative 2006; PubMed Scopus Google Scholar of has been A SH et SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral a 2013; Full Text Full Text PDF PubMed Scopus Google Scholar to in of and but without in SOD1 the In of the T S G et of SOD1 through disease onset and progression in a mouse of 11: PubMed Scopus Google MA et mutant SOD1 using and survival in an ALS model.Nat 11: PubMed Scopus Google Yamanaka K Gage FH et in amyotrophic lateral PubMed Scopus Google Scholar SOD1 disease onset by injection the or of a virus T S G et of SOD1 through disease onset and progression in a mouse of 11: PubMed Scopus Google MA et mutant SOD1 using and survival in an ALS model.Nat 11: PubMed Scopus Google Scholar These led to of in survival or motor on the of as as of SOD1 in the spinal of the were from clinical there have been SOD1 suppression T S G et of SOD1 through disease onset and progression in a mouse of 11: PubMed Scopus Google MA et mutant SOD1 using and survival in an ALS model.Nat 11: PubMed Scopus Google P delivery SOD1: does alter disease progression in 2008; 16: Full Text Full Text PDF PubMed Scopus Google P by gene therapy mutant SOD1 in of motor neurons does therapeutic in 2011; Full Text Full Text PDF PubMed Scopus Google gene 2011; Full Text Full Text PDF PubMed Scopus Google Scholar have the therapeutic of the that AAV9 the and motor neurons and A AAV9 neurons and Biotechnol. 2009; PubMed Scopus Google S T L AM et of AAV9 delivery to motor 2009; Full Text Full Text PDF PubMed Scopus Google Scholar that of is and in wild-type with the of after our efforts with have one of the in survival reported in the rapidly progressive mouse of ALS survival by is Even more markedly slowed disease progression is AAV9 therapy to reduce mutant SOD1 synthesis is after disease onset in significantly survival. the delivery in mice a of that mutant SOD1 after disease onset be in both rapid and more progressive models of ALS in that robust and suppression of SOD1 through AAV9-mediated delivery of shRNA is in slowing disease progression in mouse models of ALS, is after in SOD1 protein stage in both and mouse models and the in of the virus and the of in SOD1 in both and in mice the that in the cells with reduced SOD1 a survival over with from et MA et mutant SOD1 using and survival in an ALS model.Nat 11: PubMed Scopus Google Scholar using of SOD1 and from et et neurons in Cu/Zn superoxide mice develop but death after Genet. PubMed Scopus Google Scholar in SOD1 with evidence that SOD1 to in or G M L dismutase 1 in human PubMed Scopus Google Scholar findings that to SOD1 be in a in the of cells to SOD1 on or as of in the rapid in Valentine JS dismutase is for survival in of toxic in PubMed Scopus Google Scholar A to SOD1 be with that has in the mouse K M H et in amyotrophic lateral sclerosis in 2007; PubMed Scopus Google Y A et mutations of in a familial ALS 2008; Google Scholar and in the mouse S Yamanaka K Lobsiger CS Copeland NG Jenkins NA Kassiotis G et al.Onset and progression in inherited ALS determined by motor neurons and microglia.Science. 2006; 312: 1389-1392Crossref PubMed Scopus (1277) Google Scholar ALS-associated mutations in SOD1 have been to the of by to resulting in an of from K M H et in amyotrophic lateral sclerosis in 2007; PubMed Scopus Google Y A et mutations of in a familial ALS 2008; Google Scholar of or with slowed both the onset and progression of Y A et mutations of in a familial ALS 2008; Google Scholar However, the by has been and therefore, D T L et and to significantly survival in SOD1 ALS PubMed Scopus Google Scholar Most in cells has been linked to an of protein expression by the protein to mutant SOD1 an early in the mouse M P T P et in ALS mouse glia protein with superoxide Mol Genet. 2013; PubMed Scopus Google Scholar In of protein or genetic in SOD1 mouse models the of therapeutic of misfolded SOD1. SOD1 is in sporadic disease remains have T P et of evidence of superoxide in sporadic amyotrophic lateral 2009; PubMed Scopus Google A S L et lateral sclerosis is a disease in which of SOD1 is to the familial PubMed Scopus Google Li Y D M et of a toxic of superoxide dismutase 1 protein to affected tissues in familial PubMed Scopus Google Scholar multiple recent have the that wild-type SOD1 through to the that sporadic ALS through a to that by mutant AM Hester ME Miranda CJ Meyer K Braun L Frakes A et al.Astrocytes from familial and sporadic ALS patients are toxic to motor neurons.Nat Biotechnol. 2011; 29: 824-828Crossref PubMed Scopus (571) Google M of and is associated with of SOD1 in amyotrophic lateral PubMed Scopus Google M D I et superoxide from wild-type Mol Genet. 21: PubMed Scopus Google S M DA et of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with onset a toxic with mutant PubMed Scopus Google Scholar in of evidence is our that astrocytes produced from sporadic ALS patients are toxic to motor neurons and that toxicity is by in wild-type et neurons in Cu/Zn superoxide mice develop but death after Genet. PubMed Scopus Google Scholar have SOD1 in sporadic AM Hester ME Miranda CJ Meyer K Braun L Frakes A et al.Astrocytes from familial and sporadic ALS patients are toxic to motor neurons.Nat Biotechnol. 2011; 29: 824-828Crossref PubMed Scopus (571) Google DA G Y P et and mutant SOD1 an and a in Neurosci. PubMed Scopus Google K Andersen PM D M et SOD1 in sporadic ALS PubMed Scopus Google S M DA et of superoxide dismutase found in sporadic amyotrophic lateral sclerosis with onset a toxic with mutant PubMed Scopus Google P superoxide the of all amyotrophic lateral 2007; PubMed Scopus Google A et in SOD1 for both sporadic and familial amyotrophic lateral 2007; PubMed Scopus Google M S M N et dismutase are associated with survival in patients with sporadic amyotrophic lateral 2011; PubMed Scopus Google P Andersen PM T superoxide in from amyotrophic lateral sclerosis 2011; PubMed Scopus Google Scholar no consensus has the evidence the that a proportion of sporadic ALS patients from an AAV9-mediated SOD1 that have demonstrated to be in slowing disease progression in mice that develop disease from expressing ALS-causing mutations in SOD1. for of an AAV9-mediated suppression of SOD1 synthesis to the human have determined that the the in a nonhuman SOD1 by both motor neurons and for efforts to an human by injection as S Braun L L et gene delivery in for spinal and peripheral tissues for 2011; 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