Developments in the discovery of drugs for spinal muscular atrophy: successful beginnings and future...

1. Introduction

2. Strategies for SMA drug

discovery: increasing SMN

3. Compensating for functional

consequences of SMN deficits

4. Other strategies besides

increasing SMN

5. Is SMA a single disease?

Unraveling disease mechanism

and alternative targets

6. Neuroprotection, alone or in

combination with SMN

enhancers

7. Conclusion

8. Expert opinion

Review

Developments in the discovery ofdrugs for spinal muscular atrophy:successful beginnings and futureprospectsRebecca M. PrussTrophos, SA Parc Scientifique de Luminy Case, Marseille Cedex, France

Introduction: Spinal muscular atrophy (SMA) is an autosomal recessive disease

caused by mutations in a gene that produces a protein called survival motor

neuron (SMN). SMN has an important role in snRNP assembly in all cells but

that may not be its only role; the reasons for SMN deficiency resulting in neu-

romuscular dysfunction and motor neuron degeneration remain active areas

of research. Besides increasing SMN, compensating for SMN deficiencies or

neuroprotection may be therapeutic options for SMA. Age of onset and the

rate of disease progression are variable and therapeutic strategies should be

appropriate to subtypes of SMA patients.

Areas covered: The article discusses SMA, their targets and where these targets

can be found. Additionally, the article reviews small molecules identified as

disease modifiers and how these small molecules were discovered. The article

also describes and discusses emerging concepts regarding the disease mecha-

nisms. The author compiled this review using scientific literature, patent

databases, company and patient association and government websites.

Expert opinion: Small molecules targeting various processes implicated in

SMA are reaching the clinic. These molecules and targets, although not yet

validated, are providing insight into the complexity of a ‘simple’ genetic dis-

ease such as SMA. SMA is not a single disease and so various therapeutic strat-

egies are needed. Biomarkers and regulatory guidelines are required to select

patients for clinical trials, decide when to initiate treatment and how to

develop combinations of investigational drugs.

Keywords: biomarkers, disease mechanisms, drug discovery, neurodegeneration,

neuromuscular disease

Expert Opin. Drug Discov. (2011) 6(8):827-837

1. Introduction

Spinal muscular atrophy (SMA) is a rare neuromuscular disease that presents ininfants and young children. SMA is an autosomal recessive disease affecting an esti-mated 1 in 6000 births and is caused by progressive degeneration of motor neuronsinnervating proximal skeletal muscles due to deletions or mutations in a gene cod-ing for a protein called survival motor neuron (SMN) [1]. Independently, and nearlysimultaneously, the SMN protein, which is expressed in all cells, was discovered tobe a central scaffolding element necessary for the assembly of small nuclear ribonu-clear protein (snRNP) complexes involved in mRNA splicing [2-4]. Discovering boththe gene responsible for SMA and a function for the encoded protein was a remark-able coincidence, making SMA an exception with respect to other inherited neuro-degenerative diseases such as Huntington’s disease and familial forms of Parkinson’sdisease, Alzheimer’s disease or amyotrophic lateral sclerosis (ALS).

10.1517/17460441.2011.586692 © 2011 Informa UK, Ltd. ISSN 1746-0441 827All rights reserved: reproduction in whole or in part not permitted

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Because of its role in snRNP assembly in all cells, homozy-gous mutations or deletions in the SMN gene are lethal inother species besides humans, who benefit from a secondnearly identical gene resulting from a duplication in chromo-some region 5q13 where the SMN gene is located. A singlecopy of the centromeric SMN2 gene prevents embryoniclethality that would ordinarily result from homozygous lossof the telomeric SMN1 gene in SMA patients. Indeed, SMAdisease severity decreases with increasing SMN protein, whichin turn is related to SMN2 copy number [5-8]. Even so, up tofour copies of SMN2 cannot fully prevent SMA becauseSMN levels remain suboptimal. This is due to nucleotidechanges that produce differential splicing of exon 7, which isabsent in the majority of SMN2 transcripts [9]. The resultingprotein referred to as SMND7 has an altered C-terminal:16 amino acids coded by exon 7 are replaced by fouramino acids derived from exon 8. SMND7 levels remain lowbecause it is rapidly degraded by the ubiquitin-proteosomepathway, at least partially related to decreased capacity tooligomerize compared to full length SMN (FL-SMN) orbecause the new C-terminal creates a degradation signal [10,11].The combined understanding of the genetic basis ofSMA as well as the structure and function of SMN proteinhas led to various strategies to discover and developSMA therapeutics.Because SMA is due to SMN deficiency, increasing SMN

levels by various means is considered a promising approachto treat SMA. This includes gene and cell therapy as well assmall molecule pharmacotherapy. Besides increasing SMNprotein, compensating for the functional deficits due toSMN deficiency is another potential approach. Ultimately,like most complex diseases, combination therapeutics may

be required to fully restore SMN protein levels, provideneuroprotection and treat symptoms as well as disease mech-anisms. Here, we consider only small molecules including oli-gonucleotides that have been discovered for SMA, howthey were identified and discuss potential future trends fordiscovering and developing SMA therapeutics.

2. Strategies for SMA drug discovery:increasing SMN

Three major mechanisms to produce more FL-SMN fromthe SMN2 gene have been targeted: i) increasing exon 7inclusion in SMN2 transcripts, ii) increasing transcriptionfrom the SMN2 promoter and iii) stabilizing SMN or theSMND7 protein.

For all these strategies, small molecule screening campaignscan be said to have been successful. While their clinical valida-tion still remains to be demonstrated, these compounds serveas chemical genetic tools that are helping to understand theSMA disease process and identify further therapeutic strate-gies. Knowing the full potential of any one strategy will prob-ably guide development of combination therapies in order toachieve maximal clinical efficacy.

Table 1 lists a number of small molecules that affectone or more of these steps. One of the first approachesused to increase SMN from SMN2 was splicing modulation.Hydroxyurea and sodium butyrate were two of the first com-pounds to be identified using SMA patient cell lines to assayfor increased exon 7 inclusion in SMN2 transcripts; bothwere found to increase SMN protein as well as amelioratedisease phenotype in SMA mouse models [12-14]. Because oftheir structural similarity to butyrate (and because theywere already used clinically to treat children), phenylbutyrateand valproic acid were tested and found active in similarassays both increasing SMN2 expression and exon7 inclusion [15-17]. The ability of butyrate and valproic acidto inhibit HDAC was thought to lead to increased expres-sion of certain splicing and transcription factors that regulatethe expression of SMN. Potent and specific HDAC inhibi-tors are currently approved or under development to treatvarious types of cancer and a number of them have beentested and found to increase SMN levels in cells and mousemodels of SMA (Table 1). However, their broad epigeneticmodulatory activity makes them unlikely candidates to treata chronic, pediatric-onset disease such as SMA. Whethersufficiently selective and potent HDAC inhibitors can befound to eliminate these concerns remains to be determined.In addition, their mechanism of action needs further study.The splicing factor Sfrs10 (also known as Htra2-b1) hasbeen implicated in SMN2 exon 7 inclusion and is increasedby HDAC inhibitors [15,18]. However, a recent study foundthat Sfrs10 deletion, although lethal, had little effect onSMN2 splicing and that motor neuron-specific deletionhad no phenotypic consequences [19]. Besides calling intoquestion the validity of in vitro assay systems, these results

Article highlights.

. Screening strategies targeting mechanisms implicated inspinal muscular atrophy (SMA) have been successful;although not yet clinically validated, they are providinguseful tools to investigate SMA disease mechanisms andidentify new targets and strategies.

. Survival motor neuron (SMN) has cell-specific functionsbesides splicing; understanding these functions and theconsequence of SMN deficits will provide new targetsfor SMA drug discovery.

. SMA is not a single disease and so therapeutic strategiesand options will need to be designed for SMA subtypesbased on age of onset, disease severity and prognosis.

. Biomarkers related to pharmacology and diseaseprogression are needed; they should provide surrogateefficacy end points.

. Products targeting different mechanisms will benecessary and guidelines are required in order to decidewhen to start treatment and how to evaluatecombinations of investigational drugs for SMA.

This box summarizes key points contained in the article.

Developments in the discovery of drugs for spinal muscular atrophy: successful beginnings and future prospects

828 Expert Opin. Drug Discov. (2011) 6(8)

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have implications on the development of potential pharma-codynamic biomarkers based on splicing factor levels as sur-rogate markers for compounds that favor exon 7 inclusion.Further understanding of the complexity of the splicingmechanism involved in SMN2 exon 7 inclusion includingthe role of post-translational modification of splicing factorscould provide further insight into potential strategies andtargets [20,21].

Other screening assays specifically targeting each of thepossible routes for increasing SMN have used reporter genesor cell-free assays to detect SMN2 splicing modulators,SMN2 expression enhancers, SMN or SMND7 stabilizersand SMN-dependent snRNP assembly modulators (Table 2).Because SMN levels actually affect SMN2 splicing, com-pounds having even a modest direct effect on SMNlevels could have a larger impact on SMN expression overtime [22].

2.1 Methods targeting increased SMN2 exon

7 inclusionThe complexity of SMN2 splicing and various approaches todiscover SMA therapies targeting exon 7 inclusion (alongwith other SMA drug discovery strategies) has recently beenreviewed [23]. Besides HDAC inhibitors, assays designedspecifically to screen for enhancers of SMN2 exon 7 inclusion

have identified two additional classes of compounds,tetracycline derivatives and antisense oligonucleotides.

2.1.1 TetracyclinesAclarubicin, an anthracycline chemotherapeutic, was discov-ered as a splicing modulator in a cell-based screen usingSMA patient fibroblasts and confirmed in a neuronal cellline stably expressing a mini-gene reporter for exon 7 inclu-sion [24]. Although other similar compounds such as doxoru-bicin and or tetracyclines were not active in these whole cellassays, a cell free splicing assay found that structurally similartetracyclines promoted exon 7 inclusion (although aclarubicinwas not active in this assay) [25]. Paratek is now developing oneof the compounds active in this cell-free assay, PTK-SMA1,which is selective for SMN2 exon 7 inclusion at low concen-trations although it affects splicing of other transcripts athigher concentrations. This means dose finding studies willbe needed to optimize the therapeutic effects while avoidingpotential side effects when this class of compounds enters clin-ical development. Biomarkers related to the pharmacologicaleffect of the compounds could be developed (e.g.,SMN2 transcripts versus other gene transcripts) to serve thispurpose. Ideally, they would be able to be measured in bloodcells or serum taken from healthy subjects as well as SMApatients. How tetracyclines modulate SMN2 transcript

Table 1. Drugs and drug candidates that increase SMN production from the SMN2 gene.

Drug Mechanism of action Ref.

Expression Splicing Stability

Sodium butyrate + [12]

Vanadate + [83]

Valproic acid + + [15]

Phenylbutyrate + + [16]

Hydroxyurea + + [13,14]

HDAC inhibitorsVorinostat (SAHA)Trichostatin AM344Romidepsin (FK-228)LBH589

+ + [84,85]

[86]

[87]

[88]

[89]

Tetracyclines +AclarubicinPTK-SMA1

[24]

[25]

Oligonucleotides + [26]

PRO105ISIS-SMNRx

[27]

[28]

Salbutamol + [90]

AminoglycosidesTobramycinTC007

+ [40]

[91]

[42,43]

QuinazolinesD156844, D157495 or RG3039

+ [31,33-38]

Indoprofen and analogsALB-X

+ [44,92,93]

[45,94,95]

SMN: Survival motor neuron.

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Expert Opin. Drug Discov. (2011) 6(8) 829

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splicing is not actually known, although their activity in a cell-free assay indicates that they interact with some component ofthe splicing reaction, the RNA or protein splicing factors.Further understanding of the mechanism could reveal targetsor chemical strategies to improve this approach.

2.1.2 OligonucleotidesOligonucleotides that were designed to hybridize to a spliceo-some recognition site in intron 7 were found to efficientlyblock SMN2 exon 7 excision in vitro [26]. This highly selec-tive approach has been further developed and found to beeffective in mouse models of SMA [27,28]. Other RNA anti-sense approaches using viral vector delivery have beenreviewed recently [23]. Small synthetic oligonucleotides arecurrently being developed as SMA therapeutics by Prosensa(PRO105) [29] and ISIS in collaboration with Genzyme(ISIS-SMARx) [30]. The current products are in preclinicaldevelopment stages. Issues that are being addressed includebetter understanding of where, when, how often and howmuch to deliver, what side effects are possible (e.g., pro-inflammatory effects) and what delivery systems will beneeded to get oligonucleotides across the BBB.

2.2 Increasing SMN2 expressionIncreased SMN2 copy number is correlated with reduceddisease severity because some SMN2 transcripts give rise toFL-SMN. Therefore, increasing expression from a patient’sSMN2 genes is another possible strategy to increase SMNproduction. Two groups have reported the development of areporter gene assay driven by the SMN2 promoter [31,32] using

neuronal cell lines in order to mimic as close as possible thetranscriptional regulation present in motor neurons. One ofthese screening strategies has led to the discovery of a familyof C-5 substituted quinazolines that increase SMN expres-sion [31,33,34]. Chemical optimization of the hits comingfrom this screening campaign lead to the identification of apotential drug candidate, D156844, that is orally active,crosses the BBB and is active in a mouse model ofSMA [35,36]. Proteomic methods found that the compoundtargets and inhibits DcpS, an mRNA decapping enzyme [37].It is thought that this stabilizes SMN2 transcripts and therebyincreases SMN protein. However, it is not clear whether orhow DcpS inhibition selectively increases SMN (activity inthe original reporter gene assay suggests not) and if not,what side effects could result. This novel target and mecha-nism that were uncovered using a cell-based assay is a goodexample of biological serendipity: you may not get what youwant, but if you try, sometimes you get what you need!Whether the effects in SMA animal models are due to thismechanism or to an increase in SMN remain to be deter-mined (however, this is the case for other compounds thatare active in SMA animal models). This drug discovery pro-gram was financed by a patient organization, Familiesof SMA, and was recently licensed to Repligen; the leadmolecule, RG3039 (or D157495), is in the preclinical stageof development [38].

2.3 Promoting SMN stabilityStabilizing either FL-SMN or SMND7 is expected to increaseprotein levels. SMN occurs in oligomers and oligomerization

Table 2. Screening approaches used to identify small molecule SMA therapeutics.

Assay type Compounds identified Ref.

Phenotypic screening on whole cells measuring SMN protein or FL-SMN transcriptsSMA patient lymphoblasts Hydroxyurea, sodium butyrate [12,13]

SMA patient fibroblasts Valproic acid [15]

Phenylbutyrate [16]

Aminoglycosides [40]

Forskolin [39]

Reporter gene assaysMinigene SMN2-exon 6 -- 8-luciferase Vanadate

Indoprofen analogsPolyphenols: curcumin, resveratrol

[83]

[44,92,93]

[96]

Minigene SMN2-exon 6 -- 8-b lactamase Aclarubicin [24]

SMN2 promoter-b lactamase Quinazolines [31]

SMN2 promoter-secreted alkaline phosphatase Taxol [32]

Cell-free assaysCell free splicing assay Tetracyclines [25]

SMN-dependent snRNP assembly Antioxidants [46-48]

Pluripotent stem cellsSMA patient-derived iPSCs Valproic acid

Tobramycin[57]

Phenotypic screen for neuroprotectionTrophic factor-deprived primary motor neurons Cholesterol oximes: olesoxime [75,76]

FL-SMN: Full length SMN; iPSC: Induced pluripotent stem cell; SMA: Spinal muscular atrophy; SMN: Survival motor neuron; snRNP: Small nuclear

ribonuclear protein.



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stabilizes both SMN and SMND7. SMND7 has a shorter half-life than SMN [10] and this was shown to be associated with areduced ability to oligomerize and form complexes comparedto FL-SMN [39]. Oligomerization appears to protect SMNand SMND7 from degradation by the ubiquitin-proteosomepathway since proteosome inhibitors slowed their degrada-tion [39]. Besides a general impact on conformation affectingoligomerization, the new C-terminal of SMND7 appearsto create a ‘degron’ or degradation signal that acceleratesproteosome-mediated turnover [11]. SMN oligomerizationand stability can be increased by cAMP and protein kinase Aactivation suggesting that pharmacological approaches couldbe used to increase SMN levels by this mechanism [39].

While SMND7 is unstable, replacing the C-terminal tail byother sequences or extending it by suppressing a terminationcodon in exon 8 with aminoglycoside antibiotics producesstable, correctly targeted and functional protein [40,41]. Subcu-taneous delivery of a novel derivative of this class of antibiot-ics, TC007 [42], improved the size of muscle fibers and motorfunction in an SMA mouse model although neither this newermolecule nor G418 improved survival of these mice [41,43].Poor BBB penetration may be the reason for poor efficacyof these compounds. PTC Therapeutics, which has beendeveloping stop codon suppressing compounds for musculardystrophies and other indications, has also reported workingon compounds of this type for SMA.

Indoprofen analogs also appear to increase SMN stability.Although originally discovered in a high-throughput screenusing the minigene reporter assay designed to identifySMN2 splicing modulators (Table 2), indoprofen did not affectSMN2 transcript profile in SMA patient fibroblasts but didincrease SMN levels [44]. The effects of indoprofen were notfound with other COX inhibitors. The Spinal Muscular Atro-phy Project, financed by the NIH, has been developing opti-mized indoprofen analogs to eliminate effects on COX,increase potency and allow passage across the BBB. The mech-anism of action of these compounds appears to be, likeaminoglycosides, due to stop codon readthrough [45].

3. Compensating for functional consequencesof SMN deficits

3.1 SMN, snRNPs and spliceosome functionBecause snRNP assembly and spliceosome activity areimpaired by SMN deficits, assays focused on the discoveryof small molecules that enhance snRNP assembly could be avalid and interesting drug discovery strategy. Indeed, novelhigh-throughput screens have been developed that specificallymeasure snRNP competent SMN in cell extracts [46-48].Although these assays have so far only identified inhibitors,they served to discover that oxidative stress severely impairsSMN-mediated snRNP assembly. Following up on this obser-vation, a study in SMA patients treated for up to 24 weekswith various antioxidant formulations found a progressiveincrease in snRNP assembly competence in their cells [48].

3.2 Other functions of SMNAlthough SMN deficits impair snRNP assembly andspliceosome activity, this affects all cells so it remains to bedetermined why motor neuron degeneration is the major con-sequence in SMA. Intellect and other neurological functionsare spared as are most other tissues and organs (with the pos-sible exception of cardiac tissue; see Section 5.1 below). Otherpossible roles of SMN, particularly in neurons, are nowemerging. There is mounting evidence that SMN participatesin other ribonuclear protein complexes and in particular ispresent in axon granules that transport RNA cargo to nerveterminals [49-52]. Such neuron-specific functions may explainwhy motor neurons with axons extending from the spinalcord to proximal muscles are particularly vulnerable toSMN deficiency compared to other cells.

4. Other strategies besides increasing SMN

Other genes besides SMN2 are able to modify SMA diseaseseverity. Both clinical genetics and studies in various SMAmodelsystems have identified candidate genes and pathways. Geneticanalysis of families with a child affected by SMA found thatsome of their unaffected siblings had the same SMN1 deletions.Further investigation led to the discovery that Plastin 3 overex-pression can prevent SMA disease onset [53,54]. Plastin 3 is anactin-regulating protein implicated in axonogenesis and canincrease neurite outgrowth in cultured motor neurons fromSMAmice or in a zebrafishmodel of SMA [54]. Cytoskeletal per-turbations have been found in SMA model systems. Knockingdown SMN expression in PC12 cells activates RhoA, a regulatorof actin dynamics that can cause neuronal growth cone collapse.Treating SMA mice with Y-27632 which inhibits ROCK, adownstream effecter of RhoA, improved maturation ofneuromuscular junctions (NMJs), increased muscle fiber sizeand prolonged survival [55]. Similar knockdown studies inNSC34 neuronal cells discovered that stathmin, a microtubuledestabilizing protein, is increased. Stathmin increases were thenfound to be present in SMA mice as well as perturbations inmicrotubule structure and dynamics in their motor neurons [56].Perturbations in microtubule-mediated organelle transport andneurofilament organization are also noted in SMA models (seebelow). These results suggest that treatments affecting cytoskele-tal dynamics may be therapeutically useful in SMA even if theydo not change SMN protein levels. Recent use of induced plu-ripotent stem cells derived from SMA patient show that theymay be useful for developing newmodels to explore the functionof SMN protein and disease mechanisms [57].

5. Is SMA a single disease? Unravelingdisease mechanism and alternative targets

5.1 What leads to neuromuscular pathology and

motor neuron cell death?Motor neuron cell death occurs naturally during developmentand is programmed in relation to muscle innervation. Motor

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neuron survival depends on the extent of branching and syn-apse formation that facilitates interaction with the basal lam-ina and uptake of trophic factors supplied by muscle andSchwann cells. Connections between motor nerve presynapticterminals and the basal lamina facilitate access to trophic fac-tors and appear to be mediated by the interaction betweenlaminin subunits and synaptic vesicle proteoglycans exposedduring neurotransmitter release [58]. This implies that theamount of synaptic vesicle fusion occurring with motor neu-ron firing plays an important role in neuromuscular junction(NMJ) formation and stabilization. NMJ abnormalities arefound in most if not all SMA mouse models [59-65]. Theseinclude disorganized neurofilaments and synaptic vesicles inpresynaptic nerve terminals and immature postsynaptic struc-tures. These abnormalities are associated with decreased neu-romuscular transmission that is attributed to decreasedprobability of synaptic vesicle fusion and reduced quantalrelease [62,63,65]. Two recent descriptions of rescue or recoveryof SMA disease phenotype in mice suggest that early deficitsin synaptic vesicle release are reversible. A single intravenousadministration of a viral vector delivering the SMN gene atthe time of birth rescues severely affected SMA mice and theirrecovery is associated with normalized motor endplate currentindicative of increased quantal release [63]. Similarly, a recentlydescribed SMA mouse model in which SMN deficiency is tar-geted to motor neurons displays neuromuscular weaknessduring the first week of life but recovers during the secondweek so that ~ 70% live > 1 year [65]. Recovery was associatedwith normalization of quantal release at motor endplates.Even though these mice recover and survive, there is evidenceof simultaneous atrophy and hypertrophy in their skeletalmuscle fibers showing SMN depletion in motor neuronsalone is sufficient to generate muscle abnormalities. Neverthe-less, SMN may be important in other tissues as well: cardiacabnormalities have been noted both in severely affectedSMA patients [66] and SMA mice [67].

5.2 SMA: a spectrum of disease severity requires a

variety of therapeutic optionsDisease severity and progression in SMA patients areextremely variable and classified by type according to func-tional milestones the individual can achieve. Type 1 patientscan never sit independently, type 2 patients can sit but neverwalk independently and type 3 patients develop the abilityto walk but eventually lose this capability. These classifica-tions, as cited previously, are correlated with SMN proteinlevel and SMN2 copy number, which in turn correlates withthe rate of disease progression and the rate and extent ofdenervation as shown in a natural history study [8]. Remark-ably, in a few type 1 and type 2 SMA patients identified byprenatal testing who could be studied from birth, denervationas measured by the decrease in compound muscle actionpotential (CMAP) amplitude occurred very rapidly startingfrom nearly normal levels at birth [8]. Denervation intype 3 patients on the other hand was less marked and

CMAP was rather stable with time. Muscle fibers in biopsiestaken from SMA patients or normal controls have also foundmarked differences between type 1 and type 3 SMA patients.While muscle transcripts from type 3 patients were closerto those in normal muscle, transcripts from type 1 patientswere drastically different. There was evidence for simulta-neous atrophy and hypertrophy in type 3 SMA muscle fibersassociated with various signaling pathways that were notpresent in type 1 SMA fibers [68].

Because of the difference in disease severity and progres-sion, SMA may not be a single disease and therapies need tobe designed and developed for various subpopulations. Exceptfor the mouse model in which SMN depletion is targeted tomotor neurons and the type III SMA mice generated byback-crossing founders onto a C57Bl/6 background [69],

most SMA mouse models have a severe phenotype andare considered models of type 1 SMA. These models as wellas clinical evidence that denervation occurs soon after birthin severe forms of SMA suggest that the window of oppor-tunity for treatment may be very narrow in type 1 SMApatients [8,63,70,71]. Introduction of newborn screening forSMA will be essential to initiate treatment during thiscritical period.

In less severe SMA models, mice have a nearly normal lifespan and so far do not appear to offer similar end points toassess disease progression as those in type 2 and type 3 SMApatients. This makes identification and validation of therapiesfor less severely affected SMA patients, for whom preservingexisting neuromuscular function rather than reversing diseasewill be the objective, more challenging both conceptually andpractically. A porcine model of SMA is currently in develop-ment with the hope that it may be closer (both in size and dis-ease phenotype) to human SMA patients [72,73] and provide ameans to test therapies for mild as well as severe forms ofSMA. Because no suitable animal model exists to test thera-pies for type 2 or type 3 SMA patients, there are severalopen questions about whether treatments that increase SMNwill be effective in these patients, when the treatment(s)should be administered and how their effects will be mea-sured. New ethical issues regarding how and when to treatchildren who may have milder forms of SMA will also needto be addressed. In the future, should newborn screening beavailable, will it be possible to accurately predict the time ofdisease onset? SMN2 copy number is not sufficiently preciseeither to detect all type 1 patients who should be treatedimmediately or to predict the time of disease onset or severityif three or more copies of SMN2 are present. Will it be ethicalto initiate treatment before symptom onset? And if not, will itthen be too late?

6. Neuroprotection, alone or in combinationwith SMN enhancers

Besides increasing SMN, neuroprotection might havetherapeutic potential. Indeed the two approaches may be



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complementary. This approach was recently tested bycombining a nucleic acid SMN2 splicing modulator withIGF1 in a single viral vector and comparing the effects ofthis combination vector with those of individual vectors deliv-ering either a splicing enhancer or IGF1 in a severe SMAmouse model [74]. Interestingly, a significant effect on survivalwas produced by delivering IGF1 alone while the combinedvector was only slightly but not significantly more effective.Delivering the splicing enhancer alone had a small but insig-nificant effect although SMN protein expression wasenhanced. These results suggest that neuroprotection alone,which may have had little or no effect on SMN levels or splic-ing deficits resulting from SMN deficiency, could be a highlyeffective therapeutic option in SMA.

Neurotrophic factors, besides increasing neuronal sur-vival, enhance neurite outgrowth, which may improve neu-ronal cytoskeleton abnormalities resulting from SMNdeficits as mentioned previously (Plastin 3, RhoA, stath-min). Olesoxime (also called TRO19622) is a compoundcurrently being studied in clinical trials in type 2 andtype 3 SMA patients as well as in patients with ALS. Itsdiscovery and development for SMA was financed by theAssociation Francaise contres les Myopathies, a Frenchpatient organization. Olesoxime was discovered in a cell-based screen for compounds that rescue trophic factordeprived motor neurons; it favors both motor neuronsurvival and neurite outgrowth, has shown beneficial effectsin mouse models of ALS and SMA [75,76] and rescues othertypes of target deprived neurons [77]. The neuroprotectiveeffects of olesoxime are attributed to modulation of oxida-tive stress-induced mitochondrial permeability transitionthrough interaction with two outer mitochondrial mem-brane proteins, TSPO and VDAC. In addition, olesoximepromotes microtubule dynamics and preserves neuritesfrom toxic effects of microtubule-targeting chemotherapeu-tic drugs [78], which may explain its ability to promote neu-rite outgrowth. Despite having no effect on SMN levels,olesoxime appears to have a combination of actions thatcould be beneficial in SMA either alone or in the futurein combination with SMN enhancing therapies.

7. Conclusion

Since the discovery that SMA was due to mutations inSMN1 and that it could be possible to increase SMN produc-tion from a patient’s SMN2 gene, enormous effort and prog-ress have been made in identifying mechanisms, smallmolecules and gene therapies that could fulfill this objective.Screening models have been established that have identifiedsmall molecules with potential to be developed into drug can-didates focused on SMN production as well as neuroprotec-tion. New screening strategies are likely to evolve withfurther understanding of cell type specific functions ofSMN in neurons, skeletal muscle and cardiac tissue, all ofwhich may be affected in SMA. Assays based on SMA

patient-derived pluripotent stem cells may provide bettermodels to discover and evaluate new disease modifyingapproaches. iPierian has created a drug discovery screeningplatform based on patient-derived pluripotent stem cells andis tackling SMA therapeutics.

8. Expert opinion

The first clinical trials of SMN2 modulating therapies withhydroxyurea, valproic acid or sodium phenylbutyrate wereconducted because these drugs had already been approvedfor use in children; ongoing trials of additional compoundsor ones expected to start in the next few years were coveredin a recent review [32]. The trial results that have been reportedso far have not validated the SMN2 modulation strategy per-haps for methodological reasons (dose tested, trial duration,number of patients studied) although responder subpopula-tions could benefit from these treatments [79,80]. These andother trials have paved the way by establishing clinical trialmethodology showing that SMA disease progression is slowand variable and so trial duration will need to be long (twoor more years) to detect a disease modifying effect using anumber of motor function scales. Identifying and validatingbiomarkers both related to the product’s expected pharmacol-ogy (e.g., for the SMN approach, SMN protein, SMN2transcripts, snRNP assembly) should be used for dose selec-tion and to evaluate acute and long-term effects; they couldalso be used to screen and select possible responders for along-term clinical trial for efficacy. A clear understanding ofdisease progression, particularly in different patient subpopu-lations and disease-associated biomarkers, is also criticallyneeded. These could be more sensitive and less subjectivemeasures of disease progression and provide surrogate endpoints for efficacy earlier than an effect on motor functioncan be detected. Electromyography to assess CMAP andmotor unit number estimation has been shown to be feasiblein clinical trials even in young children and appears to becorrelated with disease severity and functional outcomemeasures; it appears to be promising as an early biomarkerindicative of efficacy and should be performed in SMA clini-cal trials to validate this hypothesis [8,81]. It will also be neces-sary to consider co-development of complementary therapies:combinations of products increasing SMN by differentmechanisms, combinations of neuroprotective therapies andproducts increasing SMN, or other strategies that mayemerge. Regulatory agencies are beginning to address how toevaluate combinations of investigational drugs in certain indi-cations [82]. The more we know about SMN and SMA diseasepathology, the more we understand that SMA is not such asimple monogenetic disease with only one therapeutic targetor strategy. The molecular tools developed so far are wideningour understanding and the scope of possibilities. Theseemerging novel ideas need to be explored. Because SMA is arare disease, preclinical and clinical research funding haslargely been provided by patient organizations and

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government agencies. Priorities should include identificationof alternative SMN functions, neuroprotection, improvingneuronal cytoskeletal dynamics, reducing oxidative stress andother targets and mechanisms as they emerge from furtherunderstanding of disease mechanism. Identification and vali-dation of pharmacodynamic biomarkers for each therapeutic

strategy as well as SMA disease progression will alsobe indispensable.

Declaration of interest

RM Pruss is an employee of Trophos.

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AffiliationRebecca M. Pruss PhD

Chief Scientific Officer,

Trophos, SA Parc Scientifique de

Luminy Case 931,

13288 Marseille Cedex 9, France

Tel: +1 33 0 491 828281;

Fax: +1 33 0 491 828289;

E-mail: [email protected]

Pruss


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Developments in the discovery of drugs for spinal muscular atrophy: successful beginnings and future...

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