Research ALS Today, Spring 2015

INSIDE

RESEARCH ALS TODAY

THE ALS ASSOCIATION

VOLUME 16

Using Motor Neurons Sheila Essey Award Drosophilia About CReATe Tirasemtic Clinical Research Recipient Journal News

1-3 3 4-5 6 6-7 7 8

SPRING 2015

Using Motor Neurons Derived from Induced Pluripotent Stem Cells to Understand ALS

Dhruv Sareen1, Robert Baloh1,2 and Clive N. Svendsen1 1 Board of Governors, Regenerative Medicine Institute and

Department of Biomedical Sciences, Cedars-Sinai Medical Center 2 Department of Neurology, Cedars-Sinai Medical Center

We need better models to understand amyotrophic lateral sclerosis (ALS) Our present understanding of the underlying biological basis for ALS-associated neurodegeneration in humans is primarily derived from rodent and other cellular models of familial ALS or from post-mortem patient tissue. However, mice have a fundamentally different biology than humans, and patient tissue limits the study to only after death. Furthermore, because we do not know the cause of sporadic ALS, which accounts for 90 percent of the ALS population, it is impossible to model this in animals. But new cutting-edge stem cell technology circumvents this problem. Cells drawn from a patient’s blood can be sent back in time to a very early stage using a few specific chemicals. These cells, termed induced pluripotent stem cells (iPSCs), can be grown indefinitely. When given specific signals, they can then be made into the motor neurons that die in ALS. iPSCs capture the patient’s exact genetic material and provide an unlimited supply of cells that can be studied in the dish, i.e., the ALS patient’s “avatar.” Our focus is to use this unique source of motor neurons to try to understand why they do not function normally in ALS. We will also generate “clinical grade” iPSC lines that will provide a new source of replacement tissue for autologous or allogeneic cell therapies currently under development at Cedars-Sinai Medical Center (CSMC).

Quality The quality of motor neurons generated from the iPSCs will affect the value of the derived information. As such, we are developing optimized techniques that can turn patient iPSCs into specifically the spinal lower motor neurons that are affected in ALS (see figure). Most published motor neuron differentiation methods focus on the generation of spinal motor neurons; however, the cortical upper motor neurons also play a crucial role in ALS. By using principles from human nervous system development, we will also establish ways to produce upper motor neurons to examine this specific population. The limitless quantity of iPSCs

A rapid and directed motor neuron differentiation protocol with high-efficiency differentiation of iPSCs into spinal lower motor neurons.

permits their use in multiple experiments. The iPSC technology will allow us to create a patient’s avatar-in-a-dish, which can be used to correlate patient clinical parameters such as site of onset and severity with any phenotypic changes in the same patient’s motor neurons. Selected patient iPSC lines will also be genetically modified to develop a toolbox of “reporter” cell lines. When motor neurons are generated from iPSCs, they will produce markers that can be seen under the microscope to allow other members of the Neuro Collaborative to identify the motor neurons and track their health over time, and see how they respond to new drug treatments.

Collaboration is key for success The Neuro Collaborative (three leading California labs headed by Drs. Cleveland, Finkbeiner and Svendsen and funded

by The ALS Association) is not working in isolation. We are also working closely with companies, such as Emulate, which are developing more physiological Continued on page 2

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VOL.16

Using Stem Cells to Understand ALS Continued from page 1

Multiple Research Strategies Reinforce One Another The effort to understand the causes of ALS, and hence develop appropriate therapies, is happening on multiple fronts, as the updates in this issue of Research ALS Today illustrate. That multi-pronged approach maximizes the chances of success against a complex problem. More importantly, the multiple research strategies supported by The ALS Association complement and synergize with one another, each making the other more powerful, and all combining to accelerate the search for new treatments.

Lucie Bruijn, Ph.D., M.B.A. Chief Scientist The ALS Association

The creation of a new core facility dedicated to induced pluripotent stem cells (iPSCs) promises to accelerate the understanding of the cellular basis of ALS, not in a single cell line but in dozens or hundreds, each derived from a single individual with ALS. Comparing the disease process among them—each a slightly different model of the same disease—is likely to reveal major new insights into the commonalities that unite and the variations that distinguish ALS in the patient population. These cell lines can be used to screen candidate drugs based on either the common or the unique disease mechanisms within each line.

Insights from cell-to-cell comparisons can also be used to stratify enrollment in clinical trials, targeting experimental treatments to those most likely to benefit from them based on their specific disease mechanism.

Improving the odds of success in clinical trials will also be aided by more precise biomarkers of disease activity and by a deeper knowledge of the genetics and epidemiology of ALS, which are the two efforts spearheaded by this year’s recipients of the Sheila Essey Award. At the same time, CReATe, a new multi-center consortium funded by the National Institutes of Health, is undertaking a major effort to correlate genotype with clinical phenotype—including patterns and rate of progression—in 700 people with ALS or one of a set of related disorders, including frontotemporal degeneration. Results from this work should allow us to gain a more detailed understanding of the multiple forms of ALS and thus shape clinical trials to match the underlying natural history of specific subgroups of patients. We continue to invest in clinician scientists, in partnership with The American Academy of Neurology, who are critical to move the research field forward and congratulate Hristelina Ilieva, M.D., Ph.D., of the Department of Neurology at the Johns Hopkins School of Medicine, Baltimore, Maryland, this year’s recipient of the Clinical Research Training Fellowship.

The search for new treatments for ALS is challenging, but the efforts supported

by The ALS Association are building a dynamic foundation for progress. We look forward to important advances in the coming year.

–Lucie Bruijn, Ph.D., M.B.A.

The Neuro Collaborative iPS cell line initiative at Cedars-Sinai Medical Center.

systems of studying cells that involve micro fluidic devices. Cells are seeded into small “chips” that have neurons and blood vessels in close apposition, mimicking the human body. This should encourage more maturation of the cells and a better models of a disease––and possibly “ALS on a chip” for drug discovery and a better understanding of the disorder. In addition, we are integrated with a number of other studies attempting to address similar problems using iPSC technology and will share resources and ideas as the project progresses. Two of these are co-funded

by The ALS Association, allowing us to combine funding in a synergistic but non-overlapping way. The first is funded by the NIH and The ALS Association and called NeuroLINCS. Here we will share the lines generated

by CSMC with a number of other institutions

Credit: Dhruv Sareen

around the country. Motor neurons will be challenged with specific molecules to better understand how they respond––and how this response may change in ALS. All of the data will be collected and analyzed together using “Big Data” techniques, and then made available to the ALS community through the NIH. In another initiative ALS ACT, funded

by The ALS Association and the new foundation ALS Finding a Cure, CSMC will be generating 50 new iPSC lines from ALS patients who are being followed over time using advanced neuroimaging techniques and novel markers of disease that are being developed by General Electric Healthcare.

This will allow us to compare “disease in a dish” with “disease in the patient” and further establish whether these new methods have predictive value in ALS. Continued on page 3

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Using Stem Cells to Understand ALS Continued from page 2

The future looks bright Important inroads to understanding the molecular pathways involved in neurodegenerative aspects of all forms of ALS have been made using rodent models. However, further elucidating these mechanisms in humans will benefit greatly from utilizing large numbers of ALS patient iPSCs and their differentiated motor neurons.

We recognize the importance and ease of access for sharing iPSC lines, so those created with The ALS Association funding will be made available to the research community using the Universal Biological Material Transfer Agreement (UBMTA), to which most major institutions already subscribe. This will allow easy transfer of the cells between ALS research groups. The ALS Association’s support of the Neuro Collaborative and other related initiatives described here will provide not only a unique collection of iPSC lines, but also begin to standardize and share methods of differentiation, gene targeting and omics profiles that will be crucial to move the field forward rapidly. Eventually it may be possible to use these models to test large numbers of novel drugs entirely in the petri dish over short periods time. If the cells in the dish give any indication of where in the nervous system the disease may be most severe, how quickly it will progress or how it may respond to treatment, it will be an enormous leap forward for the field. Finally, developing and characterizing ALS in the dish will give us a new way of exploring what goes wrong in the disease––and with this knowledge enable us to develop new treatments.

Robert Bowser, Ph.D., and Adriano Chiò, M.D., Receive the Sheila Essey Award Each year, The ALS Association and the American Academy of Neurology (AAN) present the Sheila Essey Award for ALS Research to acknowledge and honor an individual who is making significant contributions in research for the cause, treatment, prevention or cure for ALS. The award is made possible through the generosity of the Essey Family Fund, in memory of Sheila Essey, who battled ALS for 10 years and died from the disease in 2004. Past recipients have used the funds to continue ALS research or to support promising young scientists on their research teams.

2015 marks the 20th year of the Sheila Essey Award. The ALS Association and the AAN are deeply grateful for the unwavering commitment of Richard Essey in continuing to support this important honor.

This year, The Association and the AAN are very pleased to give this award to two scientists, Robert Bowser, Ph.D., and Adriano Chiò, M.D., who have each made major contributions to the understanding of ALS and the search for new therapies.

The awards will be presented in April at the Annual Meeting of the American Academy of Neurology in Washington, D.C.

Robert Bowser, Ph.D.

Adriano Chiò, M.D.

Dr. Bowser is Director of the ALS and Neuromuscular Research Center at Barrow Neurological Institute in Phoenix, Arizona. His lab is dedicated to the discovery of biomarkers for ALS, which can be used to improve diagnosis, track progression and monitor response to therapy. In 2011, he and his colleagues announced that the cerebrospinal fluid ratio of the levels of two proteins, phosphorylated neurofilament heavy chain and complement C3, was a highly sensitive and specific indicator of ALS.

Dr. Chiò is Professor of Neurology at the University of Turin, Italy, and is Medical and Scientific Director of the Turin ALS Center, the tertiary ALS care center for northwestern Italy.

Dr. Chiò leads clinical, epidemiological and genetic research on ALS. He has contributed to the discovery of several ALS genes and the development of new clinical tools for tracking disease progression and predicting outcome. As both a clinician and a researcher, Dr. Chiò has been a leader in the understanding of ALS and the testing of new therapies.

“I am truly honored to be a recipient of this year’s Shelia Essey Award for ALS Research,” Dr. Bowser said. “It is a privilege to be included with my many esteemed colleagues who have been awarded this honor in the past, and I remain committed to my research efforts to help define the pathophysiology of ALS and find improved drug treatments for ALS.”

“I feel greatly honored and thankful to be the 2015 co-recipient of the Sheila Essey Award,” Dr. Chiò said. “This prestigious award is a greatly welcomed recognition of the hard work done by my team at the University of Turin in the struggle against ALS. The award, while representing an acknowledgment for our research, is a strong motivation to proceed with even more dedication toward the goal of a world without ALS.”

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Drosophila Genetics:

A Fruitful Approach for ALS Research Fen-Biao Gao, Ph.D., Department of Neurology, University of Massachusetts Medical School, Worcester, Mass.

J. Paul Taylor, M.D., Ph.D., Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, Tenn.

T

he tiny flies lingering around overripe fruits on the kitchen countertop

may be annoying, but one species—Drosophila melanogaster—has

been a powerful force in biomedical research for more than a century.

In 1910, Dr. Thomas H. Morgan discovered a gene in Drosophila melanogaster that he named white. When the white gene is mutated, the red color of fly eyes turns into white. The next year, in a paper published in Science, Dr. Morgan concluded that the genes responsible for specific traits are located on chromosomes—an insight for which he received a Nobel Prize in 1933. In 1995, another Nobel Prize went to Drosophila geneticists Drs. Edward B. Lewis, Christiane Nüsslein-Volhard and Eric F. Wieschaus for discovering through genetic screens many key genes important for animal development. Remarkably, these fruit fly genes are conserved all the way to humans. In part because of this evolutionary conservation at the molecular level and the wide range of powerful tools for genetic analysis, Drosophila has been widely used over the last 20 years as a model organism for studies on human neurodegenerative diseases, including the devastating motor neuron disease ALS. In fact, Drosophila was used to study ALS long before fly models became fashionable for many other neurodegenerative diseases. As early as 1995, Drosophila served as an in vivo model system to examine the biophysical properties of normal and mutant versions of SOD1—the first gene linked to ALS. Over the following decade, several groups used Drosophila to study the effects of familial ALS-associated mutations in SOD1 and VAPB on lifespan and motor neuron biology. Thanks to breathtaking discoveries of new genes in ALS pathogenesis, achieved through biochemical approaches or human genetics studies over the past 10 years, Drosophila models have come into even wider use for ALS research.

A groundbreaking discovery in understanding the origins of ALS was the identification of the

RNA-binding protein TDP43 as a prominent component of the cellular pathology characteristic of ALS and related diseases such as frontotemporal dementia (FTD). Normally TDP-43 is largely localized in the J. Paul Taylor, Ph.D. Fen-Biao Gao, Ph.D. cell’s nucleus; but in ALS, it is often found redistributed to the periphery of the cell where it forms pathological clumps in diseased neurons. The extent to which loss of TDP-43 function in the nucleus versus the potential toxicity of these clumps in contributing to disease remains unclear. Subsequent identification of ALS-causing mutations in both TDP-43 and another RNA–binding protein, FUS, led to the appreciation that defective RNA metabolism is a major culprit in ALS. In dissecting these mechanisms, fruit flies were of fundamental importance because of their relatively simple, yet well-characterized nervous system. Moreover, due to the ease of genetic manipulation, fruit flies are well suited for loss-of-function and gain-of-function studies in-vivo. For instance, examination of the neuronal functions of endogenous TDP-43 using “knockout” flies identified defects in the morphology and function of synapses at the neuromuscular junction and in dendritic branching. Transgenic fly lines generated in many labs have revealed various aspects of the pathogenic mechanisms of mutant TDP-43. Other important in vivo functions of TDP-43, including that in the miRNA pathway and axonal transport, were also revealed in fly models.

Similar genetic approaches in Drosophila have been quite informative for investigating other ALS genes, such as the endogenous functions of FUS in lifespan and synaptic functions Continued on page 5

1985: The ALS Association funds study of inherited motor neuron disease

TIMELINE 1869: French neurologist Jean-Martin Charcot identifies ALS

50s: DNA structure solved 50s: Nerve growth factor (NFG) identified–protective, growth promoting factor for nerve cells

1968: SOD1 enzyme identified

70s: Programmed cell death in motor neurons demonstrated

1986: Genes for muscular dystrophy identified

1990: Congress declares the 1990s the “Decade of the Brain”

1989: The ALS Association funds search for a common genetic link to ALS

1990: Growth factor CNTF is found to increase survival of motor neurons

1991: Researchers link familial ALS to Chromosome 21

1860 1950 1960 1970 1980 1990 1991

The ALS Association begins workshops Glutamate transporter shown to be defective in ALS Growth factor BDNF found to increase survival of motor neurons

1992

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Drosophila Genetics Continued from page 4

at the neuromuscular junction, the toxic properties of ectopically expressed mutant FUS, and effects of disease mutations in hnRNPA2 and hNRNPA1 on stress granule formation.

Synapses at the Drosophila neuromuscular junction, a popular model for studying ALS and other neurodegenerative diseases.

Flies have another major advantage—the ability to rapidly reveal genetic interactions between distinct ALS genes, illuminating common biological pathways involved in disease. Indeed, several studies uncovered the genetic interactions between TDP-43 and FUS, TDP-43 and VCP (a chaperone involved in both FTD and ALS), VCP and FUS, and TDP-43 and FMRP (an RNA-binding protein missing in fragile X syndrome). Studies in flies also played a pivotal role in identifying Ataxin-2 as a strong modifier of TDP-43 toxicity and risk factor for ALS. More recent work pointed out the potential involvement of TDP-43 in neurotoxicity induced by CGG repeats. Thus, extensive studies in Drosophila and other model organisms strongly suggest that mutations in at least a subset of ALS genes directly compromise the same genetic pathways. Therapeutic approaches targeting these core pathways may be feasible.

The power of Drosophila genetics is also well suited for identifying genetic modifiers of ALS genes through unbiased screens. For instance, an RNAi screen identified the type 1 inositol triphosphate receptor as a modifier of TDP-43. The more commonly performed deficiency screens identified TDP-43 and two other RNA-binding proteins as dominant suppressors of mutant VCP toxicity. Deficiency screens also showed that syntaxin 13 has a novel function in autophagy as a strong enhancer of the toxicity of mutant CHMP2B (an FTD gene that may also contribute to ALS) and that p38 and JNK have opposing roles in TDP-43 toxicity. Candidate gene screen is another productive approach, as in the identification of arginine methyltransferases 1/8 and transportin as modifiers of FUS toxicity in Drosophila.

The latest breakthrough is the discovery of C9orf72 GGGGCC repeat expansion as the most common genetic mutation in both ALS and FTD—a discovery that has caused a great excitement in the field of neurodegeneration research, in part because expansion of different repeat sequences also causes many other age-dependent neurological diseases, such as fragile X syndrome, Huntington’s disease and myotonic dystrophy. Three widely discussed potential

TIMELINE cont.

SOD1 gene mutation (chromosome 21) discovered in familial ALS Trials using glutamate blocker riluzole begin

Animal studies combining CNTF and BDNF demonstrate decreased motor neuron loss GDNF rescues degenerating motor neurons during development in an in vitro experiment

FDA approves riluzole

Toxic properties of the SOD1 enzyme discovered and linked to familial ALS

Nuclear RNA foci in a glial cell, showing one of the key pathological features of C9ALS/FTD can be recapitulated in Drosophila.

Red: RNA foci; to Blue: DNA marker.

Provided by Nam Chul Kim

Provided by Helene Tran

pathogenic mechanisms in C9ALS/FTD are C9orf72 haploinsufficiency, GGGGCC repeat RNA toxicity and abnormal polydipeptides produced through an unusual mode of translation: repeat-associated non-AUG (RAN) translation. Unlike TDP-43 and FUS, C9orf72 has no Drosophila homolog, so this model organism is not useful to test the haploinsufficiency hypothesis. However, Drosophila genetics once again offers a great opportunity to examine both gain of toxic RNA function and polydipeptide toxicity. Indeed, expanded GGGGCC repeats as part of a messenger RNA, when expressed in Drosophila neuronal systems, such as photoreceptor neurons in the eye, cause degenerative phenotypes. These phenotypes likely reflect the production of pathogenic polydipeptides, because overexpression of poly(GR) and poly(PR) alone is highly toxic in flies.

These early exciting observations have raised more interesting questions than answers, as in any other burgeoning young field of research. For instance, how do long stretches of intronic GGGGCC repeat RNAs themselves, which in patients can reach to thousands of copies, cause toxicity? It is certain that other fly models of C9orf72 repeat expansion will be informative.

Moreover, considering the complexity of the potential pathogenic mechanisms underlying C9ALS/FTD, there is no doubt that the time-tested genetic approaches in Drosophila, such as unbiased genetic screens, will once again prove to be highly fruitful.

RNAi discovered by Craig Mello and Andrew Fire

A transgenic rat is designed; efforts start on fly model

The ALS Association co-sponsors workshop on high-throughput drug screening with NINDS NINDS issues first ever RFA (request for applications) specifically for ALS research

Attention turns to support cells of nerve tissue to find role in ALS Inflammation and programmed cell death gather research interest ALS2 gene (alsin protein) linked to juvenile ALS The ALS Association/NINDS collaborative effort begins screening drugs

The ALS Association holds scientific workshop on “Environmental Factors and Genetic Susceptibility” Aggressive search for new ALS genes funded by The ALS Association Scientists complete map of mouse genome Agency of Toxic Substances and Disease Registries awards five grants focused on ALS Department of Defense approves funding for ALS-specific research

1993 1994 1995 1996 1998 2000 2001 2002

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About

The CReATe (Clinical Research in ALS and related disorders for Therapeutic Development) consortium is a collaborative effort to advance therapy development in ALS, frontotemporal dementia, progressive muscular atrophy, primary lateral sclerosis. and hereditary spastic paraplegia.

The goal of CReATe is to discover links between clinical phenotype and underlying genotype, and to discover and develop biomarkers.

The consortium is recruiting 700 patients. The ALS Association will support the collection of biospecimens for ALS. CReATe is led by Michael Benatar, M.D., Ph.D., of the University of Miami, Florida, and involves multiple clinical sites including Duke University, Durham, North Carolina; University of California San Diego; University of California San Francisco; University of Kansas, Kansas City; Mayo Clinic, Jacksonville, Florida; St. Jude Children’s Research Hospital, Memphis, Tennessee; and Tubingen, Germany. It is part of the Rare Diseases Clinical Research Network and is funded by the National Institutes of Health.

More information is available at: https://www.rarediseasesnetwork.org/CREATE/index.htm

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VOL.16

Tirasemtiv

Improving Muscle Function as a Potential Therapeutic Strategy for Amyotrophic Lateral Sclerosis

By Jinsy Andrews, M.D., MSc and Jeremy Shefner M.D., Ph.D.

Changes from baseline in percent predicted slow vital capacity in BENEFIT-ALS

ALS is a complex and heterogenous disease; the possibilities of different causes and pathways suggest that many different disease modifying approaches may be needed.

However, the final common pathway in ALS primarily involves progressive weakness, so a novel way to address function in ALS is to target the muscle. Tirasemtiv is a first in class small molecule that improves the efficiency of muscle contraction. Through this mechanism, tirasemtiv may enhance the response of a muscle weakened due to diminished nerve input in ALS regardless of variable clinical presentations and etiologies.

Mechanism of Action

Tirasemtiv is a selective fast skeletal muscle troponin activator that increases the affinity of troponin C for calcium and sensitizes the sarcomere to calcium, shifting the calciumforce relationship and amplifying the muscle

TIMELINE cont.

Study shows surrounding support cells play key role in ALS Study shows that human embryonic stem cells can be stimulated to produce motor neurons Gulf War study shows that vets deployed to Persian Gulf in 1991 developed ALS at twice the rate of those not deployed there IGF-1 gene therapy study proves beneficial in mice with ALS VEGF gene abnormalities shown to be potential factor in ALS The ALS Association collaborates with U.S. Department of Veterans Affairs to enroll all vets with ALS in registry Early tests of ceftriaxone appear to increase survival in mice with ALS Combination of creatine and minocycline prove more effective together in mouse model than either drug alone

response to submaximal nerve stimulation.

This results in increasing muscle force and decreasing muscle fatigability, presumably by reducing the energetic requirements of calcium cycling during muscle contraction 1.

Clinical Studies Phase 1 Three Phase 1 clinical studies with tirasemtiv were completed in healthy volunteers. Tirasemtiv was generally safe and well tolerated. The pharmacodynamic effects of tirasemtiv were explored by measuring the force of foot dorsiflexion during peroneal nerve stimulation at various stimulation frequencies before and following dosing with tirasemtiv. Skeletal muscle force increased in relation to both the dose and the plasma concentration of tirasemtiv with the effect being most prominent in the middle of the range of stimulation frequencies tested 2.

Phase 2 Three Phase 2a hypothesis generating clinical studies in ALS patients were completed with tirasemtiv CY 4021(n=67) was a doubleblind, randomized, three-period, crossover study using single doses, one week apart, in random order of tirasemtiv at 250 and 500 mg versus placebo. CY 4024 (n=49) was a double-blind, randomized, four parallel group study using tirasemtiv at 125, 250 or 375 mg once daily for 14 days versus placebo. In CY 4025 (n=27), patients taking riluzole 50 mg daily were titrated from 125 mg twice daily to 250 mg twice daily over three weeks in a double-blind, randomized fashion in two parallel groups.

In these studies, tirasemtiv appeared generally safe and well tolerated. In CY 4021, both patients and investigators perceived a dose-dependent improvement in patients’ overall status six hours after single doses Continued on page 7

Study implicates smoking as likely risk factor in sporadic ALS Study releases evidence that mitochondrial malfunction may play an important role in ALS Study funded by The ALS Association to find biomarkers in cerebrospinal fluid and blood

Ceftriaxone increases levels of the glutamate transporter GLT1 in a mouse model of ALS First international workshop on frontotemporal dementia discusses link to ALS Stem cells engineered to make GDNF survive when transplanted into rats modeling ALS Early data suggests that mutant SOD1 may be secreted by and may activate microglia Launch of TREAT ALS initiative (Translational Research Advancing Therapies for ALS) to accelerate clinical trials in ALS VEGF increases survival in a rat model of ALS while improving motor

ALS patient samples collected to NINDS ALS Repository Repository samples allow genome analysis for sporadic ALS First TREAT ALS clinical trials funded First TREAT ALS clinical trials begun TDP-43 discovered as a common link in FTD, ALS Chromosome 9 region intense focus for FTD

Stem cell study shows SOD1 mutant support cells can kill any motor neuron ALS U.S. registry efforts gaining ground in Congress Fish model of ALS: Progress reported SOD1 in altered form common to both sporadic and inherited ALS Engineered stem cells making GDNF help motor neurons survive in SOD1 mutant rats First genome screening data published based on NINDS ALS Repository

2003

2004 2005 2006 2007

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Tirasemtiv

Additionally, in a three-period, crossover, Phase 2a clinical study in patients with Continued from page 6 myasthenia gravis receiving single doses of of tirasemtiv. In CY 4024 and CY 4025, tirasemtiv (250 and 500 mg) and placebo, encouraging trends in the ALS Functional forced vital capacity increased from baseline Rating Scale-Revised score (ALSFRS-R) and by 7.1±2.1% (250 mg, p=0.0012) and respiratory measures were observed.

4.5±2.1% (500 mg, p=0.034) compared to Based on observations from these studies in placebo4. These studies provide preliminary ALS, a larger and longer duration, Phase 2b evidence that tirasemtiv may preserve respiclinical trial, BENEFIT-ALS, was conduct- ratory function by preserving the function of the diaphragm, the only continuously ed in 711 patients with ALS. In this study, exercising skeletal muscle in the human body. tirasemtiv was administered twice daily at each patient’s maximum tolerated dose, up Next Steps to 500 mg daily for 12 weeks.

The statistically significant effects to reduce The primary endpoint, the average change the decline in vital capacity and muscle in ALSFRS-R, was not significantly differstrength in BENEFIT-ALS suggest that ent between groups; however, two prespeci- tirasemtiv has a biological and potentially fied secondary endpoints reflecting skeletal clinically important effect in ALS. Based muscle function revealed significant effects on these observations, a multi-national, with tirasemtiv. There was a statistically double-blind, randomized, placebosignificant reduction in the decline of controlled, Phase 3 study with tirasemtiv percent predicted slow vital capacity (SVC) treatment in patients with ALS is planned on tirasemtiv (see figure on p. 2). The to begin in the second quarter of this year percent change from baseline in Muscle with the primary objective of assessing the Strength Mega-Score, a combined measure effects of tirasemtiv on respiratory function of strength from several muscles, also in patients with ALS. declined more slowly on tirasemtiv.

The dramatic effect of tirasemtiv on vital capacity suggests a clinically important effect on diaphragm function. Other studies provide some clues to this observation.

Chronic treatment with tirasemtiv in the dy2J mouse model of congenital muscular dystrophy had significantly higher diaphragm muscle fiber cross-sectional area and better respiratory response to CO stimulation compared to placebo3.

References:

1. Russell AJ, Hartman JJ, Hinken AC, Muci AR, Kawas R, Driscoll L, Godinez G, Lee KH, Marquez D, Browne WF 4th, Chen MM, Clarke D, Collibee SE, Garard M, Hansen R, Jia Z, Lu PP, Rodriguez H, Saikali KG, Schaletzky J, Vijayakumar V, Albertus DL, Claflin DR, Morgans DJ, Morgan BP, Malik FI. Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases. Nat Med. 2012 Feb 19; 18(3):452-5.

2. Hansen R, Saikali KG, Chou W, Russell AJ, Chen MM, Vijayakumar V, Stoltz RR, Baudry S, Enoka RM, Morgans DJ, Wolff AA, Malik FI. Tirasemtiv amplifies skeletal muscle response to nerve activation in humans. Muscle Nerve. 2014 Dec; 50(6):925-31. [4011 paper] 3. Miciak JJ1, Warsing LC, Tibbs ME, Jasper JR, Jampel SB, Malik FI, Tankersley C, Wagner KR. Fast skeletal muscle troponin activator in the dy2J muscular dystrophy model. Muscle Nerve. 2013 Aug; 48(2):279-85.

4. Sanders DB, Rosenfeld J, Dimachkie MM, Meng L, Malik FI; for the Tirasemtiv in Myasthenia Gravis Study Group. A DoubleBlinded, Randomized, Placebo-Controlled Trial to Evaluate Efficacy, Safety, and Tolerability of Single Doses of Tirasemtiv in Patients with Acetylcholine Receptor-Binding Antibody-Positive Myasthenia Gravis. Neurotherapeutics. 2015 Mar 6.

TIMELINE cont.

Stem cells generated from ALS patients Discovery of DPP6 in two genomewide association studies in ALS Mutations in TDP-43 linked to familial and sporadic ALS Induced Pluripotent Stem Cell Technology opens up new avenues for ALS

Identification of new gene linked to familial ALS, Fused in Sarcoma (FUS) on Chromosome 16 FDA approval of SOD1 antisense and stem cell trials in U.S.

First patients enrolled for antisense and stem cell trials in U.S.

2008

2009

2010

Clinical Research Training Fellow Named The ALS Association and the American Academy of Neurology (AAN) are pleased to announce that Hristelina Ilieva, M.D., Ph.D., of the Department of Neurology at the Johns Hopkins School of Medicine, Baltimore, Maryland, is this year’s recipient for the Clinical Research Training Fellowship, which is supported by The Association’s ALS Research Institute. The purpose of the award is to recruit talented and promising young clinicians who propose innovative clinical research and to foster their development to make significant contributions to ALS clinical research.

ALS is a progressive neurodegenerative disease that affects neurons (nerve cells) in the brain and the spinal cord. Eventually, people with ALS lose the ability to initiate and control muscle movement, which often leads to total paralysis and death within two-to-five years of diagnosis.

There is no cure and no life-prolonging treatments for the disease.

Dr. Ilieva’s research will examine a group of proteins in the cerebrospinal fluid (CSF) in people with ALS due to mutation in the C9orf72 gene, the most common genetic cause of ALS. The proteins to be studied include the so-called RAN peptides, which are unique products of the gene mutation, as well as other disease-related proteins that may be found in other forms of ALS as well. By characterizing the relative amounts, distribution, and life cycles of these proteins, Dr. Ilieva’s work will contribute to the development of accurate biomarkers for disease onset, severity, progression and response to therapy. Such biomarkers are critical for advancing clinical trials of ALS treatments.

Hristelina Ilieva, M.D., Ph.D., Department of Neurology at Johns Hopkins School of Medicine, Baltimore, Maryland

Dr. Ilieva will work under the mentorship of Jeffrey Rothstein, M.D., Ph.D., one of the leading researchers in the field of ALS biology. “Dr. Ilieva is simply an outstanding neurologist as well as a neuroscientist,” said Dr. Rothstein. Prior to beginning her work at Johns Hopkins, Dr. Ilieva performed research in the labs of two other prominent ALS researchers, Don Cleveland, Ph.D., at the University of California at San Diego, and Stanley Appel, M.D., at Methodist Hospital Neurological Institute in Houston, Texas.

“I am very grateful for the opportunity that the Clinical Research Training Fellowship brings,” Dr. Ilieva said. “With the help of the award, I hope to acquire new skills and knowledge that will allow me to become a stronger clinician scientist. At the end of the day, we are all indebted to the patients with ALS. Their courage in fighting this disease is a strong motivator for me and the people I work with to do our best.”

Ubiquilin-2 discovery; C9orf72 discovery

March: The ALS Association hosts 2nd Drug Discovery Workshop for ALS September: Researchers find genetic region influencing age at which people develop ALS

February: Identification of C9RAN translated peptide March: First human antisense trial published–– approach safe in people with ALS November: Progress in understanding effects of C9orf72 gene in ALS

March: Mutations in Matrin 3 identified linked to ALS September: Mutations in mitochondrial gene CHCHD10 linked to ALS October: Mutations in microtubule associated gene TUB4A linked to ALS

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2012

2013

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VOL.16

JOURNAL NEWS GENES Discovery of TBK1 Highlights Autophagy Pathway in ALS A gene that may cause more than 1% of all cases of ALS has been discovered through whole-exome sequencing of 2,874 people with ALS. The study, led

by David Goldstein, Ph.D., of Columbia University, drew on the combined efforts over two dozen laboratories in six countries, highlighting the global and collaborative nature of ALS research today.

Mutations in TBK1 were found in about 1.5% of ALS cases and only 0.2% of controls. The protein encoded by TBK1 interacts with two other proteins implicated in ALS, optineurin and p62, which also have roles in the autophagy pathway.

“This important discovery points strongly at the autophagy pathway, which has also been implicated from other studies,” said ALS Association Chief Scientist Lucie Bruijn, Ph.D., M.B.A. “That will help us focus our efforts on understanding that process as a possible target for therapy.”

survival in the SOD1G93A rat model of amyotrophic lateral sclerosis after suppression of mutant SOD1 in the motor cortex. J Neurosci. 2014 Nov 19;34(47):15587-600. http://www.ncbi.nlm.nih.gov/pubmed/25411487

Use of angiotensin-converting enzyme (ACE) inhibitors is associated with a reduced risk of ALS, according to an epidemiological study of more than 700 cases and more than 14,000 controls from the general medical populaMicroRNAs (miR) are a new class of gene regulatory RNAs with widespread tion in Taiwan. After controlling for use of multiple other drugs, ALS risk was and still largely unknown functions. Here, researchers showed that expres- reduced by 57% in those receiving more than 449 defined daily doses of ACE sion of miR-155 was upregulated in ALS mice and in the spinal cords of both inhibitors in four years. familial and sporadic ALS patients. In mice, increase of miR-155 was associ- Lin FC, Tsai CP, Kuang-Wu Lee J, Wu MT, Tzu-Chi Lee C. Angiotensin-converting enzyme inated with dysregulation of microglia homeostasis and loss of multiple sur- hibitors and amyotrophic lateral sclerosis risk: a total population-based case-control study. vival factors. Genetic ablation of miR-155 mitigated microglia dysfunction JAMA Neurol. 2015 Jan 1;72(1):40-8. http://www.ncbi.nlm.nih.gov/pubmed/25383557 and increased survival of ALS mice, with a stronger effect in females than males, for unknown reasons. “These findings identify miR-155 as a thera- Therapeutics peutic target for the treatment of ALS,” the authors concluded.

Improving CNS Penetration of Riluzole

Davoli A, Greco V, Spalloni A, Guatteo E, Neri C, Ricciardo Rizzo G, Cordella A, Romigi A, Cortese C, Bernardini S, Sarchielli P, Cardaioli G, Calabresi P, Mercuri NB, Urbani A, Longone P. Evidence of hydrogen sulphide involvement in amyotrophic lateral sclerosis. Ann Neurol. 2015 Jan 27. http://www.ncbi.nlm.nih.gov/pubmed/25627240

More than one-quarter of people with sporadic ALS, and almost two-thirds of those with familial ALS, harbor rare mutations in more than one known ALS gene, according to this study. Those with these mutations had disease onset 10 years earlier than those with only one or no identified mutation, GDNF-producing Astrocytes Slow Decline in ALS Model supporting the concept of oligogenic mutation as a contributor to ALS. Astrocytes in rats exhibit an age-dependent senescence that impairs their Cady J, Allred P, Bali T, Pestronk A, Goate A, Miller TM, Mitra RD, Ravits J, Harms MB, Baloh ability to support neurons, according to this study. The rate of decline in supRH. Amyotrophic lateral sclerosis onset is influenced by the burden of rare variants port is accelerated in the SOD1 rat model and is mitigated by administration in known amyotrophic lateral sclerosis genes. Ann Neurol. 2015 Jan;77(1):100-13. of glial cell-derived neurotrophic factor (GDNF). “Replacing aging astrocytes http://www.ncbi.nlm.nih.gov/pubmed/25382069 with young ones producing GDNF may therefore have a significant survival promoting effect on aging motor neurons and those lost through diseases DISEASE MECHANISMS such as ALS,” the authors suggest.

Therapy for Upper Motor Neurons Aids Lower Motor Neurons Das MM, Svendsen CN. Astrocytes show reduced support of motor neurons with agALS affects both upper and lower motor neurons. New research shows that reducing the level of the disease-causing mutant SOD1 protein in upper motor neurons alone in ALS rats delayed disease onset, prolonged survival and enhanced the survival of lower motor neurons, even though the lower motor neurons continued to have harmful levels of the protein. The results suggest that the survival of lower motor neurons depends in part on upper motor neurons, and that therapies targeting upper motor neurons may be especially helpful.

Thomsen GM, Gowing G, Latter J, Chen M, Vit JP, Staggenborg K, Avalos P, Alkaslasi M, Ferraiuolo L, Likhite S, Kaspar BK, Svendsen CN. Delayed disease onset and extended National Office

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ACE Inhibitors May Reduce ALS Risk

Role for MicroRNA in ALS Pathogenesis

Butovsky O, Jedrychowski MP, Cialic R, Krasemann S, Murugaiyan G, Fanek Z, Greco DJ, Wu PM, Doykan CE, Kiner O, Lawson RJ, Frosch MP, Pochet N, Fatimy RE, Krichevsky AM, Gygi SP, Lassmann H, Berry J, Cudkowicz ME, Weiner HL. Targeting miR-155 restores abnormal microglia and attenuates disease in SOD1 mice. Ann Neurol. 2015 Jan;77(1):75-99.

Cirulli ET, Lasseigne BN, Petrovski S, Sapp PC, Dion PA, Leblond CS, Couthouis J, Lu YF, Wang http://www.ncbi.nlm.nih.gov/pubmed/25381879 Q, Krueger BJ, Ren Z, Keebler J, Han Y, Levy SE, Boone BE, Wimbish JR, Waite LL, Jones AL, Carulli JP, Day-Williams AG, Staropoli JF, Xin WW, Chesi A, Raphael AR, McKenna-Yasek D, Non-neuronal Cells Cady J, Vianney de Jong JM, Kenna KP, Smith BN, Topp S, Miller J, Gkazi A; FALS Sequencing Consortium, Al-Chalabi A, van den Berg LH, Veldink J, Silani V, Ticozzi N, Shaw CE, Baloh Glia-produced H2S Promotes Inflammation, is Elevated in ALS CSF RH, Appel S, Simpson E, Lagier-Tourenne C, Pulst SM, Gibson S, Trojanowski JQ, Elman L, McCluskey L, Grossman M, Shneider NA, Chung WK, Ravits JM, Glass JD, Sims KB, Van Deerlin Hydrogen sulfide, H2S, is released from astrocytes and microglia and harms VM, Maniatis T, Hayes SD, Ordureau A, Swarup S, Landers J, Baas F, Allen AS, Bedlack RS, motor neurons in culture, according to a new study, which also showed elHarper JW, Gitler AD, Rouleau GA, Brown R, Harms MB, Cooper GM, Harris T, Myers RM, evated levels of H2S in the CSF of ALS patients. “We propose that H2S, as a Goldstein DB. Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and glial-released inflammatory factor, contributes to the ALS-mediated motor pathways. Science. 2015 Feb 19. http://www.ncbi.nlm.nih.gov/pubmed/25700176 neuron death,” the authors concluded.

Multiple Rare Mutations Mean Early Age of ALS Onset

Acknowledgement: Richard Robinson, Science Writer

Epidemiology

ing that is accelerated in a rodent model of ALS. Neurobiol Aging. 2015 Feb;36(2):1130-9. http://www.ncbi.nlm.nih.gov/pubmed/25443290

Stem Cell-Derived Astrocytes from ALS Patients Cause Motor Deficits in Model Neural progenitor cells, derived from either human embryonic stem cells or induced pluripotent stem cells (iPS cells) implanted into the spinal cords of adult mice developed into astrocytes, migrated away from the site of implantation and integrated into the nervous system. Neural progenitors derived from people with ALS also migrated and integrated, but caused motor deficits similar to those seen in genetically created ALS mouse models. This transplant model may provide important opportunities to study the role of astrocytes in disease onset and progression.

Acquired pharmacoresistance may help explain the limited therapeutic value of riluzole, according to a new study. The drug efflux transporters P-glycoprotein and breast cancer-resistant protein have been previously shown to be selectively upregulated at the blood-brain and spinal cord barrier in ALS mice and patients. Here, the same researchers showed that blocking these transporters increased CNS penetration of riluzole and slowed disease progression in ALS mice, even when given at symptom onset, when the drug is normally ineffective in the model.

Jablonski MR, Markandaiah SS, Jacob D, Meng NJ, Li K, Gennaro V, Lepore AC, Trotti D, Pasinelli P. Inhibiting drug efflux transporters improves efficacy of ALS therapeutics. Ann Clin Transl Neurol. 2014 Dec;1(12):996-1005. http://www.ncbi.nlm.nih.gov/pubmed/25574474 (free online)

Stem Cell Transplant Update In an update on the first trial of human stem cell transplants in ALS, researchers reported results of six autopsies. Donor DNA was identified in all cases, comprising up to 5% of total tissue DNA. Transplanted cells survived up to 2.5 years post-transplant. Both neuronal and astrocytic markers were detected among the transplanted cells, indicating some degree of post-transplant differentiation. The authors note that the role of continued immunosuppression in the success of transplantation is not clear, in that there was no correlation between graft survival and duration of immunosuppressive treatment.

Tadesse T, Gearing M, Senitzer D, Saxe D, Brat DJ, Bray R, Gebel H, Hill C, Boulis N, Riley J, Feldman E, Johe K, Hazel T, Polak M, Bordeau J, Federici T, Glass JD. Analysis of graft survival in a trial of stem cell transplant in ALS. Ann Clin Transl Neurol. 2014 Nov;1(11):900-8. http://www.ncbi.nlm.nih.gov/pubmed/25540804 (free online).

Case Study: Ten-year ALS Treatment Costs Exceed $1.4 Million In work supported by The ALS Association, researchers have shown that the 10-year cost for treatment of a person with ALS was more than $1.4 million dollars. The data collected in the study represented all disease-related costs incurred by one family whose 18-year-old son was diagnosed with ALS in 2002. Over 10 years, total costs were $1,433,992. The highest costs were for in-home caregivers ($669,150), tracheostomy ventilation ($212,430) and hospital care ($114,558). Insurance reimbursed 85% of the costs, while the family paid 9%, and 6% was paid by non-profit charities. “While these data reflect costs only for a single individual, they indicate major cost drivers in ALS,” according to lead study author Mary Lyon, RN, MN, Vice President for Patient Services for The ALS Association.

Chen H, Qian K, Chen W, Hu B, Blackbourn LW 4th, Du Z, Ma L, Liu H, Knobel KM, Ayala M, Obermann M, Lyon M. Financial cost of amyotrophic lateral sclerosis: A case study. AmyoZhang SC. Human-derived neural progenitors functionally replace astrocytes in adult mice. J troph Lateral Scler Frontotemporal Degener. 2014 Sep 23:1-4. http://www.ncbi.nlm.nih.gov/pubmed/25245119 Clin Invest. 2015 Feb 2. http://www.ncbi.nlm.nih.gov/pubmed/25642771