Spinal Muscular Atrophy: Disease Mechanisms and Therapy

Spinal Muscular Atrophy

Disease Mechanisms and Therapy

Editors

Charlotte J. Sumner

Johns Hopkins University School of Medicine, Baltimore, MD, United States

Sergey Paushkin

Spinal Muscular Atrophy (SMA) Foundation, New York, NY, United States

Chien-Ping Ko

University of Southern California, Los Angeles, CA, United States

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Preface

Perspectives

Sixty Years of Spinal Muscular Atrophy: A Personal Odyssey

Advances in Spinal Muscular Atrophy Research

From Mapping Survival Motor Neuron to Treatment of Spinal Muscular Atrophy

The Path to Therapeutics Development for Spinal Muscular Atrophy

Section I. Clinical Features and Diagnosis of SMA

Chapter 1. Spinal Muscular Atrophy: 125 Years Later and on the Verge of a Cure

Introduction

The First 100 Years: Understanding the Spinal Muscular Atrophy Clinical Spectrum

The Mapping of the SMA Gene and Creation of Animal Models

Case Example: Spinal Muscular Atrophy, Type III

Spinal Muscular Atrophy Clinical Trials and the Development of Outcome Measures

Conclusion and Future Directions

Chapter 2. Developmental Aspects and Pathological Findings in Spinal Muscular Atrophy

Introduction

Development of the Neuromuscular System

Neuropathological Aspects of Spinal Muscular Atrophy

Ultrasound Assessments of Fetal Spinal Muscular Atrophy Patients

Conclusions

Chapter 3. Standard of Care for Spinal Muscular Atrophy

Introduction

Respiratory Management

Nutritional Support

Endocrine Concerns and Intermediary Metabolism

Orthopedics

Acute Illness, Anesthesia, and Postoperative Care

Obstetrics

Physical Therapy and Assistive Devices

Conclusion

Chapter 4. Strategy for the Molecular Testing of Spinal Muscular Atrophy

Diagnostic Testing

Newborn Screening

Carrier Testing

SMN2 Testing

Conclusion

Section II. Cellular and Molecular Mechanisms of the Disease

Chapter 5. Transcriptional and Splicing Regulation of Spinal Muscular Atrophy Genes

Introduction

Organization of Survival Motor Neuron Gene

Role of Exonic Regulatory Elements in Survival Motor Neuron Exon 7 Splicing

In Vivo Selection of Exon 7 Revealed Additional Regulatory Elements

Role of Negative Intronic Regulatory Elements

Role of Positive Intronic Regulatory Elements

Role of RNA Structure and Long-Distance Interactions in Survival Motor Neuron Exon 7 Splicing

Role of Other Splicing Factors

Transcription and Transcription-Coupled Splicing Regulation

Concluding Remarks

Chapter 6. The Function of Survival Motor Neuron Complex and Its Role in Spinal Muscular Atrophy Pathogenesis

Early Studies on the Biochemistry of Survival Motor Neurons

Biogenesis of Spliceosomal Small Nuclear Ribonucleoproteins

The Survival Motor Neuron Complex As a Chaperone for Small Nuclear Ribonucleoproteins Assembly

Survival Motor Neuron

Gemin2

Gemins 3 and 4

Gemin5

Gemins 6 and 7

Gemin8

Unrip

The Methylosome/PRMT5 Complex

Survival Motor Neuron Complex Subunits and Intermediates in a Stepwise Sm Core Assembly Pathway

Regulation of Survival Motor Neuron Complex Activity

Survival Motor Neuron As a Redox Sensitive Machine

Why Does Survival Motor Neuron Loss of Function Cause Spinal Muscular Atrophy?

Potential Role for the Survival Motor Neuron Complex in Other Neurodegenerative Diseases

Chapter 7. RNA-Processing Dysfunction in Spinal Muscular Atrophy

The SMN Complex: An Assembly Machine for Ribonucleoprotein Complexes

Ribonucleoprotein Assembly Defects in Spinal Muscular Atrophy

Disease-Relevant SMN Functions: Lessons From Spinal Muscular Atrophy–Linked Mutations of SMN

SMN–Mediated Small Nuclear Ribonucleoprotein Assembly and Its Requirement in Time and Space

RNA-Processing Defects in Spinal Muscular Atrophy

SMN–Dependent RNA-Processing Events Linked to Spinal Muscular Atrophy

Outstanding Questions and Perspectives for Future Investigation

Chapter 8. Axonal and Neuromuscular Junction Pathology in Spinal Muscular Atrophy

Structure, Development, and Function of the Mammalian Neuromuscular Junction

Structural and Functional Neuromuscular Junction Pathology in Animal Models of Spinal Muscular Atrophy

Delayed Maturation of the Neuromuscular Junction in Animal Models of Spinal Muscular Atrophy

Cellular Components of the Neuromuscular Junction and Their Distinct Contribution to Neuromuscular Pathology in Spinal Muscular Atrophy

Evidence for Neuromuscular Junction Pathology in Human Spinal Muscular Atrophy Patients

Utilizing Neuromuscular Junction Pathology as a Readout in Preclinical Studies

Dysregulation of Developmental Pathways and Their Contribution to Neuromuscular Pathology in Spinal Muscular Atrophy

Selective Vulnerability of Different Motor Neuron Pools and Neuromuscular Junction Populations in Spinal Muscular Atrophy

Potential Cellular and Molecular Mechanisms Underlying Neuromuscular Junction Pathology in Spinal Muscular Atrophy

Future Perspectives and Conclusion

Chapter 9. Motor Circuit Dysfunction in Spinal Muscular Atrophy

Introduction

Plasticity of the Central Nervous System

Spinal Muscular Atrophy

The Role of Proprioception in Spinal Muscular Atrophy

Insights From Studies Using Animal Models

Glutamate Mishandling in Spinal Muscular Atrophy

Summary

Chapter 10. Contributions of Different Cell Types to Spinal Muscular Atrophy Pathogenesis

Introduction

Role of Astrocytes in Central Nervous System Disorders

Transgenic Models Provide Insight Into Tissue-Specific Function

Spinal Muscular Atrophy Astrocyte Pathology

Muscle Contribution to Spinal Muscular Atrophy Pathogenesis

Muscles Versus Motor Neurons: Delineating the Interactions of the Motor Unit and the Contribution of Muscle to Spinal Muscular Atrophy Pathogenesis

Myogenesis and Muscle Stem Cell Defects in Spinal Muscular Atrophy

Anabolism and Catabolism in Spinal Muscular Atrophy

Cytoskeletal Dynamic and Force Production in Muscles From Spinal Muscular Atrophy Mice

Cardiac Defects in Spinal Muscular Atrophy

Could Altered Metabolism Be Involved in Spinal Muscular Atrophy Pathogenesis?

Liver Defects in Spinal Muscular Atrophy

Pancreatic Defects in Spinal Muscular Atrophy

Perspective and Therapeutic Implications

Chapter 11. Temporal Requirements for the Survival Motor Neuron Protein

Introduction

Temporal Requirements for the Survival Motor Neuron Protein: Lessons From Model Organisms

Conclusions and Future Directions

Chapter 12. Spinal Muscular Atrophy Disease Modifiers

Survival Motor Neuron–Dependent Modifiers

Survival Motor Neuron‒Independent Modifiers

Conclusion

Section III. Cell and Animal SMA Models

Chapter 13. Cell Culture Models of Spinal Muscular Atrophy

Introduction

Probing Survival Motor Neuron Biology in Non–Motor Neuron Cultures

Two Sources of Pluripotent Stem Cells for Disease Modeling—Embryonic Stem Cells and Induced Pluripotent Stem Cells

Disease Modeling Using Patient Induced Pluripotent Stem Cells–Derived Motor Neurons

How to Better Model Spinal Muscular Atrophy—Looking at Non–Motor Neuron Aspects of the Disease

Concluding Remarks

Chapter 14. Nonmammalian Animal Models of Spinal Muscular Atrophy

A Brief History of Drosophila, Caenorhabditis elegans, and Zebrafish Model Systems

Survival of Motor Neuron Gene Conservation

Modeling Spinal Muscular Atrophy in the Fly

Modeling Spinal Muscular Atrophy in Caenorhabditis elegans

Modeling Spinal Muscular Atrophy in Zebrafish

Impact on the Spinal Muscular Atrophy Field

Chapter 15. Mammalian Models of Spinal Muscular Atrophy

Introduction

Mouse Models of Spinal Muscular Atrophy

Severe Spinal Muscular Atrophy Mouse Models

The Use of Severe Spinal Muscular Atrophy Mouse Models to Determine Where High Survival Motor Neuron Expression Is Required

The Use of Severe Spinal Muscular Atrophy Mouse Models to Determine When High Survival Motor Neuron Expression Is Required

Mild Spinal Muscular Atrophy Mouse Models

How Does Mouse Pathology Relate to Spinal Muscular Atrophy Patients?

Electrophysiological Measures in Mouse Models

Genetic Missense Mutations of Survival Motor Neuron

Genetic Background Check

Mouse Models Use in Therapeutic Testing and Large Mammalian Models of Spinal Muscular Atrophy

Conclusions

Section IV. Therapeutic Development

Chapter 16. Spinal Muscular Atrophy Therapeutics Development

Upregulation of Survival Motor Neuron Protein

Neuroprotection

Stem Cell Therapy

Enhancement of Muscle

Targeting Modifiers

Lessons Learned

Conclusions

Chapter 17. Small Molecule Approaches to Upregulate SMN Expression From the SMN2 Locus

Introduction

Discussion

Chapter 18. Antisense-Oligonucleotide Modulation of SMN2 Pre-mRNA Splicing

Introduction

Antisense Technology

Early Strategies in the Design of Antisense Oligonucleotides to Promote SMN2 Exon 7 Inclusion

Simultaneous Identification of Splicing Silencers and Lead Antisense Oligonucleotides by Antisense Oligonucleotide Walks

Mechanism of Action of ASO10-27

Preclinical Studies of ASO10-27

Ongoing Clinical Trials of Nusinersen

Concluding Remarks and Future Perspectives

Chapter 19. Gene Transfer in Spinal Muscular Atrophy

Rationale for Gene Therapy in Spinal Muscular Atrophy

SMNΔ7 Mouse Model

In Vivo Gene Therapy and Spinal Muscular Atrophy

Beyond Retrograde Transport

The Advent of Blood–Brain Barrier Penetrating Viral Vectors

Systemic Gene Delivery for Spinal Muscular Atrophy

Gene Delivery to Cerebrospinal Fluid for Spinal Muscular Atrophy

Gene Therapy Trial in Spinal Muscular Atrophy Type 1

Concluding Remarks and Perspectives

Chapter 20. Neuroprotection As a Therapeutic Approach for Spinal Muscular Atrophy

Introduction

What Is Neuroprotection?

How Do Survival Motor Neuron Deficits Cause Neurodegeneration?

Neuroprotection: Intervening in a Constant Battle Between Life and Death Targeting Mitochondria

Therapeutic Approaches to Providing Neuroprotection

Discussion

Chapter 21. Skeletal Muscle in Spinal Muscular Atrophy As an Opportunity for Therapeutic Intervention

Skeletal Muscle Physiology

Mechanisms Regulating Skeletal Muscle Mass

Muscle Pathology in Spinal Muscular Atrophy

The Role of Survival Motor Neuron Protein in Skeletal Muscle: Implications for Therapeutic Development for Spinal Muscular Atrophy

Spinal Muscular Atrophy Muscle As an Opportunity for Therapeutic Intervention

Conclusion

Chapter 22. Addressing Cell Therapy for Spinal Muscular Atrophy: Open Issues and Future Perspectives

Introduction

Why Stem Cell Therapy for Spinal Muscular Atrophy?

Motor Neuron Precursor Transplantation

Neural Stem Cell Transplantation

Other Strategies: Targeting Nonneuronal Cell Populations and In Vivo Reprogramming

Open Issues

Conclusions

Section V. Clinical Research

Chapter 23. Spinal Muscular Atrophy Motor Functional Scales and Measures of Pulmonary Function

Introduction

Part 1: Functional Motor Scales As Clinical Tools

Part 2: Pulmonary Function

Part 3: Selecting Outcome Measures for Natural History Studies and Clinical Trials

Chapter 24. Development and Testing of Biomarkers in Spinal Muscular Atrophy

Introduction

Overview of Types of Biomarkers

Biomarkers in Spinal Muscular Atrophy

Survival Motor Neuron–Related Biomarkers

Non-Survival Motor Neuron–Related Biomarkers

Physiological and Imaging Biomarkers

Conclusions

Chapter 25. Natural History of Spinal Muscular Atrophy

Introduction

Natural History Studies in Spinal Muscular Atrophy Type I Patients

Natural History Studies in Spinal Muscular Atrophy Type II/III Patients

Trends and Insights

Conclusions

Chapter 26. Spinal Muscular Atrophy Clinical Trials: Lessons Learned

Introduction

Inclusion Criteria

Enrollment

Impact of Clinical Networks and Databases

Retention

Stratification

Outcome Measures and Personnel Support/Cost

Conclusion

Appendix 1. SMA Types, Summary

Appendix 2. SMN1 and SMN2 Copy Numbers of Commercially Available Spinal Muscular Atrophy Fibroblast and Lymphoblastoid Cell Lines

Appendix 3. Transacting Factors and cis-Elements Involved in Modulation of SMN2 Exon 7 Alternative Splicing

Appendix 4. Sequence Alignment of the SMN Proteins From Diverse Organisms and List of SMN1 Mutations Identified in Humans

Appendix 5. SMN Role in the snRNP Assembly

Appendix 6. Select SMN-Dependent and SMN-Independent Modifiers

Appendix 7. Mouse Models of SMA and Mice Used in SMA Research

Appendix 8. SMA Strains for Testing Site-Specific Smn Expression

Appendix 9. Functional Scales Used in SMA

Appendix 10. Select SMA Organizations Around the World

Index

Copyright

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Cover design by Arya C. Singh

Front cover, top image: Proprioceptive sensory fibers making synaptic contacts with somata and dendrites of motor neurons in the spinal cord of a wild-type mouse. Proprioceptive fibers were labeled with Fluorescein dextran (in green) and the motor neurons were labeled with Texas Red dextran (in red) by retrograde fill from the dorsal root and ventral root, respectively.

Front and back cover, bottom image: Spinal motor neurons in the 1st, 2nd, and 3rd lumbar segments (labeled respectively in blue with Cascade Blue dextran, in red with Texas Red dextran, and in green with Alexa-488 dextran) in the rostro-caudal aspect visualized by retrograde fill from each perspective ventral root.

Images courtesy of Dr. George Mentis, Motor Neuron Center, Columbia University, New York, NY.

Back cover photo: Emily Lozina, type 1, 2/11/11 – 12/18/15, courtesy of Jennifer Lozina.

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List of Contributors

E.J. Androphy,     Indiana University School of Medicine, Indianapolis, IN, United States

W.D. Arnold,     The Ohio State University, Columbus, OH, United States

C.E. Beattie,     Ohio State University, Columbus, OH, United States

T. Bordet,     Biotherapies Institute for Rare Diseases, Evry, France

P.J. Boyd,     University of Edinburgh, Edinburgh, United Kingdom

A.H.M. Burghes,     The Ohio State University, Columbus, OH, United States

M.E.R. Butchbach,     Nemours Alfred I. duPont Hospital for Children, Wilmington, DE, United States

A.N. Calder,     Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, United States

K.S. Chen,     Spinal Muscular Atrophy (SMA) Foundation, New York, NY, United States

J.J. Cherry,     RaNA Therapeutics, Cambridge, MA, United States

S.P. Corti,     IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy

T.O. Crawford,     Johns Hopkins University School of Medicine, Baltimore, MD, United States

B.T. Darras,     Boston Children’s Hospital, Boston, MA, United States

D.C. De Vivo,     Columbia University Medical Center, New York, NY, United States

M.O. Deguise

Ottawa Hospital Research Institute, Ottawa, ON, Canada

University of Ottawa, Ottawa, ON, Canada

C.J. DiDonato

Feinberg School of Medicine, Chicago, IL, United States

Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, United States

G. Dreyfuss,     HHMI University of Pennsylvania School of Medicine, Philadelphia, PA, United States

V. Dubowitz,     UCL Institute of Child Health, London, United Kingdom

A.D. Ebert,     Medical College of Wisconsin, Milwaukee, WI, United States

I. Faravelli,     IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy

R.S. Finkel,     Nemours Children’s Hospital, Orlando, FL, United States

K.H. Fischbeck,     National Institute of Neurological Disorders and Stroke (NINDS), NIH, Bethesda, MD, United States

E.V. Fletcher,     Columbia University Medical Center, New York, NY, United States

K.D. Foust,     The Ohio State University, Columbus, OH, United States

E.L. Garcia,     University of North Carolina, Chapel Hill, NC, United States

T.H. Gillingwater,     University of Edinburgh, Edinburgh, United Kingdom

D. Hammers,     University of Florida, Gainesville, FL, United States

L.T. Hao,     Ohio State University, Columbus, OH, United States

A.C. Hart,     Brown University, Providence, RI, United States

K.J. Hodgetts,     Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, United States

M.D. Howell,     Iowa State University, Ames, IA, United States

Y. Hua

Shantou University Medical College, Shantou, Guangdong, China

Soochow University, Suzhou, Jiangsu, China

S.T. Iannaccone,     University of Texas Southwestern Medical Center, Dallas, TX, United States

J. Jarecki,     Cure SMA, Elk Grove Village, IL, United States

B.K. Kaspar,     The Ohio State University, Columbus, OH, United States

S.J. Kolb,     The Ohio State University, Columbus, OH, United States

L. Kong,     Johns Hopkins University School of Medicine, Baltimore, MD, United States

R. Kothary

Ottawa Hospital Research Institute, Ottawa, ON, Canada

University of Ottawa, Ottawa, ON, Canada

A.R. Krainer,     Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, United States

V. Le Verche,     Spinal Muscular Atrophy (SMA) Foundation, New York, NY, United States

C.L. Lorson,     University of Missouri, Columbia, MO, United States

C.M. Lutz,     The Jackson Laboratory, Bar Harbor, ME, United States

A. MacKenzie,     CHEO Research Institute, Ottawa, ON, Canada

A.G. Matera,     University of North Carolina, Chapel Hill, NC, United States

E.S. Mazzone,     Università Cattolica del Sacro Cuore, Largo Gemelli, Rome, Italy

V.L. McGovern,     The Ohio State University, Columbus, OH, United States

J. Melki,     University of Paris Saclay, Le Kremlin-Bicêtre, Paris, France

N. Mendoza-Ferreira,     University of Cologne, Cologne, Germany

G.Z. Mentis,     Columbia University Medical Center, New York, NY, United States

E. Mercuri,     Università Cattolica del Sacro Cuore, Largo Gemelli, Rome, Italy

S. Messina,     University of Messina, Messina, Italy

U.R. Monani,     Columbia University Medical Center, New York, NY, United States

J. Montes,     Columbia University, New York, NY, United States

L.L. Nelson,     University of Texas Southwestern Medical Center, Dallas, TX, United States

S.Y. Ng,     Harvard University, Cambridge, MA, United States

P. O’Hern,     Brown University, Providence, RI, United States

M.A. Osborne,     The Jackson Laboratory, Bar Harbor, ME, United States

M. Oskoui,     McGill University Health Center, Montreal, QC, Canada

T.N. Patitucci,     Medical College of Wisconsin, Milwaukee, WI, United States

S. Paushkin,     Spinal Muscular Atrophy (SMA) Foundation, New York, NY, United States

L. Pellizzoni,     Columbia University Medical Center, New York, NY, United States

M.J. Pérez-García,     Vall d’Hebrón Research Institute, Barcelona, Spain

T.W. Prior,     The Ohio State University, Columbus, OH, United States

R.M. Pruss,     Windhover Biomed Ltd., London, United Kingdom

L.L. Rubin,     Harvard University, Cambridge, MA, United States

S.B. Rutkove,     Beth Israel Deaconess Medical Center, Boston, MA, United States

M.K. Schroth,     University of Wisconsin School of Medicine and Public Health, Madison, WI, United States

L.R. Simard,     University of Manitoba, Winnipeg, MB, Canada

N.N. Singh,     Iowa State University, Ames, IA, United States

R.N. Singh,     Iowa State University, Ames, IA, United States

B.R. So,     HHMI University of Pennsylvania School of Medicine, Philadelphia, PA, United States

C.J. Sumner,     Johns Hopkins University School of Medicine, Baltimore, MD, United States

S.S. Sunshine,     Spinal Muscular Atrophy (SMA) Foundation, New York, NY, United States

H.L. Sweeney,     University of Florida, Gainesville, FL, United States

S. Tisdale,     Columbia University Medical Center, New York, NY, United States

E.F. Tizzano

Vall d’Hebrón Research Institute, Barcelona, Spain

Vall d’Hebrón Hospital, Barcelona, Spain

L. Torres-Benito,     University of Cologne, Cologne, Germany

B. Wirth,     University of Cologne, Cologne, Germany

Z. Zhang,     HHMI University of Pennsylvania School of Medicine, Philadelphia, PA, United States

Preface

Since mid-1980s, we have witnessed a boom in the identification of genes underlying neurological diseases, but there are only few diseases for which this knowledge has led to efficacious treatment for patients. Spinal muscular atrophy (SMA) may soon become one such disease. SMA is the leading inherited cause of infant mortality characterized by motor neuron loss and muscle atrophy. The identification of the causative gene survival motor neuron 1 (SMN1) by Dr. Judith Melki and colleagues in 1995 galvanized the SMA community and attracted many individuals from different disciplines to join the fight against SMA. As a result, the SMA community has witnessed significant advances in both basic and clinical research. At the time of the writing of this book, there are about 20 drugs in development including six in clinical trials. Remarkably, we now stand on the brink of the first FDA-approved treatment for this devastating neurodegenerative disease. Given the complexity of the disease, the diverse research approaches involved, the unique advances in therapeutic development, and the increasing body of literature on SMA, we felt that the time has come for the first comprehensive book on SMA. We envision that this book will become the single go to textbook for graduate and medical students, as well as an essential reference for academic and biotech/pharma researchers, clinical researchers and practitioners, and patients and their advocacy organizations. We also hope that this integrated, thorough review will enable further advancements in SMA basic research, therapeutics development, and patient care. Finally, this book may be valuable to researchers and clinicians who may want to apply SMA research strategies, therapeutic approaches, and lessons learned to advance treatment for other diseases.

The book is comprised of 26 chapters organized into five sections and represents an international effort by leading SMA researchers and clinicians, whose interests and expertise encompass a wide spectrum of topics and disciplines. Chapters were written to bridge multiple disciplines and to promote effective communication between basic scientists, clinical researchers, and health care providers on the latest development in SMA. Each chapter includes an introduction, historical background, and comprehensive review of the topic, as well as outstanding questions for future investigations and key references for additional detailed study.

Section I describes the clinical features, diagnosis, and standards of care for SMA patients. It also details the development and pathology of the motor unit in SMA patients.

Section II focuses on molecular mechanisms of SMA, including transcription and splicing regulation of the SMN genes and the functions of the SMN protein. These molecular mechanisms are critical for providing both insights into the biology of SMN and the foundation for rational design of potential therapeutics. This section also covers cellular mechanisms underlying the disease, an effective therapeutic window with respect to the temporal requirement of SMN, and disease modifiers.

Section III details the cellular and animal models, which are indispensable for understanding the disease mechanisms and developing therapeutics in SMA.

Section IV is dedicated to SMA therapeutics development and covers the various strategies employed to date. This section also highlights the remarkable collaborative effort involving academic researchers, industry teams, advocacy organizations, and government, as well as SMA patients.

Section V focuses on advances in clinical research, including natural history studies, motor function scales and outcome measures, and biomarkers. The section concludes with lessons learned in clinical trials.

Acknowledgments

We are very grateful to Drs. Victor Dubowitz, Judith Melki, Arthur H. M. Burghes, and Kenneth H. Fischbeck for sharing their unique insights and perspectives on SMA. We are also honored by the overwhelming support of the contributing authors. Their willingness to share their knowledge is an illustration of the generous and collaborative spirit of the SMA community. The advances of SMA research and therapeutics development would not be possible without the participation of SMA patients and their loving families, as well as the generous support by government-funding agencies and many SMA advocacy organizations. We would like to thank Dr. George Mentis for providing the front cover picture and Jennifer Lozina for sharing a photograph of her beautiful daughter, Emily, as well as Arya Singh for her artistic design of the book cover. We would also like to thank Sara Sunshine for help with the book. Last, but not least, we much appreciate Dr. Natalie Farra, Senior Acquisitions Editor, for providing the opportunity to pursue this endeavor, as well as Kristi Anderson, Senior Editorial Project Manager, and the Elsevier/Academic Press staff, for their organizational skills that ensured the smooth and timely completion of this book.

Charlotte J. Sumner, MD

Sergey Paushkin, MD, PhD

Chien-Ping Ko, PhD

Perspectives

Sixty Years of Spinal Muscular Atrophy: A Personal Odyssey

V. Dubowitz,     UCL Institute of Child Health, London, United Kingdom

We live in exciting times, and it has been a tremendous privilege to witness the remarkable growth in interest in the neuromuscular disorders over the past half-century and the quantum leap since mid-1990s in resolving the complex genetic background of spinal muscular atrophy (SMA),now on the cusp of potential therapy.

This comprehensive, state-of-the-art textbook, covering all aspects of SMA, is very timely and should provide a springboard for further efforts and advances in the future.

I shall give a broad overview of the progress of events since mid-1950s, with an inevitable personal bias of my own journey, ranging from the recognition of the various clinical forms of the disease, through the early history, amazing clinical acumen, and descriptive prowess of our forebears, to the genetic revelations in the past few decades and the potential for gene manipulation in providing a potential therapy for the disease in the future.

My first exposure to neuromuscular disorders was in a 3-week temporary locum post in 1957 at Queen Mary’s Hospital for Children, south of London, where they had 2 wards of 16 beds each for long-term care of muscular dystrophy and associated disorders. I came for 3  weeks and stayed for 3  years and became committed to a career in pediatrics and a lifetime focus on muscle disorders.

At that time there was a well-defined severe infantile SMA, which had already been described at the turn of the century and had the eponymous title of Werdnig–Hoffmann disease.¹,² Onset was at birth or in the first weeks of life and death usually in the first year.

Half a century later, a late-onset, adolescent or early-adult, ambulant form of SMA was documented in the 1950s and given the eponymous title of Kugelberg–Welander disease.³

From early on I noted several cases with a late infantile onset after the child had achieved the ability to sit. They then seemed to develop severe weakness of the legs and never achieved the ability to stand independently. I documented 12 such cases in 1964,⁴ and it was of particular interest that several of them had had an affected younger sibling who had died in the first year of life with a classical severe infantile SMA, confirmed in some at autopsy.

In addition, the first case I diagnosed of this intermediate severity had a younger sister who had a similar onset with reluctance to stand or walk, but her parents encouraged her, and she achieved the ability to stand and to walk and then remained ambulant and fairly stable until her first pregnancy at 21  years of age, after which she lost the ability to walk independently. Of special interest as well was how static many of these intermediate cases were, with no obvious deterioration. I personally followed this initial case for 33  years and, apart from some increase in scoliosis and a need for intermittent supportive oxygen therapy, he had shown very little change over the years.⁵(p334)

At a guest lecture at the Children’s Hospital of Washington in 1967, I suggested that the severe infantile SMA and the mild late-onset ambulant form might be opposite ends of a continuous spectrum of severity, given the intermediate group with variable severity from the severe to the mild end and the fact that clinical variants of the disease could occur in the same sibship.⁶

Classification

The medical profession has an obsession with trying to pigeonhole diseases into compartments and draw up classifications, which have no advantage unless they clarify rather than confuse.

There are no specific types of SMA, and the introduction of types 1, 2, and 3 was suggested by a committee of clinicians and geneticists in 1990 to promote collaborative studies between different centers and to identify the genes for SMA.⁷ Their classification was based primarily on the age of onset and the age of death. I disagreed with this approach, as the age of onset was extremely variable in relation to individual severity and patients do not normally present at clinic with a death certificate. I tried to convince the committee that we should base the diagnosis entirely on the severity of the individual patient, so there would be a severe group, an intermediate group, and a mild group, and the broad distinctions could be the ability to sit unaided and the ability to stand and walk unaided. This was (somewhat reluctantly) added on to the types. This is a classic example of a committee trying to design a horse and ending up with a camel.

I also tried to get the committee to include in the diagnostic criteria for the severe form, the one cardinal feature, which is sparing of the diaphragm in association with the severe weakness of the intercostal muscles. This results in the classical abdominal breathing and the bell-shaped chest with very little expansion. SMA is, in fact, the only neuromuscular disease that spares the diaphragm and affects the intercostal muscles and can be confidently diagnosed on this one clinical sign. I failed to convince them.

Of additional clinical interest is that in contrast to the consistent involvement of the limbs, there is sparing of the facial muscles so that these infants have a normal expression and facial mobility and also have a normal intellect. The committee was prepared to include the presence of facial weakness as an exclusion criterion.

In order to vent my frustration, I then wrote a commentary on the chaos in the classification of SMA.⁸ I subsequently had several discussions with my neurology colleagues, and on one occasion I suddenly had a eureka moment, If you want to designate patients on a numerical basis, perhaps your 1, 2, 3 division is not sufficiently sensitive and you should perhaps go into a decimal classification to concede the range of change within each group. After giving it some further thought and introducing it at our clinic, it became apparent that it was a very practical way of documenting very accurately the grade of involvement of the patient. I then wrote a sequel to my chaos paper.⁹

I also recalled two remarkable children I had seen during the 1950s with horrendous scoliosis, were able to sit in a semi-recumbent position, and were borderline between the severe and the intermediate group. They survived 8 and 10  years, respectively, and had a remarkable sparing of the face as well as normal intellect (see illustrations⁵(p340)).

As with any other assessment of particular features in infants, one should first define the middle of the range and then it becomes easy to complete the rest. Therefore, if one looks at type 1.5 as the child who is floppy from early infancy and has mainly abdominal breathing with collapse of the chest but no spontaneous respiratory difficulties, apart from when there is a superadded chest infection, then type 1.1 would be an infant who was severely paralyzed at birth, had severe respiratory involvement, and might require ventilator support.

At the other end of the scale a 1.9 would be a child almost able to sit independently but not quite and needing some support, and having much better respiratory function.

In the intermediate group, type 2.5 would be middle of the range for the intermediate group, sitting with stability and good posture; whereas 2.1 would be a child barely able to maintain a sitting position and type 2.9 would be a child with ability to stand with support but not independently.

In the ambulant group, 3.5 would be a reasonably steady ambulation; whereas 3.1 would be just able to stand and walk independently and 3.9 would be on the verge of normal but perhaps unable to walk fast, to hop on one leg, or to run.

Type 4.0 would be normal and there is no real advantage in trying to subdivide further on the basis of age between an earlier-onset type 3 up to the age of 20  years and a later-onset type 4, beyond 20  years, and possibly dichotomizing siblings on either side of the artificial divide.

Since that time, we have consistently used this scoring system within our muscle unit and found it to be particularly sensitive to assessing the specific status of an individual patient. It should also be of great use when conducting clinical trials. Because of the relatively stable situation in SMA (of all severities), one would need to see an actual improvement in function to be confident of a response. This would be quite impossible to base on a type related to age of onset as this is extremely variable, and about a third of cases of the classical severe SMA (types 1.2–1.5) may have an antenatal onset and not require ventilation at birth.

The disease is relatively stable but some patients may show deterioration with time and lose ability to sit independently or to walk, but should still retain their original designation. In the severe group one also notes marked deterioration at times following an acute respiratory infection, leaving more severe and general weakness, including the face. Marked loss of function may also follow severe infections in the intermediate group.

Another interesting clinical feature that we frequently observed in the intermediate SMA children, was a very fine tremor of the hands. We did routine electrocardiograms on all children attending our neuromuscular clinic and frequently got a note from the cardiology department, apologizing for the artifact in the baseline of the ECG. This actually reflected the fine tremor in the muscles and was a potential marker for intermediate SMA.⁵(p335)

Another remarkable feature is the relatively acute and rapid onset of the severe cases and then a fairly steady state with no obvious loss of any residual function that they have. I had one personal experience of this. In Sheffield during the 1960s a woman was admitted in labor, who had had three previous infants affected by SMA and dying in first year of life. I saw the infant soon after birth and he seemed fully active with no apparent weakness and normal muscle tone and a normal response of the Moro and other neonatal reflexes. I reviewed him every day through the first week and there was no change, so discharged him. At routine follow-up at 6  weeks he was completely paralyzed, with the classical distribution of severe SMA. His mother related that this had come on fairly suddenly one night, and there was no concern when put to sleep but in the morning was completely paralyzed.

A more severe group with marked in utero weakness and needing ventilator support at birth and with either death in utero or in the early infancy, was designated as prenatal SMA.¹⁰ But this again is confusing, based on time of onset rather than severity, given that a proportion of classical type 1 cases may have a prenatal onset. I suggested that it might be more appropriate to designate this very severe group as SMA 0 and one could then define a 0.1 as death in utero and a 0.9 as born with severe respiratory difficulties but almost managing without ventilation. A 0.5 would have severe respiratory difficulty at birth requiring ventilator support and also more severe weakness, which can be more generalized and also associated with facial weakness, as well as more marked contractures of limbs.¹¹

A Ramble Through the History of Spinal Muscular Atrophy

In addition to my interest in the clinical spectrum and management of SMA, I also have a special interest in medical history and have spent many an interesting few hours in the bowels of the Royal Society of Medicine of London Library, poring into the superb descriptions by our forebears of these interesting neuromuscular diseases.

In a 2009 review of the history of SMA¹² I came to three main conclusions:

1. If someone’s name is attached to a disease, he or she is usually the second person to have described the disease.

2. If you discover something new, do not read the early literature. It has all been described before. Far safer to just stick to Pub-Med, which covers the past 40–50  years.

3. If you quote historical papers always read the originals.

Although the names of Werdnig and Hoffmann have been associated with the severe infantile form of SMA, they did not describe the severe form but the milder, intermediate form.

In 1891 Guido Werdnig of the Department of Pathological Anatomy in the University of Graz in Austria described two brothers with onset of weakness around 10  months.¹ One had complicating pertussis and hydrocephalus and died at 3  years. The other survived till 6  years. At autopsy he found degeneration of the anterior horn cells of the cord. Werdnig wrote a further review in 1894,¹³ but subsequently made very little further contribution to the medical literature.

In contrast, Johan Hoffmann, a protégé and later successor of Erb in the medical clinic in Heidelberg, was a prolific medical author on a wide range of subjects, including SMA.

In a series of three seminal papers on SMA in the Deutsches Archiv fur Nervenheilkunde in 1893, 1897, and 1900,²,¹⁴,¹⁵ he reviewed Werdnig’s two cases and added seven of his own from three further families. He also included excellent illustrations of the histology of the muscle and central nervous system and showed the degeneration of the anterior horn cells of the spinal cord. In his 1897 paper, Hoffmann included three sequential pictures of a girl with intermediate severity SMA, which he appropriately acknowledged to come from a paper by Thomson and Bruce in the first issue of the newly established Edinburgh Hospital Reports in 1893.¹⁶

Their case is a typical intermediate severity SMA, with ability to sit unaided and the sequential pictures show the progression of her scoliosis. When I revisited the original Thomson and Bruce publication, I was also interested to note in the first picture of the child that it clearly shows hyperextension of her fingers, which I thought I may have been the first to observe and highlight in my 1964 series of cases, together with joint laxity, which might account for the rapid progression of scoliosis.⁴

Therefore, although Werdnig was indeed the first to publish a case of SMA of intermediate severity, Thomson and Bruce had also followed their case in parallel with Werdnig from 1889 and wrote a more extended and better illustrated report, both clinically and pathologically.¹⁶

The first detailed description of the severe form of SMA was by Beevor in Brain in 1903.¹⁷ Beevor’s case was the fourth affected infant in a family of eight children and the three previous cases had all died by 6  months. He drew attention to the associated intercostal weakness and sparing of the diaphragm. The diagnosis was confirmed at autopsy with degeneration of the anterior horn cells of the cord.

The first clinical picture I have found of the severe form of SMA, showing the classical jug-handle posture of the arms with flexion and internal rotation, together with the wasting and retraction of the intercostals and activity of the diaphragm, was in a personal copy of the second edition of the pediatric textbook of Jonathan Hutchinson published in 1910.¹⁸ I was able to verify the inclusion of the same figure in the first edition of the book in 1904.

The mild ambulant form of SMA was comprehensively documented by Kugelberg and Welander³ in an American journal, Archives of Neurology and Psychiatry (Chicago), in a series of 12 cases presenting like a limb girdle muscular dystrophy but shown to be neurogenic on electromyography and muscle biopsy. An almost equivalent series from three families was published by another Swedish group, Wohlfart, Eliasson, and Fex a year earlier¹⁹ in a Scandinavian journal, Acta Psychiatrica et Neurologica (Kjobenhavn). They found associated neurogenic changes in the muscle and suggested this might be a mild variant of Werdnig–Hoffmann disease. Indeed, Kugelberg and Welander referred to the publication of Wohlfart’s group and both groups had also presented cases at several clinical meetings prior to the two definitive publications.

Another key publication in the SMA field was a monograph written by Sven Brandt in 1950²⁰ for his medical doctorate thesis and comprising 112 cases from 89 Danish families, with 97 being under one year. A large proportion were of the severe form but some also conformed to the intermediate. He also included a number of classical pictures of the severe form.

Finally, there is one more twist to the tale. In 1899 Sylvestre²¹ presented a single case at the pediatric society of Paris of a 2-month-old infant with flaccid paralysis of all four limbs and trunk since birth and weakness of the intercostals, but sparing of the diaphragm. This was the sixth child in the family and the third and fifth had been similarly affected and died at 3 and 5  months. The case report, without any illustrations, was published in the first issue of the newly established Bulletins de la Société de Pédiatrie de Paris. Had it not been for the sparing of the diaphragm one would not have been sure of this being an SMA rather than a congenital dystrophy or one of the severe congenital myopathies.

The New Dawn

The next important milestone in SMA was the location in 1990 of the gene for SMA initially by Gilliam’s group in New York in an Amish group of large families with the mild variant²² and soon after by Melki’s group in Paris.²³ It was shortly after that the same gene locus could be confirmed for the severe form both by Gilliam²⁴ (with whom we collaborated) and also by Melki.²⁵ In the same year a working group of clinicians and geneticists from both sides of the Atlantic was convened in New York to define the clinical criteria for diagnosis and exclusion of SMA and to coordinate the collaborative molecular genetic studies, which I referred to earlier.⁷

It took another 5  years until the gene was isolated and characterized by Melki’s group,²⁶ a novel gene which they named survival motor neuron (SMN) gene. It was a complex gene as this part of chromosome 5 is duplicated and a normal individual has two copies of the gene, an active SMN1 gene and an inactive SMN2. Severe cases have a deletion of exon 7 of the active gene and no change in SMN2. Milder cases also have deletion of exon 7 of SMN1 but have an increased copy number of SMN2 which provides some compensation for the deficit in SMN1.

One of the spin-offs of the discovery of the gene was that the geneticists were now able to diagnose absence of the SMN1 gene in cases previously unrecognized as SMA, as they had an atypical presentation with exclusion criteria such as generalized weakness, facial weakness, and an in utero onset with even more severe and generalized weakness, and respiratory failure than the classical severe SMA.

In the current era of therapeutic trials, we have to accept that SMA is a relatively static disease (at all levels of severity) and that one would have to demonstrate an improvement in individual cases, irrespective of their severity. From a prognostic point of view the only single sign of relevance in the severe cases is the intercostal weakness and compromise of respiratory reserve. Any beneficial therapy would have to show improvement in the shape and mobility of the chest in addition to the respiratory function.

And One Final Afterthought …

There has recently been a trend in talking of the change in natural history of a disease following the introduction of supportive and potentially therapeutic treatment. I have suggested that this is a desecration of the Queen’s English.²⁷ The natural history of a disease is the natural history, that is the course of a disease without intervention. If one is going to have a changing natural history, we shall soon be describing the natural history in each country depending on resources, in each part of a country and each center depending on facilities and expertise. What changes in relation to treatment is the prognosis for the disease or the course or outcome. Life is difficult enough getting consensus in the medical profession without introducing shifting goalposts.

References

1. Werdnig G. Zwei frühinfantile hereditäre Fälle von progressiver Muskelatrophie unter dem Bilde der Dystrophie, aber auf neurotischer Grundlage. Arch fur Psychiatr Nervenkrankh. 1891;22:437–481 Berlin.

2. Hoffmann J. Über chronische spinale Muskelatrophie im Kindesalter auf familiärer Basis. Deut Zeitsch Nervenheilkd. 1893;3:427–470.

3. Kugelberg E, Welander L. Heredofamilial juvenile muscular atrophy simulating muscular dystrophy. Arch Neurol Psychiatry (Chic). 1986;75:500–509.

4. Dubowitz V. Infantile muscular atrophy. A prospective study with particular reference to a slowly progressive variety. Brain. 1964;87:707–718.

5. Dubowitz V. Muscle Disorders in Childhood. 2nd ed. Saunders; 1995.

6. Dubowitz V. Infantile muscular atrophy – a broad spectrum. Clin Proc Child Hosp WA. 1967;23:223–239.

7. Munsat T.L. Workshop report: international SMA collaboration. Neuromuscul Disord. 1991;1:81.

8. Dubowitz V. Chaos in classification of the spinal muscular atrophies of childhood. Neuromuscul Disord. 1991;1:77–80.

9. Dubowitz V. Chaos in the classification of SMA: a possible resolution. Neuromuscul Disord. 1995;5:3–5.

10. Macleod M.J, Taylor J.E, Lunt P.W, Mathew C.G, Robb S.A. Prenatal onset spinal muscular atrophy. Eur J Paediatr Neurol. 1999;3:65–72.

11. Dubowitz V. Very severe spinal muscular atrophy (SMA type 0): an expanding clinical phenotype. Eur J Paediatr Neurol. 1999;3:49–51.

12. Ramblings in the history of spinal muscular atrophy. Neuromuscul Disord. 2009;19:69–73.

13. Werdnig G. Die frühinfantile progressive spinale Amyotrophie. Arch fur Psychiatr Nervenkrankh. 1894;26:707–744 Berlin.

14. Hoffmann J. Weiterer Beiträge zur Lehre von der hereditären progressiven spinalen Muskelatrophie im Kindesalter. Deut Zeitsch Nervenheilkd. 1897;10:292–320.

15. Hoffmann J. Dritter Eitrag zur Lehre von der hereditären progressiven spinalen Muskelatrophie im Kindesalter. Deut Zeitsch Nervenheilkd. 1900;18:217–224.

16. Thomson J, Bruce A. Progressive muscular atrophy in a child with a spinal lesion. Edinb Hosp Rep. 1893;1:372.

17. Beevor C.E. A case of congenital spinal muscular atrophy (family type) and a case of hemorrhage into the spinal cord at birth, giving similar symptoms. Brain. 1902;25:85–108.

18. Hutchinson R. Lectures on Diseases of Children. 2nd ed. London: Edward Arnold; 1910:276 Fig. 32 (plate).

19. Wohlfart G, Fex J, Eliasson S. Hereditary proximal spinal muscular atrophy – a clinical entity simulating progressive muscular dystrophy. Acta Psychiatr Neurol (Kjobenhavn). 1955;30:395–406.

20. Brandt S. Werdnig–Hoffmann’s infantile progressive muscular atrophy. Opera ex domo biologiae hereditariae humanae universitatis hafniensis. 22. Copenhagen: Ejnar Munksgaard; 1950.

21. Sylvestre M. Paralysie flasque de quatre membres et des muscles du tronc (sauf le diaphragme) chez un nouveau-né. Bull Soc Pediatr Paris. 1899;1:3–10.

22. Brzustowicz L.M, Lehner T, Castilla L.H, et al. Genetic mapping of chronic childhood-onset spinal muscular atrophy to chromosome 5q11.2-13.3. Nature. 1990;344:540–541.

23. Melki J, Abdelhak S, Sheth P, et al. Gene for chronic proximal spinal muscular atrophies maps to chromosome 5q. Nature. 1990;344:767–768.

24. Gilliam T.C, Brzustowicz L.M, Castilla L.H, et al. Genetic homogeneity between acute and chronic forms of spinal muscular atrophy. Nature. 1990;345:823–825.

25. Melki J, Sheth P, Abdelhak S, et al. Mapping of acute (type 1) spinal muscular atrophy to chromosome 5q12-q14. Lancet. 1990;336:271–273.

26. Lefebvre S, Burglen L, Reboullet S, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell. 1995;80(1):155–165.

27. Dubowitz V. Unnatural natural history of Duchenne muscular dystrophy. Neuromuscul Disord. 2015;25:936.

Advances in Spinal Muscular Atrophy Research

J. Melki,     University of Paris Saclay, Le Kremlin-Bicêtre, Paris, France

Spinal muscular atrophy (SMA) is a genetically heterogeneous group of inherited neuromuscular disorders characterized by defect of motor neurons of the spinal cord leading to progressive atrophy and weakness of skeletal muscles. When Professors Jean Frezal and Arnold Munnich proposed to me to run a project aiming at identifying the genetic basis of autosomal recessive SMA, with the support of the Association Française contre les Myopathies (AFM), I was very enthusiastic and hoped that this project should solve the following challenges facing the field at that time (1) at the clinical level, invasive procedures were necessary to support the diagnosis of SMA including electromyographic study and/or muscle biopsy; (2) carriers of the disease had a recurrent risk of 25% of having another child affected without possible prenatal or pre-implantation genetic diagnosis to help families; and (3) no therapeutic options. Our team identified the survival motor neuron 1 (SMN1) gene as responsible, when mutated, for SMA in 1995,¹ after an extensive search due to the complexity of the 5q13 region, which is characterized by an inverted duplication of a large element (about 500  kb) containing several genes. The telomeric copy contains the SMN1 gene and the centromeric copy contains a highly homologous gene called SMN2; both genes are transcribed. SMA is caused by deletion or conversion of SMN1 gene in 93% of cases. The only functionally relevant difference between the two genes identified to date is a silent C  →  T mutation in exon 7 of SMN2, which determines an alternative spliced isoform that predominantly excludes exon 7¹ through a disruption of an exon splicing enhancer and the creation of an exon splicing silencer element.²,³ The truncated transcript lacking exon 7 encodes a putative shorter and in vivo unstable protein.⁴ To prove that this gene was indeed the disease gene, the identification of intragenic SMN1 mutations was essential and was reported in our initial report¹ as well as thanks to a fruitful collaboration with Montserrat Baiget⁵ reporting the first frameshift and premature stop codon in SMN1 gene in unrelated patients. The identification of SMN1 gene greatly improved diagnostic testing avoiding invasive procedure and family-planning options of SMA patients and carriers. Then we provided the first evidence for a tight inverted correlation between the SMN2 copy number (and therefore the amount of the full-length protein encoded by this gene) and the clinical severity of human SMA disease thanks to the collaboration with Gideon Dreyfuss leading us to define SMN2 as an attractive target for therapy in SMA.⁶ The discovery that Gems are associated with Cajal bodies, nuclear domains implicated in the assembly, and modification of RNPs, provided the first link of SMN to RNA processing, which remains the most likely affected pathway.⁷–⁹

Currently, the main targets for therapeutics in SMA are aimed at increasing SMN levels by regulating SMN2 transcription or splicing, or by using gene therapy. These approaches include gene therapy for SMN replacement,¹⁰,¹¹ antisense oligonucleotides (ASOs) to modify SMN2 splicing,¹²,¹³ small molecules to modify SMN2 splicing,¹⁴ to increase the stability of SMN protein, or to activate the SMN2 promoter.¹⁵ Other therapeutic possibilities such as stem cells that can differentiate into motor neurons,¹⁶ neuroprotective agents,¹⁷ and the use of targets downstream of SMN (once defined) are alternative attractive therapeutic strategies.

What will be the best targets for SMA therapy? Potential therapeutic benefit in SMA may depend on several factors. What is the capacity of the remaining mutant motor neurons to reinnervate skeletal muscle fibers? The functional diversity of motor neurons is well known, but little is known about the subgroups of motor neurons targeted by SMN protein deficiency as shown by the marked involvement of intercostal and axial muscles and the sparing of the diaphragm. Another question is whether there is a defect of motor neurons development, a progressive loss of motor neurons or both. In addition, animal models provided evidence that SMA pathology is not restricted to motor neurons but rather is a composite of pathology involving, in addition to motor neurons, skeletal muscle, neuromuscular junctions,¹⁸–²⁰ and sensory-motor neurotransmission.²¹ Finally, what is/are the main targets of SMN leading to SMA? These questions are critical for the design of therapeutic strategies: when, how, and which cell types should be targeted? This reinforces the need, in parallel to therapeutic research and development, to fill the gap between SMN1 gene defect, RNA metabolism, the unknown relevant target(s) to date, and the resulting SMA phenotype.

This book is written by experts who contribute to major progress in the fields of SMA from the clinical features, molecular mechanisms, animal models, therapeutic developments to clinical trials. I have no doubt that this book will become an indispensable resource for clinicians and scientists having the goal to cure SMA.

References

1. Lefebvre S, Bürglen L, Reboullet S, Clermont O, Burlet P, Viollet L, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell. 1995;80:155–165.

2. Cartegni L, Krainer A.R. Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet. 2002;30:377–384.

3. Kashima T, Manley J.L. A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy. Nat Genet. 2002;34:460–463.

4. Vitte J, Fassier C, Tiziano F.D, Dalard C, Soave S, Roblot N, et al. Refined characterization of the expression and stability of the SMN gene products. Am J Pathol. 2007;171:1269–1280.

5. Bussaglia E, Clermont O, Tizzano E, Lefebvre S, Bürglen L, Cruaud C, et al. A frame-shift deletion in the survival motor neuron gene in Spanish spinal muscular atrophy patients. Nat Genet. 1995;11:335–337.

6. Lefebvre S, Burlet P, Liu Q, Bertrandy S, Clermont O, Munnich A, et al. Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet. 1997;16:265–269.

7. Liu Q, Dreyfuss G. A novel nuclear structure containing the survival of motor neurons protein. EMBO J. 1996;15:3555–3565.

8. Liu Q, Fischer U, Wang F, Dreyfuss G. The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins. Cell. 1997;90:1013–1021.

9. Fischer U, Liu Q, Dreyfuss G. The SMN-SIP1 complex has an essential role in spliceosomal snRNP biogenesis. Cell. 1997;90:1023–1029.

10. Foust K.D, Wang X, McGovern V.L, Braun L, Bevan A.K, Haidet A.M, et al. Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat Biotechnol. 2010;28:271–274.

11. Valori C.F, Ning K, Wyles M, Mead R.J, Grierson A.J, Shaw P.J, et al. Systemic delivery of scAAV9 expressing SMN prolongs survival in a model of spinal muscular atrophy. Sci Transl Med. 2010;2:35–42.

12. Singh N.K, Singh N.N, Androphy E.J, Singh R.N. Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron. Mol Cell Biol. 2006;26:1333–1346.

13. Hua Y, Vickers T.A, Okunola H.L, Bennett C.F, Krainer A.R. Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice. Am J Hum Genet. 2008;82:834–848.

14. Naryshkin N.A, Weetall M, Dakka A, Narasimhan J, Zhao X, Feng Z, et al. Motor neuron disease. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy. Science. 2014;345:688–693.

15. Jarecki J, Chen X, Bernardino A, Coovert D.D, Whitney M, Burghes A, et al. Diverse small-molecule modulators of SMN expression found by high-throughput compound screening: early leads towards a therapeutic for spinal muscular atrophy. Hum Mol Genet. 2005;14:2003–2018.

16. Frattini E, Ruggieri M, Salani S, Faravelli I, Zanetta C, Nizzardo M, et al. Pluripotent stem cell-based models of spinal muscular atrophy. Mol Cell Neurosci. 2015;64:44–50.

17. Bordet T, Buisson B, Michaud M, Drouot C, Galéa P, Delaage P, et al. Identification and characterization of cholest-4-en-3-one, oxime (TRO19622), a novel drug candidate for amyotrophic lateral sclerosis. J Pharmacol Exp Ther. 2007;322:709–720.

18. Cifuentes-Diaz C, Nicole S, Velasco M.E, Borra-Cebrian C, Panozzo C, Frugier T, et al. Neurofilament accumulation at the motor endplate and lack of axonal sprouting in a spinal muscular atrophy mouse model. Hum Mol Genet. 2002;11:1439–1447.

19. Kariya S, Park G.H, Maeno-Hikichi Y, Leykekhman O, Lutz C, Arkovitz M.S, et al. Reduced SMN protein impairs maturation of the neuromuscular junctions in mouse models of spinal muscular atrophy. Hum Mol Genet. 2008;17:2552–2569.

20. Martinez T.L, Kong L, Wang X, Osborne M.A, Crowder M.E, Van Meerbeke J.P, et al. Survival motor neuron protein in motor neurons determines synaptic integrity in spinal muscular atrophy. J Neurosci. 2012;32:8703–8715.

21. Mentis G.Z, Blivis D, Liu W, Drobac E, Crowder M.E, Kong L, et al. Early functional impairment of sensory-motor connectivity in a mouse model of spinal muscular atrophy. Neuron. 2011;69:453–467.

From Mapping Survival Motor Neuron to Treatment of Spinal Muscular Atrophy

A.H.M. Burghes,     The Ohio State University, Columbus, OH, United States

As with many disease genes, the positional cloning of the spinal muscular atrophy (SMA) gene started with mapping using linkage analysis. The remarkable thing in the SMA story is how rapidly the field has advanced in the short time since the cloning of the gene to the development of true therapeutic options. Here I describe some of the events that occurred behind the scenes during these developments. By necessity this is a personal perspective, and I am sure I have missed many critical points either due to memory lapse or the fact that I was not there. I apologize at the beginning for any oversights. This is not meant to be a comprehensive account but more a sketch of some of the events that occurred.

I first became aware of SMA during the late 1970s and early 1980s while working at the Jerry Lewis Muscle Research Center at the Hammersmith Hospital in west London (which is adjacent to Her Majesty’s Prison Service Facility of Wormwood Scrubs of which I did not attend). At the time SMA had only a clinical description. The muscle pathology of fiber-type grouping showing the effects of denervation and electrophysiology (EMG) were used diagnostically. However, there was virtually no research, either genetic or biochemical, on the disease. Linkage mapping had been performed in Duchenne muscular dystrophy, and it was clear that with an increase in the number of markers it would be possible to perform linkage analysis in other genetic disorders. I thus became interested in mapping SMA. Prior to leaving the Hammersmith Hospital where I completed my Ph.D., the director of the unit Dr. Victor Dubowitz asked me what I would work on if I stayed. My answer was SMA, and thus Victor went on to collect SMA families in collaboration with Kay Davies in Oxford for mapping the SMA gene. I started my laboratory at the Ohio State University in August 1988 and began collecting SMA families for mapping with the aid of Dr. Jerry Mendell. Prior to that, Dr. Don Wood of the Muscular Dystrophy Association (MDA) and Dr. Conrad Gilliam, who was moving to Columbia, had started to collect SMA families. In France, Dr. Judith Melki with Dr. Arnold Munich and others collected families during early 1988 and rapidly obtained sufficient numbers for linkage analysis as described in her editorial in this book. There were two schools of thought at the time: one believed that type 1 SMA was due to mutations in a different gene from types 2 and 3, and the other believed that SMA types 1, 2, and 3 were all caused by mutations in the same gene. I was of the latter view because there were pedigrees that contained different types of SMA in the same family. Linkage to SMA was identified by Conrad Gilliam’s group in collaboration with laboratories from around the world as well as Judith’s group using both French and North African families. Quickly both groups showed that type 1 SMA mapped to the same loci. As evidenced by the European Neuromuscular Center (ENMC) sponsored meeting held in June 1992, as well as MDA sponsored meetings, there were now a series of groups working on obtaining closer markers and physical maps of the SMA gene. Prior to 1992, Dr. Louis Kunkel and Dr. Gilliam presented markers in MAP1B at an MDA meeting. Initially it was hoped that MAP1B was a candidate SMA gene, but recombinants showed that this was not the case. As in any classical positional cloning experiment the task was to find new probes closer to the gene. Thus, the race for the SMA gene was on.

When we started on the journey to map the gene, we and others were not aware of the horrific nature of the arrangement of the genome in the area containing the survival motor neuron (SMN) genes (5q13)—namely, the duplication or multiplication of everything, and partial genes and exons in this region that produce mRNA but not functional protein. When we obtained markers closer to the gene the markers invariably showed more than two alleles indicating multiple copies of the marker. In addition, the region contained many bits of genes including some exons of β-cadherin and β-glucoronidase that produce RNA but not a functional protein. This caused much confusion with at least one of my graduates saying, If there was ever another gene hunt in the Burghes laboratory, they were not taking part because it was guaranteed to be a nightmare. Of course the individual in question enjoyed the challenge and is a very successful researcher today.

The seemingly endless possible arrangement of genes over this large region was unprecedented. Pulse-field gel electrophoresis showed that the genes, markers, and other elements in the SMA region are not the same in every individual. Thus, the depiction of the SMN1 and SMN2 genes in an inverted duplication often displayed in textbooks represents just one of many possible arrangements and copy number of those elements. Even now the structure of this genomic region is not fully understood. To date no real SMN deletion junction has been defined. We also do not know if the macrostructure of the region confers risk of de novo mutation (deletion), although we suspect it does. Furthermore, while we know that two SMN1 genes can occur on the same chromosome, no duplication junction has been reported. One difficulty in finding junction fragments is that the human genome database contains only one possible arrangement. An example of the complexity of this region is shown in the data from the 1000 Genomes Project. Alignment of 1000 genomes to one possible arrangement has resulted in 1000 misalignments and one giant mess in the SMN region.

We collaborated with a number of other laboratories including Dr. John McPherson in the Wasmuth Laboratory to obtain markers and their physical placement using radiation hybrids, Dr. Alex Mackenzie’s laboratory on analysis of CAAT1/C161, and Dr. Louise Simard and Dr. Brunhilde Wirth on Ag1-CA/C272 analysis. It soon became clear that the SMA gene fell right where our markers were. Eventually we realized that the possibility of two genes with one being deleted was likely. However, it seemed at the time most likely that one copy of the gene was nonfunctional and actually a pseudogene. In 1995, three groups reported deletions of NAIP (XS2G3 was part of the NAIP gene) and SMN1 in SMA patients. The detection of small mutations in various types of SMA helped to make the distinction between NAIP and SMN1 as the gene responsible for SMA. Type 1 SMA patients with small SMN1 mutations and an intact NAIP gene seemed to be just as affected as SMA patients lacking the NAIP gene. It was the realization by Melki’s group that SMN1 and SMN2 were almost identical except for a critical change in exon 7, along with the detection of small mutations, that cemented the cloning of the SMN gene in 1995 as described in the editorial by Judith Melki in this book.

The scientific meetings to that date had served to exchange information specific to getting the gene. After the cloning of the gene there was a need to bring people from a variety of disciplines together to study the function of SMN and therapeutic approaches to SMA. But now the topics got much broader and needed a different setting. Two sets of meetings were born out of this: a broad, international meeting with Families of SMA (FSMA, now Cure SMA) and a smaller, more focused meeting with Andrew’s Buddies (now FightSMA). The first meeting of FSMA occurred in Schaumburg, Illinois outside of Chicago in 1996. In 1997, international experts were recruited to this meeting after discussions with Audrey Lewis, the founder of FSMA. The first FSMA meeting had a group of people who could be incorporated around a boardroom table. It is now much larger with more than 300 researchers and multiple companies in attendance, but there is still an open forum for exchange. Both FightSMA and Cure SMA as well as MDA funded many critical projects at early stages that have allowed ideas to move the field forward in many ways. Audrey Lewis and the founder of Fight SMA Martha Slay were instrumental in advancing research and knowledge in SMA. In addition, I hope that both Alex Mackenzie and I have managed to guide the science so that the two groups complement but do not overlap in their research efforts. In the case of Cure SMA (while I know the name is now Cure SMA I do have to say I would not allow Audrey to use the cure word in those days because we simply did not know if it was possible), we chose to make this an international meeting that encouraged all scientists to discuss unpublished material. As such the abstracts were not published on the Internet