Evaluation of Therapeutic Response in Spinal Muscular Atrophy Using Multispectral Optoacoustic Tomography (MSOT) and Magnetic Resonance Imaging (MRI)
Phase: NA
Conditions: Muscular Diseases, Spinal Muscular Atrophy
Sponsor: University of Erlangen-Nürnberg Medical School

Evaluation of Therapeutic Response in Spinal Muscular Atrophy using 
Multispectral Optoacoustic Tomography (MSOT) and Magnetic Resonance 
Imaging (MRI) 
  
  
Unique Protocol ID: 453_19B  
ClinicalTrials.gov Identifier: NCT04262570  
  
  
Sponsor:   
ELAN-Fonds, Interdisciplinary Center for Clinical Research (IZKF), Friedrich-Alexander 
University (FAU) Erlangen-Nürnberg  
and  
Department of Pediatrics and Adolescent Medicine  
University Hospital Erlangen  
Friedrich-Alexander-University (FAU) Erlangen-Nürnberg  
Pediatric Experimental and Translational Imaging Laboratory (PETI_Lab)  
Dr. med. Ferdinand Knieling  
Loschgestr. 15  
91054 Erlangen  
Germany  
Tel.: +49 9131 85-33118  
E-mail: ferdinand.knieling@uk-erlangen.de   
   
 
 
 
 
 
 
 
 
Protocol Version 1.3 – 21.02.2020 
 
 

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  Study protocol  
 
 
 
 
TheraponSMA 
 
Evaluation of therapeutic response in spinal muscular atrophy using multispectral 
optoacoustic tomography (MSOT) and magnetic resonance imaging (MRI) 

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Table of content 
 
 
Table of content .............................................................................................................. 2 
 
Study titel, version number, version date ........................................................................ 3 
 
Project summary ............................................................................................................. 4 
 
Responsabilities ............................................................................................................. 5 
 
Scientific background ...................................................................................................... 6 
 
Study objectives ............................................................................................................11 
 
Target parameters .........................................................................................................12 
 
Study design ..................................................................................................................14 
 
Study population ............................................................................................................15 
 Study course .................................................................................................................17 
 Risk-benefit analysis .....................................................................................................19 
 Biometrics .....................................................................................................................24 
 Data management and data protection .........................................................................25 
 Handling of biomaterials ...............................................................................................26 
 Insurance ......................................................................................................................26 
 Signatures ....................................................................................................................27 
 
 
 

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Study titel, version number, version date 
 
Study title 
Evaluation of therapeutic response in spinal muscular atrophy using multispectral 
optoacoustic tomography (MSOT) and magnetic resonance imaging (MRI) 
 
Version number 
Version 1.3 
 
Version date 
21.02.2020 
 
 
Protocol versions 
Date 
Version 
Status 
Changes 
17.12.2019 
1.0 
Final application 
 
14.01.2019 
1.1 
Amendment 
Specification of 
recruitment for control 
patients 
11.02.2020 
1.2 
Final 
 
21.02.2020 
1.3 
Amendment 
Specification of clinical 
routine testing 
 
 
 

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Project summary  
Neuromuscular diseases are caused by numerous pathologies of the central nervous system 
(brain and spinal cord), the peripheral nervous system or the skeletal muscle. Spinal muscular 
atrophy (SMA) is characterized as a pathology of the central nervous system characterized by 
the degeneration of motor anterior horn nerve cells in the spinal cord or lower brain stem, 
leading to progressive muscle weakness and atrophy. The clinical spectrum of all subtypes of 
the disease is broad, but in almost all cases the therapeutic options are limited to supportive 
therapy and palliative measures to prevent or alleviate complications such as spinal 
deformities or respiratory insufficiency. In the course of the disease, patients lose their motor 
neurons and thus their original skeletal muscle mass. In the muscle itself, a histological loss of 
the nerve and muscle fibers as well as a fatty remodeling can be observed. Most patients show 
normal cognition and consciously experience the course of their disease.  
Since 2016 (USA) and 2017 (European Union), the drug Nusinersen (Spinraza®, Biogen) has 
been available for the treatment of children and adults with SMA. It contains an antisense 
oligonucleotide, which is supposed to increase the expression of the usually in SMA reduced 
"survival motor neuron protein". In the ENDEAR study, the study used for FDA approval, 40% 
of children in the treatment arm achieved significant improvements in the area of motor 
milestones, while no child in the control group achieved this. Further positive effects were also 
reported in older children and adolescents as a result of the CHERISH study. The drug is 
administered intrathecally (in the spinal fluid). The first three doses were administered at 
intervals of 14 days and the fourth at intervals of 30 days. Subsequently, maintenance therapy 
is administered at intervals of 4 months. According to the Joint Federal Committee, the annual 
therapy costs currently amount to 310,877.58 € to 310,942.95 € - internationally, Spinraza® 
ranks second among the most expensive drugs in the world. In addition, the first gene therapy 
(Zolgensma®) has already been approved in the USA at a price of over $2,000,0000. Whether 
patients benefit from this therapy can only be assessed clinically so far. Our preliminary work 
shows that new methods such as multispectral optoacoustic tomography (MSOT) and 
magnetic resonance imaging detect tissue changes very sensitively. With both methods, the 
molecular composition of muscle tissue can be determined non-invasively and quantitatively 
at the same time. This first pilot study will now investigate whether these differences can also 
be displayed in the course of therapy.  
 
 

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Responsabilities 
Study director 
Dr. Ferdinand Knieling 
Prof. Dr. Regina Trollmann 
Department of Pediatrics  
University Hospital Erlangen  
Loschgestr. 15 
91054 Erlangen  
Germany 
Tel. 09131 8533118 
Mail: ferdinand.knieling@uk-erlangen.de  
Dr. Matthias Türk 
Department of Neurology  
University Hospital Erlangen  
Schwabachanlage 6 
91054 Erlangen 
Germany 
Tel. 09131 8533001 
Mail: matthias.tuerk@uk-erlangen.de  
Prof. Dr. Michael Uder 
Prof. Dr. Armin Nagel 
Department of Radiology 
Universitity Hospital Erlangen 
Maximiliansplatz 1 
91054 Erlangen 
Germany  
Tel.: 09131 85 36065 
Email:  michael.uder@uk-erlangen.de  
Other facilities possibly involved (e.g. laboratory, imaging, etc.) 
Prof. Dr. Maximilian J. Waldner (technical realization) 
Deparment of Medicine 1  
University Hospital Erlangen 
Ulmenweg 18 
91054 Erlangen 
Germany  
Tel.: 09131 8545025 
Mail: maximilian.waldner@uk-erlangen.de 
 
Sponsoring  
Children’s Hospital Erlangen, Else-Kröner-Fresenius Foundation (Else-Kröner-Fresenius 
Memorial Scholarship Ferdinand Knieling) 
 
 

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 Scientific background  
Spinal muscular atrophy (SMA) is an autosomal-recessive disorder, characterized by 
progressive muscle weakness and atrophy. Spinal muscle atrophy has an incidence of 
1/10,000 with an estimated carrier frequency of 1/50 (Sugarman 2012, McAndrew 1997, Pearn 
1978). The condition is caused by a homozygous deletion or mutation in the survival motor 
neuron 1 (SMN1), resulting in reduced expression of the survival motor neuron (SMN) protein. 
This leads to the degeneration of motor neurons in the spinal cord and brain stem (Lefebvre 
1995, Prior 2010). A nearby related gene, survival motor neuron 2 (SMN2), also produces the 
SMN protein, but due to aberrant splicing around 85-90% of translated protein are truncated 
and non-functional and rapidly degraded in the cell (Mailman 2002); remaining 10-15% are 
functional (Butchbach 2016), partially compensating the loss of SMN1 by SMN2 protein 
synthesis (Hsieh-Li 2000). Individuals with a higher copy number of SMN2 do in general have 
a milder phenotype (Mailman 2002, Butchbach 2016).  
The various forms of SMA are differentiated according to distribution pattern, onset of disease, 
disease severity and hereditary pattern and are generally designated according to the muscle 
groups mainly affected (Pearn 1973, https://www.dgm.org/muskelerkrankungen/spinale-
muskelatrophie). A large number of similar, much rarer forms are known that can be associated 
with additional dysfunctions. The non-proximal spinal muscle atrophies are also very rare and 
do usually not lead to significant limitations of vital functions at the onset of adulthood. 
However, the vast majority (approx. 90%) of patients belong to the group of so-called proximal 
SMA, which is characterized by the onset of muscle weakness in muscle groups close to the 
trunk (above all thigh and hip muscles, later also arm and shoulder girdle involvement). 
Proximal SMA is divided into different subtypes, which are primarily defined according to the 
onset of the disease, the learned motor skills and life expectancy. 
Spinal muscular atrophy type 0, also called prenatal onset SMA, describes the most severe 
form of SMA as it presents before birth with decreased fetal movements. Neonates are born 
with severe hypotonia and weakness, areflexia, facial weakness, joint contractures, difficulty 
swallowing and respiratory failure on examination. Life-expectancy is greatly reduced, most 
patients die within the first 6 months of age (Dubowitz 1999, MacLeod 1999). 
Spinal muscular atrophy type 1, also called Werdnig-Hoffman disease or infantile SMA, is 
the most common type of SMA. Symptoms usually exhibit at birth or before 6 months of age. 
Infants present with hypotonia (proximal more than distal limbs, legs more affected than arms) 
with poor head control (“floppy baby”), reduced or absent tendon reflexes and are never able 
to sit unassisted. Patients may show a bell-shaped thorax, paradoxical breathing, swallowing 

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weakness and tongue fasciculations. Cognition is not affected. Disease is usually fatal prior to 
2 years of life due to respiratory failure (Finkel 2014, Thomas 1994). 
Spinal muscular atrophy type 2, also called Dubowitz disease or intermediate SMA, presents 
usually between 6-18 months of age. This form tends to manifest with progressive leg 
weakness, which is worse than weakness in the arms, hypotonia and in a great percentage of 
patients areflexia. Comorbidities are related to muscular weakness and include scoliosis and 
joint contractures, as well as possible restrictive lung disease due to intercostal muscle 
weakness. Cognition is normal in affected patients (von Gontard 2002). Children with SMA 
type 2 are able to sit unassisted but are never able to walk independently. Life expectancy is 
reduced, but most patients live until adulthood (Fried 1971, Hausmanowa-Petrusewicz 1985, 
Imai 1995). 
Spinal muscular atrophy type 3, also called Kugelberg–Welander disease or juvenile SMA, 
usually manifests after 12 months of age. Patients suffer from a proximal muscle weakness, 
which is more pronounced in the legs than in the arms. Individuals are able to walk unassisted, 
however at some point a wheelchair may be needed. Unlike SMA type 2, these patients do 
usually not suffer from orthopedic comorbidities or respiratory muscle weakness. Cognition 
and life expectancy are normal within this group. (Kugelberg 1956, Meadows 1969, Zerres 
1997).  
Spinal muscular atrophy type 4, is the mildest form of disease with an onset in adulthood, 
often at age 30 or later. Patients present with mild muscle weakness in the legs, that may 
procced to the arms. A small number of patients may require wheelchair assistance. Cognition 
and life expectancy is normal (Pearn 1978, Tsukagoshi 1965, Piepers 2008). 
Extensive research has been done over the last two decades to develop an effective treatment 
strategy for SMA. Nusinersen (Spinraza®, Biogen), the first approved drug for the treatment 
of SMA, has been available since December 2016. This is an antisense oligonucleotide drug 
that leads to an increased production of full-fledged functional SMN protein. It is administered 
directly intrathecally as it does not cross the blood-brain barrier. Several studies showed 
improvements in motor function and survival in patients treated with Nusinersen, where early 
treatment was necessary to maximize the benefit of the drug. Long-term data are still pending. 
Several other promising therapeutic approaches, including gene therapy for SMA, are currently 
under development. 
 
 

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There are currently no prospective markers available that can predict early muscle 
degeneration, disease progression, therapy response or therapeutic changes. Since 2017, the 
University Hospital Erlangen (Department of Medicine 1, Department of Pediatrics) has a 
multispectral optoacoustic tomography (MSOT) funded by the DFG. This allows non-invasive, 
quantitative imaging of the composition of target tissues.  
In MSOT, similar to conventional sonography, a transducer is placed on the skin and instead 
of sound, energy is supplied to the tissue by means of pulsed laser light. This leads to a 
constant change of minimal expansions and contractions (thermoelastic expansion) of 
individual tissue components or molecules. The resulting sound waves can then be detected 
by the same examination unit. Previous studies have shown that the quantitative determination 
of hemoglobin can be used to obtain information on blood flow and inflammatory activity in the 
intestine from patients with Crohn's 
disease (Waldner, Knieling et al. 
2016, Knieling, Neufert et al. 2017) 
(Waldner 2016, Knieling 2017). This 
principle 
has 
already 
been 
successfully used in a follow-up 
study on pediatric patients with 
Duchenne 
muscular 
dystrophy 
(Regensburger 2019, ESMI Young 
Investigator 
Award 
2019). 
In 
addition to this technology, MRI 
imaging has advanced in the field of 
muscle diseases. The first example 
is the 23Na-MRI: while standard 
MRI is based on the imaging of 
protons (1H), mainly water and 
hydrocarbon compounds in the 
human body, the 23Na-MRI gets its 
signal from the sodium atoms of the 
human 
body. 
With 
this 
measurement technique, valuable 
information about cellular sodium 
homeostasis in muscular sodium 
channel diseases can be obtained, 
which has meanwhile contributed to the development of successful interdisciplinary 
therapeutic concepts for some of these channelopathies (e.g. (Amarteifio, Nagel et al. 2012).  
Abbildung 1 –  MSOT Prinzip (a), experimentelle Vorarbeiten (b), und 
erstmalig Darstellung von Kollagen in vivo (c) 

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Hollingsworth et al (Hollingsworth, de Sousa et al. 2012) recommend the following MRI 
examination of skeletal muscles: T1-weighted imaging with lipid-water separation to quantify 
lipid infiltration and T2-mapping to examine inflammation and edema. However, many new 
MRI measurement techniques have not yet been used to examine rare muscle diseases. Our 
preliminary work shows that both technologies map different molecular structures but similar 
muscular pathologies (Regensburger et al. 2019). In this first pilot study on patients with SMA, 
we will now investigate whether the differences in muscle composition of SMA patients with or 
without therapy can be quantified and whether they can be used simultaneously as markers 
during therapy with nusinersen. Ideally, both techniques can complement or validate each 
other. In the future, this could generate a completely new, non-invasive method for evaluating 
endogenous biomarkers for therapy response.  
 
Literatur 
Sugarman EA, Nagan N, Zhu H, et al. Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular 
atrophy: clinical laboratory analysis of >72,400 specimens. Eur J Hum Genet 2012;20:27-32 
 
McAndrew PE, Parsons DW, Simard LR, Rochette C, Ray PN, Mendell JR, Prior TW, Burghes AHM. Identification 
of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number. Am 
J Hum Genet 1997; 60: 1411–1422. 
 
Pearn J. Incidence, prevalence, and gene frequency studies of chronic childhood spinal muscular atrophy. J Med 
Genet 1978; 15:409. 
 
Lefebvre S, Bürglen L, Reboullet S, et al. Identification and characterization of a spinal muscular atrophy-
determining gene. Cell 1995; 80:155. 
 
Prior TW. Spinal muscular atrophy: a time for screening. Curr Opin Pediatr 2010;22:696-702. 
Mailman MD, Heinz JW, Papp AC, et al. Molecular analysis of spinal muscular atrophy and modification of the 
phenotype by SMN2. Genet Med 2002;4:20-26 
 
Butchbach ME. Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular 
Atrophy and Other Neurodegenerative Diseases. Front Mol Biosci 2016; 3:7. 
 
Hsieh-Li HM, Chang JG, Jong YJ, et al. A mouse model for spinal muscular atrophy. Nat Genet 2000; 24:66. 
 
Pearn, J. K., Carter, C. O., Wilson, J. The genetic identity of acute infantile spinal muscular atrophy. Brain 96: 463-
470, 1973. 
 
Dubowitz V. Very severe spinal muscular atrophy (SMA type 0): an expanding clinical phenotype. Eur J Paediatr 
Neurol. 1999;3(2):49–51. 
 
MacLeod MJ, et al. Prenatal onset spinal muscular atrophy. Eur J Paediatr Neurol. 1999;3(2):65–72. 
 
Finkel RS, et al. Observational study of spinal muscular atrophy type I and implications for clinical trials. Neurology. 
2014;83(9):810–817. 
 
Thomas NH, Dubowitz V. The natural history of type I (severe) spinal muscular atrophy. Neuromuscul Disord. 
1994;4(5–6):497–502. 
 
von Gontard A, et al. Intelligence and cognitive function in children and adolescents with spinal muscular atrophy. 
Neuromuscul Disord. 2002;12(2):130–136. 
 
Fried, K., Emery, A. E. H. Spinal muscular atrophy type II. A separate genetic and clinical entity from type I (Werdnig-
Hoffmann disease) and type III (Kugelberg-Welander disease). Clin. Genet. 2: 203-209, 1971. 
 
Hausmanowa-Petrusewicz, I., Zaremba, J., Borkowska, J. Chronic proximal spinal muscular atrophy of childhood 
and adolescence: problems of classification and genetic counselling. J. Med. Genet. 22: 350-353, 1985. 

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Imai, T., Matsuya, M., Matsumoto, H., Ishikawa, Y., Minami, R. Preservation of central motor conduction in patients 
with spinal muscular atrophy type II. Brain Dev. 17: 432-435, 1995. 
 
Kugelberg, E., Welander, L. Heredofamilial juvenile muscular atrophy simulating muscular dystrophy. Arch. Neurol. 
Psychiat. 75: 500-509, 1956. 
 
Meadows, J. C., Marsden, C. D., Harriman, D. G. F. Chronic spinal muscular atrophy in adults. I. The Kugelberg-
Welander syndrome. J. Neurol. Sci. 9: 527-550, 1969. 
 
Zerres K, et al. A collaborative study on the natural history of childhood and juvenile onset proximal spinal muscular 
atrophy (type II and III SMA): 569 patients. J Neurol Sci. 1997;146(1):67–72. 
 
Knieling F et al. Multispectral Optoacoustic Tomography for Assessment of Crohn's Disease Activity. N Engl J Med. 
2017 Mar 30;376(13):1292-1294.  
 
Waldner MJ, Knieling F, et al. Multispectral Optoacoustic Tomography in Crohn's Disease: Noninvasive Imaging of 
Disease Activity. Gastroenterology. 2016 Aug;151(2):238-40. doi: 10.1053/j.gastro.2016.05.047. Epub 2016 Jun 3.  
 
Regensburger, A. P., L. M. Fonteyne, J. Jungert, A. L. Wagner, T. Gerhalter, A. M. Nagel, R. Heiss, F. Flenkenthaler, 
M. Qurashi, M. F. Neurath, N. Klymiuk, E. Kemter, T. Frohlich, M. Uder, J. Woelfle, W. Rascher, R. Trollmann, E. 
Wolf, M. J. Waldner and F. Knieling (2019). "Detection of collagens by multispectral optoacoustic tomography as 
an imaging biomarker for Duchenne muscular dystrophy." Nat Med 25(12): 1905-1915. 
Amarteifio, E., A. M. Nagel, M. A. Weber, K. Jurkat-Rott and F. Lehmann-Horn (2012). "Hyperkalemic periodic 
paralysis and permanent weakness: 3-T MR imaging depicts intracellular 23Na overload--initial results." Radiology 
264(1): 154-163. 
Hollingsworth, K. G., P. L. de Sousa, V. Straub and P. G. Carlier (2012). "Towards harmonization of protocols for 
MRI outcome measures in skeletal muscle studies: consensus recommendations from two TREAT-NMD NMR 
workshops, 2 May 2010, Stockholm, Sweden, 1-2 October 2009, Paris, France." Neuromuscul Disord 22 Suppl 2: 
S54-67. 
 
 
 

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Study objectives  
Evaluation of the muscle structure of patients with spinal muscular atrophy under Spinraza 
therapy using multispectral optoacoustic tomography (MSOT) and magnetic resonance 
imaging (MRI). 
Hypotheses: 
 
The optoacoustic spectrum of patients with and without therapy differs at 0, 2 and 
12 months  
 
The optoacoustic spectrum of patients with therapy changes over time. 
 
The quantitative fraction of lipid signal in muscles determined by MSOT differs 
between patients with and without therapy. 
 
The quantitative fraction of collagen signal in muscles determined by MSOT differs 
between patients with and without therapy. 
 
The quantitative fraction of hemo/myoglobin in muscles determined by MSOT 
differs between patients with and without therapy. 
 
The quantitative fraction of oxygenated/deoxygenated hemoglobin in muscles 
determined by MSOT differs between patients with and without therapy. 
 
There are no side differences in patients with and without therapy. 
 
T1 relaxation time of patients with and without therapy differs at 0, 2 and 12 months 
 
T2 relaxation time of patients with and without therapy differs at time 0, 2 and 12 
months 
 
Fat-water fraction of patients with and without therapy differs at the time of 0, 2 and 
12 months 
 
Sodium concentration of patients with and without therapy differs at 0, 2 and 12 
months 
 
Primary study objective: 
 
Comparison of the optoacoustic spectrum determined by MSOT in patients with SMA with 
and without treatment over time 
 
Secondary study objectives: 
 
Comparison of the quantitative lipid/collagen/haemo/myoglobin content determined by 
MSOT in patients with and without therapy 
 
Correlation of lipid/collagen/haemo/myoglobin content determined by MSOT with disease 
duration/patient age 

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 
Correlation of lipid/collagen/haemo/myoglobin content determined by MSOT with clinical 
routine testing  
 
Correlation of T1 relaxation time/T2 relaxation time/fat water portion/sodium concentration 
with clinical routine testing  
 
Correlation of T1 relaxation time/T2 relaxation time/fat water portion/sodium concentration 
and MSOT determined lipid/collagen/haemo/myoglobin content 
 
Measurement of the signal differences in the right / left comparison 
Study type 
Since no data exists so far to support the hypothesis of this study, it is an explorative study / 
pilot study. 
 
Target parameters 
The MRT measurement are performed on the lower leg muscles. 
The MSOT measurements are performed over the proximal and distal extremity muscles in 
left-right comparison (right - left). (Leg proximal: musculus quadriceps, distal: musculus triceps 
surae; arm proximal: musculus biceps, distal: forearm flexors). 
 
Primary target: 
Optoacoustic Absorption Spectrum of Muscle. 
This target is measured non-invasively by MSOT. 
 
Secondary targets: 
Quantitative lipid signal (in arbitrary units) 
Quantitative collagen signal (in arbitrary units) 
Quantitative hemo/myoglobin signal (in arbitrary units) 
Muscle oxygenation (in %) 
Ratio of lipid to hemo/myoglobin signal or collagen to hemo/myoglobin signal 
These target values are collected non-invasively using MSOT. 
 
T1 relaxation time  
T2 relaxation time  
Fat-water percentage  
Sodium concentration  
These target values are collected non-invasively using a 3T/7T MRI. 
 
 

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Clinical scores are collected according to the recommendations as standardized examinations. 
These are identical tests that, according to the University Clinic for Neurology/Department of 
Pediatrics and Adolescent Medicine, are also performed outside of the study in the regular 
clinical routine in SMA patients (see below).  
 
Clinical test 
Timeline 
 
Induction 
Maintenance 
 
d 0 
d 14 
d 28 
d 63 
every 4 
months 
Motor function  
HFMSE 
X 
 
 
X 
X 
RULM 
X 
 
 
X 
X 
MRC-Sum* 
X 
 
 
X 
X 
ALSFRS-R* 
X 
 
 
X 
X 
6-MWT 
X 
 
 
X 
X 
Dynamometer* 
X 
 
 
X 
X 
Neurophysio.* 
(Nn. med./uln./tib. on 
both sides. + F-wave) 
X 
 
 
X 
X 
Lung function 
VC/FVC/FEV1* 
X 
X 
X 
X 
X 
Muscle function tests: Expanded Hammersmith functional motor scale (HFMSE)/Revised Upper Limb Module (RULM)/6-Minute-
walking-test (6MWT)/Medical Research Council (MRC) Scale for Muscle Strength/ALS Functional Rating Scale (ALS-FRS)  
Source: Department of Neurology, University Hospital Erlangen, *not in adolescents 
These target values are clinically determined at presentation. 
Age 
Sex 
Weight 
Skin color 
Ethnic Background 
Disease duration 
Current medication 
 
These target values are either available in the electronic patient file or they are collected at 
presentation for the study. 
 

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Study design 
Monocentric / multicentric 
This is a monocentric study.  
Study arms: intervention/control 
Interventions are not planned. A comparison is made between SMA patients with and 
without therapy. The study procedure is identical for all patients. There is no change in 
the already planned therapeutic regime. 
Randomization 
Randomisation is not planned. The allocation to the groups is based on the known 
therapy. 
Blinding 
Blinding for the examination is not possible due to the small collective. Blinding takes 
place during the measurement and evaluation of the data. Blinding of the patients is 
not necessary. 
 
Graphic presentation of the study design 

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Study population 
Inclusion and exclusion criteria 
Preliminary phase 
Inclusion criteria: 
SMA patients (therapy arm): 
 
Genetically confirmed SMA type III 
 
From age 14 
 
Willingness and ability to participate, sufficient knowledge of the german language to 
understand the declaration of consent, or if not possible, information of the patient in his/her 
mother tongue or English 
 
High probability that the patients will be able to fully participate in the study (defined by the 
ability to lie still for about 1 hour and follow any breathing commands) 
 
Medical indication for Spinraza® therapy; start of study before first administration 
Spinraza® administration 
 
SMA patients (control arm): 
 
Genetically confirmed SMA type III  
 
From age 14 
 
Willingness and ability to participate, sufficient knowledge of the german language to 
understand the declaration of consent, or if not possible, information of the patient in his/her 
mother tongue or English 
 
High probability that the patients will be able to fully participate in the study (defined by the 
ability to lie still for about 1 hour and follow any breathing commands) 
 
No medical indication for Spinraza® therapy 
 
Exclusion criteria: 
SMA Patients: 
 
Pregnancy 
 
Tattoo on the skin area to be examined 
 
General contraindications for MRT examinations 
 
Electrical implants like pacemakers or perfusion pumps 
 
Pronounced claustrophobia 

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 
Study participants with ferromagnetic or electrically conductive implants such as aneurysm 
clips, surgical clips, prostheses, artificial hearts, heart valves with metal parts, metal 
splinters, tattoos next to the eye, symmetrical tattoos on the extremities or steel implants 
must consult the study physician; they may not be able to be examined (relative 
contraindications for MRI). 
 
Non-approved concomitant medication: strongly sedating medication must be excluded, as 
intensive monitoring of bodily functions during ongoing imaging cannot be guaranteed and 
the examination is dependent on the active participation of the test person. 
Patient number 
As this is a pilot study, an exact case number calculation is not possible. It is planned to 
study a total of 5 patients with SMA with therapy and 5 patients with SMA without therapy.  
Recruitment routes and measures 
Patients (and parents) will be informed about the possibility of participating in the study during 
an elective presentation at the Department of Neuropediatrics in our Pediatric and Adolescent 
Clinic (children and adolescents) or at the Neuromuscular Center of the Neurology Department 
of the University Hospital. The primary aim is to include adult patients in this study. The medical 
indication for Spinraza® therapy was established in advance by the attending physicians of 
the Pediatric and Adolescent Clinic or the Neurology Clinic.  
If patients are willing to participate, they will be fully informed about the aims and methods 
(especially about the scientific/explorative character of the study), benefits and risks and the 
revocability of their participation in the study. Patients in childhood and adolescence are 
additionally informed and educated in an age-appropriate manner about the study and its 
procedure.  
 
 
 
 
 
 

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Study course 
Procedure for informing about and obtaining consent 
Patients can only be enrolled in the study after a written consent form has been given. The 
written declaration of consent requires oral and written information of the patients, as well as 
their parents or legal guardians, about objectives and methods (including the scientific-
explorative character of the study), benefits and risks, and the revocability of study 
participation. Children and adolescents are informed by means of age-appropriate, 
comprehensible patient information sheets. By giving their written consent, the patients or their 
parents/guardians declare that they agree to the collection and storage of study-relevant data 
and their verification by monitoring or authorities. It must be clearly communicated to the study 
participant that a withdrawal of consent is possible at any time and without any disadvantage. 
Furthermore, all study participants/test persons or parents/guardians are informed that this 
study is a purely scientific study without any current diagnostic or therapeutic benefit. 
The original of the consent form is kept in the study folder at the study location. The patient or 
parents/guardians will receive a copy of the patient information and informed consent form. 
The patient information and informed consent form are attached to this study protocol. 
Measurements 
After informing the patients and parents/guardians and obtaining 
consent, the MRI examination will be performed. This takes place 
in a lying position; according to the body region to be examined 
(lower leg), the test person is positioned with its feet first on the 
equipment couch. Rigid or flexible "coils" are attached directly or 
close to the patient. These coils serve as antennas that pick up 
the signals from the body and forward them directly to the 
computer. Hearing protection must be worn during the 
examination, as MRI examinations can be very loud. In order for 
the patient to be able to draw attention to themselves, he/she 
receives a bell button shortly before the examination begins. 
During the examination, communication via an intercom system 
is possible. An intravenous administration of contrast medium 
does not take place.  
Subsequently, all study participants are imaged by MSOT on 4 
anatomical regions: thigh/lower leg, forearm/upper arm at 
predefined muscle groups. (Leg proximal: musculus quandriceps, distal: musculus triceps 
Presentation 

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surae; arm proximal: musculus biceps, distal: forearm flexors). The examination is performed 
analogous to sonography over the corresponding skin layers without further invasive 
procedures. The anatomical region can be localized by means of built-in B-scan sonography; 
subsequently, the corresponding optoacoustic signals can then be conducted. The duration 
per anatomical region is limited to 5 minutes; this corresponds to a maximum of 20 minutes for 
both upper extremities and 20 minutes for both lower extremities. Patients can remain in a 
relaxed body position during the examination; assistance in the form of breathing manoeuvres 
or similar is not necessary. Clinical routine tests scores are then collected including tests for 
muscle strength like the Expanded Hammersmith functional motor scale (HFMSE)/ Revised 
Upper Limb Module (RULM) / 6-minute-walking test (6MWT)/ Medical Research Council 
(MRC) Scale for Muscle Strength/ ALS Functional Rating Scale (ALS-FRS)/  Dynamometer as 
well as neurophysiologic testing and lung function testing.  
Recording of target parameters 
 
MRI imaging and measurement of T1 relaxation time/T2 relaxation time/fat water 
proportion/ sodium concentration 
 
Non-invasive in-vivo measurement of lipid, collagen, myo/hemoglobin content and 
oxygenation by MSOT 
 
Clinical evaluation of routine testing including muscle tests, neurophysiologic testing and 
lung function testing 
 
Determination of routine data (duration of illness, current medication) from electronic 
patient files in Soarian/Meona 
Time schedule and study duration for the individual patient/control  
For the individual patient, the duration of study participation is 180 minutes. Approximately 20 
minutes are allotted for the education of study participants and parents/guardians, 
approximately 90 minutes for the clinical routine testing, as well as 40 minutes for the MSOT 
and 60 minutes for MRI examination. The appointments are planned according to the 
therapeutic plan or the application of Spinraza®, so there is no additional travel time for the 
patients. The control group without therapy receives in addition to the annual controls (visit 1 
and 3) one additional appointment for visit 2.  
Total duration of study 
Depending on the number of patients, the expected total duration of the study until the 
inclusion of the last patient is approximately 24 months. 
 

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Risk-benefit analysis 
All study-related risks (for MSOT) 
Based on the classification criteria for medical devices (Directive 93/42/EEC, Annex IX), the 
optoacoustic system of iThera Medical corresponds to Class IIa: 
 
Active diagnostic device 
 
non-invasive 
 
Temporary use (<60 min) 
No CE certification is available for this research device (current type designation according to 
imprint: Acuity Echo). A conformity assessment procedure in the sense of the MPG is not 
intended or planned by the manufacturer at the present time. It is therefore a purely scientific 
pilot study. There is no dependency on the manufacturer, all diagnostic and analytical 
procedures are available to the study directors on site. The cooperation with the company is 
regulated in a separate contract drawn up by the legal department before the start of the study. 
 
Adherence to energy levels  
The laser safety and maximum permitted radiation dose for irradiation with laser pulses is 
regulated in the laser standards ANSI and IEC 60825. The MSOT system meets these 
standards and therefore remains below the MPE (maximum permissible exposure) limits for 
skin irradiation and is therefore considered safe. 
 
Temperature increases due to MSOT in tissue 
Optoacoustic imaging does not result in any significant temperature increase in the tissue. The 
absorption of a laser pulse in the tissue results in a local transient temperature increase of a 
few millikelvin. Depending on the duration of the examination and the skin type of the patient, 
temperature increases occur typically in the range of less than one degree Kelvin. 
 
Histological changes in tissue 
Histological changes in the target tissue and surrounding structures are neither expected nor 
have they been observed in previous preclinical and clinical studies. 
Slight, reversible redness or warming might occur in very sensitive skin.  
 
Such side effects are to be noticed at any time by the patient or doctor; the examination can 
then be interrupted or aborted. In any case, no irreversible damage is to be expected. 
 

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In general, the near infrared light used in the MSOT can lead to retinal damage if the eye is 
irradiated. In order to prevent this, test participants and examiners will wear appropriate laser 
safety glasses during the examination. 
 
Since the data obtained is not used to interpret diagnostic results, there is no risk of possible 
misdiagnosis or incorrect display of data in this exploratory pilot study. 
 
No other risks exist for this study, nor have we described any risks based on our own 
preliminary data. 
 
Magnetic resonance imaging  
In contrast to computed tomography, MRI does not use ionising radiation, so that no 
permanent side effects are expected. The risk for the planned MRI feasibility study is not 
higher than for any clinically performed native MRI examination. 
Static magnetic field 
The static magnetic field exerts forces and torques on ferromagnetic objects, which can be so 
strong that the (mostly ferromagnetic) objects fly uncontrolled towards the magnet and can hit 
patients and staff (missile effect). The magnetic forces are proportional to the field strength B 
and the field change with location (dB/dz). To protect the patients from the dangers of magnetic 
attraction of ferromagnetic objects, the operating personnel is trained to ensure that magnetic 
objects are not brought into the examination room. Magnetic fields also have general biological 
effects. For example, when movement occurs in the magnetic field, electrical voltages are 
induced in the human body. However, these effects are very small. In a study funded by the 
Federal Office for Radiation Protection, the cognitive performance of 41 healthy volunteers 
was examined at the German Cancer Research Center (DKFZ) at different magnetic field 
strengths (1.5T; 3T; 7T). During this study, the test subjects were lying on the patient table of 
the MR system. The tests were performed at rest and during a defined movement of the patient 
table. For the investigated static magnetic fields strength (up tp 7T) no significant effects on 
cognitive performance were observed.  
In summary, studies in animals and humans have shown that body temperature, blood 
pressure, heart rate, respiration or other physiological parameters are not affected by chronic 
exposure to static magnetic fields. Only a small but measurable increase in systolic blood 
pressure with increasing magnetic field strength has been shown. The maximum increase at 
8 T was about 3.6 mmHg. However, this corresponds to only half the change caused by sitting 
up from the supine position outside a magnetic field. At no time symptomatic physiological 
changes were recorded, and no complaints were reported even after 3 months. The effects of 

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prolonged exposure to high magnetic fields above 4 Tesla on mammals, however, have been 
poorly studied, so that statements on long-term safety can only be made to a limited extent. 
No measurable effects were observed in studies in rats exposed to a field of 9.4T for up to 10 
weeks. 
The gradient system 
Gradient circuits can cause the appearance of magnetic phosphenes as well as nerve and 
muscle cell stimulation. Fast switched gradients produce high magnetic field changes per time 
(dB/dt), which induce voltages in the body. If a current flows through the tissue, for example 
via nerve endings, this can lead to so-called peripheral nerve stimulation. However, the 
manufacturer of the gradient system guarantees that the limits for gradient switching times and 
amplitudes recommended in the IEC 60601-2-33 guidelines are adhered to.  Therefore, the 
nerve stimulation effects do not need to be further considered in the risk assessment of this 
study. 
Another safety-relevant effect of the gradient fields are noises caused by gradient switching, 
which develop due to current and field strength-dependent Lorentz forces in the gradient tube. 
These often unpleasant loud knocking noises are especially generated in fast imaging 
processes where high currents flow through the gradients. The test patients always wear 
hearing protection during the examination, so that the noise exposure remains well below the 
legal limits of 99 dB. 
The high frequency system 
During the MR measurement, high-frequency (HF) fields are sent into the human body, which 
are partially absorbed by the tissue and can lead to an increase in body temperature. The 
thermoregulatory response of human tissue to HF pulses has been studied for 50 years. For 
example, it has been calculated that the body temperature of lightly dressed subjects with 
undisturbed thermoregulation at room temperature increases by up to 0.6 °C (63 MHz, 1.5 
Tesla) at an RF exposure of 4 W/kg. The assumed specific absorption rate (SAR) of 4 W/kg 
body tissue corresponds to the so-called "controlled mode first level" (IEC safety guideline), 
which is also used as the upper limit in routine clinical imaging. The magnitude of the real 
temperature rise is generally even smaller because skin cooling was not taken into account in 
the calculations (worst case scenario).  
The body's own energy production at rest is about 1.2 W/kg - this corresponds to the energy 
that is conserved when wearing a thin sweater. Most healthy people are able to compensate 
15x this resting energy, and there will only be a minimal increase in core body temperature. 
Studies at 1.5T have shown that HF absorption in humans only leads to the expected cardiac 
adaptation and does not cause any negative health effects. Theoretically, a 63 kg person is 

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even able to release 1296 W to the environment through the skin by cardiac adaptation (i.e. 
maximum blood flow increase) - this would correspond to a SAR of 20.6 W/kg. 
Benefits associated with this study 
The data obtained in this study may provide important insights into the pathomechanism of 
SMA. The possible quantitatively determinable differences could be used in the future as a 
monitoring method for therapeutic response or prognosis of this disease. 
There is a potential benefit for the trial participants. With low probability, the scientific 
evaluation of the data may yield results that indicate previously unknown pathological changes 
in this patient. In this case, further investigation by a specialist would be recommended to the 
patient. 
Termination criteria  
Termination criteria for the individual participant: 
 
an event occurs that could endanger the patient or the personnel, 
 
the patient waives his/her consent to participate in the examination 
 
the patient does not follow the instructions of the examiner and the operating  
 
Termination criteria for the whole study: 
 
Intermediate results indicate that the investigations cannot achieve the 
objective of the study 
 
An examination can be interrupted by the test person/patient at any time without giving 
reasons. For this purpose, the MRI provides a so-called bell button. The activation of this button 
gives a signal to the doctor/examiner, helping the patient to draw attention to himself/herself 
even while the measurement is in progress.  
In addition, between the individual measurements/examinations, the patient is in contact with 
the doctor/examiner via microphone and headphones and can thus also verbally request the 
abortion of an examination. 
Statement of medical justifiability 
The risk of occurrence of undesired events is considered as extremely low, especially in regard 
of previous experiences made in children with Duchenne muscular dystrophy and in healthy 
volunteers. No serious event has been reported so far, neither at our site nor in the literature. 
The majority of the reported (foreseeable) problems were related either to the use of ultrasound 
gel for examination or to the necessity to wear eye protection. The use of filter glass also 

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explains the reported vision redness. This phenomenon was reversible within seconds. Table 
1 shows the reported adverse events from our study (MSOT_DMD, 67_18 B). 
 
Table – Adverse events 
In this study no central organs are examined, but only extremities are measured - this leads to 
a further significant reduction of a possible residual risk. Likewise, contrast agents are not 
applied during any examination. 
Particularly in light of the background of completely new therapeutic approaches, we hope that 
this method will provide us with a child-friendly, non-invasive diagnostic tool for the treatment 
of these complex diseases.  
 
 
 
Muscular dystrophy 
Duchenne N=10 
Healthy volunteer 
N=10 
Reversible adverse events- no.(%) 
 
 
     Pressure of safety goggles 
2 (20%) 
 
     Coolness of Ultrasound-gel 
 
4 (40%) 
     Red cast view 
 
1 (10%) 
Serious adverse events- no.(%) 
0 (0%) 
0 (0%) 

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Biometrics 
Explorative study: explanation of the statistical methodology, justification of the 
selected number of cases  
 
Case number calculation: 
As this is a pilot study and no information on the expected differences between the different 
groups is available yet, no case number calculation was performed. The number of cases given 
represents an estimate or is within the appropriate range for a pilot study.  
Statistical methods: 
Continuous variables are given as mean values with standard deviation, categorical variables 
as numbers with percentages if necessary. The MSOT parameters are compared using a two-
sided, unpaired t-test with the same deviations. If the standard deviation is unequal, a 
correction according to Welch may be applied. Furthermore, ROC analyses (Receiver 
Operator Characteristics) between patients with and without therapy are planned. Genetics 
serve as the gold standard. Correlations are indicated by the Pearson coefficient. All statistical 
tests are performed on both sides and a p-value of <0.05 is considered statistically significant. 
All analyses are performed using GraphPad Prism (version 7.00 or later, GraphPad Software, 
La Jolla, CA, USA), RStudio (version 1.1.456 or later, RStudio Inc., Boston, MA, USA) or IBM 
SPSS Statistics (version 24 or later, IBM Corp., Armonk, NY, USA). 
 
 

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Data management and data protection 
Data acquisition and storage  
All raw data, such as patient files, are source documents. Their availability is ensured for 
routine monitoring. The participation of the individual patients or test persons in the study is 
documented. The study leader maintains an independent list for the identification of the 
participating patients. This list containes the names and date of birth as well as the date of 
examination and pseudonymisation codes of the patients and subjects. The study leader is 
responsible for the quality of data collection and storage. The data storage (complete data) 
takes place on computers or specially designed network drives of the University Hospital 
Erlangen. The raw imaging data (no patient-related data) are stored on dedicated servers of 
iThera Medical GmbH. 
Pseudonymisation 
Prior to a scientific analysis of the materials and data of this study, all information will be 
pseudonymized according to the guidelines of the Federal Data Protection Act. 
Data transfer 
In this study, data transfer is only intended for the MSOT raw data. The company iThera 
Medical GmbH will work with this data to ensure adequate recording quality and to develop 
algorithms for evaluation. The data will only be passed on pseudonymously on encrypted 
physical drives. The data will not be used for later approval of the prototype used. The 
cooperation is explicitly regulated in advance by the legal department (an additional agreement 
to the service contract can be found in the appendix). 
The study results can be published anonymously, whereby it will not be possible to draw 
conclusions about the identity of the participating persons. The data will be kept for 10 years 
and then destroyed.  
Revocation, data deletion 
If the declaration of consent is revoked, data collected up to this point can be taken into 
account. The patient has the right to demand that the data be destroyed, provided that legal 
provisions do not prevent such destruction. 
 

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Handling of biomaterials 
No biomaterials are obtained.  
 
Insurance 
The participants of the study are insured via the group contract of the CCS Erlangen. 
 
 
 

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Signatures 
 
 
 
Dr. med. Ferdinand Knieling 
Study director 
Prof. Dr. med. Regina Trollmann 
Study director 
 
 
 
Prof. Dr. med. Michael Uder 
Study director 
 
 
 
Prof. Dr. rer. nat. Armin Nagel 
Study director 
 
 
 
Dr. med. Matthias Türk 
Study director 
 
 
 
Prof. Dr. med. Maximilian Waldner 
Technical implementation