# Drug Candidate BIO101 for Spinal Muscular Atrophy as Monotherapy or Combined With the Antisense Oligonucleotide ASO-10-27

Cynthia Bézier, Steve Cottin, Parvin Nazari Hashemi, Mirella El Khoury, Zoé Clerc, Christine Balducci, Delphine Sapaly, Laure Weill, René Lafont, Stanislas Veillet, et al.

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Cynthia Bézier, Steve Cottin, Parvin Nazari Hashemi, Mirella El Khoury, Zoé Clerc, et al. Drug Candidate BIO101 for Spinal Muscular Atrophy as Monotherapy or Combined With the Antisense Oligonucleotide ASO-10-27. Journal of Cachexia, Sarcopenia and Muscle, 2025, 16 (5), pp.e70104. 10.1002/jcsm.70104. hal-05453886

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ORIGINAL ARTICLE

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# Drug Candidate BIO101 for Spinal Muscular Atrophy as Monotherapy or Combined With the Antisense Oligonucleotide ASO-10-27

Cynthia Bézier<fnref n="1" />,² | Steve Cottin² | Parvin Nazari Hashemi² | Mirella El Khoury<fnref n="1" /> | Zoé Clerc<fnref n="1" /> | Christine Balducci² |
Delphine Sapaly<fnref n="1" /> | Laure Weill<fnref n="1" /> | René Lafont²,³ | Stanislas Veillet² | Pierre J. Dilda² | Frédéric Charbonnier<fnref n="1" /> |
Mathilde Latil² ID | Olivier Biondi<fnref n="1" />,⁴ ID

**Correspondence:** Mathilde Latil (mathilde.latil@biophytis.com) | Olivier Biondi (olivier.biondi@univ-evry.fr)

**Received:** 24 March 2025 | **Revised:** 8 July 2025 | **Accepted:** 7 September 2025

**Funding:** This work was essentially supported by Biophytis funding. This work was also supported by INSERM and Université Paris Cité fundings.

**Keywords:** 20-hydroxyecdysone | combinatorial therapy | motor performance | motor units | mouse models | spinal muscular atrophy

## ABSTRACT

**Background:** Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of survival of motor neuron (SMN) protein inducing progressive muscle weakness and atrophy due to motor neurons degeneration. Despite benefits of SMN restoration therapies in patients, motor defects are still persistent. We investigated the potential of BIO101, a new drug candidate promoting muscle growth by activating the protective arm of the renin-angiotensin system through the MAS receptor, as monotherapy or in combination with the SMN-based therapy ASO-10-27 (Nusinersen).

**Methods:** BIO101 was administrated daily on severe or mild Taiwanese SMA mouse models or diluted in culture medium of SMA patient-derived myoblasts. The BIO101 effects were evaluated on severe SMA mouse model in vivo (growth, survival and motor function), ex vivo (motor neuron, neuromuscular junction maturation, skeletal muscle phenotype) and on muscle SMN expression, while motor function effects were evaluated on mild SMA mouse model. The in vitro effects on proliferation, differentiation, metabolism and SMN expression of SMA patient-derived myoblasts were analysed. Effects of the combination of BIO101 with ASO-10-27 were evaluated on severe SMA mouse model, in vivo and on tissular intracellular AKT signalling and SMN expression.

**Results:** In severe SMA mice, BIO101 alone protected lateral motor neurons (+20%, *p*<0.05), limited muscular atrophy (+30%, *p*<0.01), accelerated maturation of muscular fibres (+70% for fast-twitch muscles) and neuromuscular junctions (+50% of perforated clustering, *p*<0.05) with more prominent effects on fast-twitch muscles. Those adaptations led to an improvement of muscular function, significant at 7, 9 and 10days post-natal (+2-fold for crossed squares and time of suspension, *p*<0.01), which was also observed in mild SMA mice at 8 and 9months of age (*p*<0.01). Interestingly, BIO101 treatment also improved SMA patient-derived myoblast differentiation (+20% myotube diameter and nuclei/myotube, *p*<0.05) and anaerobic performances (ECAR, + 10%; *p*≤0.05) without any impact on the proliferative state and aerobic capacities through MAS receptor activation. All BIO101 effects were independent of SMN protein expression. When combined with the ASO-10-27, BIO101 enhanced even more muscle resistance to fatigue (> 3-fold

<footnote n="1">¹Université Paris Cité & INSERM UMR-S1124, Paris Cedex 06, France | ²Biophytis, Sorbonne Université, Paris Cedex 05, France | ³Sorbonne Université, Institut de Biologie de Paris-Seine (BIOSIPE) & CNRS, Paris, France | ⁴Laboratoire de Biologie de l'Exercice pour la Performance et la Santé (LBEPS), UMR, Université d'Evry, IRBA, Université de Paris Saclay, Evry-Courcouronnes, France</footnote>

<footnote>Mathilde Latil and Olivier Biondi contributed equally to the work.</footnote>

<footnote>This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>

<footnote>© 2025 The Author(s). *Journal of Cachexia, Sarcopenia and Muscle* published by Wiley Periodicals LLC.</footnote>
over 27 days for time of suspension, *p* < 0.05) when compared with severe SMA mice treated with ASO-10-27 alone, without effects on survival through the activation of AKT intracellular pathway and independently of SMN protein expression.

**Conclusions:** We showed that BIO101 constitutes an efficient SMN-independent therapy to improve muscle performance in SMA, which could open new therapeutic avenues for patients in combination with SMN-based therapies, or as monotherapy for less severe forms.

# 1 | Introduction

Spinal muscular atrophy (SMA) is a neuromuscular disorder that stands as the leading genetic cause of death in childhood, affecting approximately 1 in 10 000 newborns [1]. The clinical phenotype encompasses a wide spectrum of severities, categorized in four types based on the age of onset and the motor symptoms. These range from type I, the most prevalent and severe form, to the adult form, the type 4 [2, S1]. SMA is caused by homozygous loss-of-function mutations of the telomeric *Survival of Motor Neuron 1* gene (*SMN1*) coding for the ubiquitously expressed SMN protein [3]. While patients possess at least one copy of the paralog gene *SMN2*, approximately 90% of its transcripts lack exon 7, resulting in a truncated and unstable SMNΔ7 protein [4, S2, S3]. Only 10% of the *SMN2* transcripts encode full-length SMN protein, enabling viability but not fully compensating for the lack of *SMN1*. Thus, variation in genomic *SMN2* copy number and expression is inversely correlated to SMA severity [5].

The discovery of this genetic context has recently led to the approval of three therapies focusing on increasing SMN expression. These include (1) modulation of *SMN2* mRNA splicing using antisense oligonucleotide (ASO) (Spinraza, synonyms: nusinersen, ISIS 396443, ISIS-SMN<sub>Rx</sub>, ASO-10-27) [6], (2) small molecule (Evrysdi, risdiplam) [7] and (3) viral vector-based gene therapy to compensate the mutated *SMN1* gene (Zolgensma, onasemnogene abeparvovec) [8]. While these therapies demonstrate significant improvements in survival and motor function, none are considered curative. Their clinical efficacy varies depending on the therapeutic modality, as well as the age and severity of the patients [9, 10, S4, S5]. This supports the need to develop novel therapeutic strategies targeting residual pathophysiological alterations and persistent neuromuscular defects that cannot be fully reversed by improving SMN expression alone [11, S6]. Most of the new approaches under development are targeting the muscle, aiming to increase muscle mass through myostatin inhibition [12, S7-S9] or to improve muscle contractility using a fast skeletal muscle troponin activator [13]. However, these approaches do not comprehensively address the muscular function in its entirety, from the muscle itself to its environment, nor the broader multi-tissue and metabolic dimensions. In SMA context, several alterations of the muscular metabolism have been reported, including glycolysis, mitochondrial function and vasculature defects, which contribute directly to muscle wasting, fatigue and disease severity. This highlights that metabolic dysregulation in muscle is both a consequence of SMN deficiency and a potential therapeutic target in SMA. To date, no drug candidates targeting muscle metabolism and function have been approved for the treatment of SMA.

In this context, 20-hydroxyecdysone (20E), the most prevalent phytoecysteroid, emerges as promising candidate to positively impact the SMA disease progression. Indeed, this molecule has a

broad spectrum of pharmacological effects in mammals [14], such as stimulation of muscle anabolism [S10], promotion of angiogenesis [S11] and modulation of energetic metabolism [S12, S13], all of which are affected in SMA and play crucial roles in muscle function [15, 16, S14]. 20E activates the MAS receptor, a key component of the renin-angiotensin system, eliciting a rapid Ca²⁺ flux leading to AKT activation and increased protein synthesis in skeletal muscle cells [17, S15]. BIO101, a pharmaceutical-grade preparation of 20-hydroxyecdysone (> 97% purity), has been developed and demonstrated a favourable safety profile in adults, as well as beneficial effects on mobility in sarcopenic patients [18].

This study utilized myoblasts from type 2 SMA patients and severe (*Smn*Δ⁷/Δ⁷; *tgSMN2*²⁰⁺, two copies) and mild (*Smn*Δ⁷/Δ⁷; *tgSMN2*²⁰⁺, four copies) Taiwanese SMA mouse models [19] to investigate BIO101 effects from molecular and cellular to behavioural levels. Severe Taiwanese SMA mouse model is characterized by critical defect in growth and motor function leading to death at around 12 days post-natal, while mild Taiwanese SMA mouse model is characterized by nonlethal distal necrosis and progressive motor neurons death (from 6 months of age) and motor defect (from 9 months of age). The results indicate that BIO101 monotherapy exerts beneficial effects on myoblasts from SMA patients, as well as on the entire motor unit of severe SMA mice (*Smn*Δ⁷/Δ⁷; *tgSMN2*²⁰⁺), leading to an enhancement of muscle function. These effects are more pronounced in mild SMA mice (*Smn*Δ⁷/Δ⁷; *tgSMN2*²⁰⁺). Interestingly, when combined with a single intrarebroventricular injection of ASO-10-27, BIO101 considerably improves muscle function of severe SMA mice without significantly affecting others in vivo parameters. All BIO101-induced benefits were independent of SMN expression, despite the expected activation of the AKT intracellular signalling. Altogether, these findings collectively support the consideration of BIO101 as a prime candidate for a wide application in SMA patients.

# 2 | Materials and Methods

## 2.1 | BIO101

BIO101, the Good Manufacturing Practice drug substance at pharmaceutical-grade of 20-hydroxyecdysone (≥ 97%), was from Patheon (Regensburg, Germany). For mice treatment, BIO101 was complexed with (2-hydroxypropyl)-β-cyclodextrin) (H107, Sigma).

## 2.2 | Mice and treatments

Animal handling and experimentation were performed in line with approved Institutional Animal Care and Use Committee protocols at the Paris-Cité University (CEEA 34, agreement number C750607), following the national authority guidelines
(Ministère de la Recherche et de la Technologie, France) on
ethical treatment of laboratory animals based on European
Union Directive 2010/63/EU (APAFIS#26872-2019062115 56
4717 v7). Transgenic mild Taiwanese SMA males (SmnΔ7/Δ7;
tgSMN2⁺⁺⁺, FVB.Cg-Smn1t<sup>tm1Hung</sup> Tg (SMN2)2Hung/J, strain
#005058) [19] were obtained from the Jackson Laboratory and
were crossed with either heterozygous Smn knock-out females
(SmnΔ7/⁺), for generating severe Taiwanese SMA (FVB/NRj-
SmnΔ7/Δ7; tgSMN2₀/⁺) and control mice (FVB/NRj-Smn⁺⁺/Δ7;
tgSMN2₀/⁺), or with heterozygous Smn knock-out females
having four copies of the human SMN2 transgene (FVB/NRj-
SmnΔ7/⁺; tgSMN2⁺⁺/⁺), for generating mild Taiwanese SMA
(FVB/NRj-SmnΔ7/Δ7; tgSMN2⁺⁺/⁺) and control mice (FVB/NRj-
SmnΔ7/⁺; tgSMN2⁺⁺/⁺).

The entire litter of severe SMA mice was assigned to one group of treatment at birth, but litters from the same female were systematically assigned to different groups of treatment. For mild Taiwanese SMA mice, animals were randomly assigned to different groups without sex discrimination. Severe Taiwanese SMA mice received either 50 mg/kg/day of BIO101 or the vehicle cyclodextrin (VH) in tap water by oral gavage from P0. When co-treated, at P0, they received in addition a single intracerebroventricular injection, as previously described [20], of either 8 µg of ASO-10-27 (5'-TCACTTTCATAATGCTGG-3') or six-mismatch control (5'-TTAGTTTAATCACGCTCG-3') produced by Eurogentec as previously described [21, S16]. Mild Taiwanese SMA mice received either only water (VH) or 50 mg/kg/day of BIO101 directly diluted in the water feeder from 6 to 9 months of age. For all molecular and tissular analyses, mice were anaesthetised at 10.5 days or 9 months of age by pentobarbital administration (EXAGON®) at 1 µL/g 1 h after the last BIO101 or VH treatment.

**2.3** | Motor Behaviour Assays

The open-field and grip tests were performed as previously de-
scribed [22], and details of these methods are described in the
*Supplemental Methods*.

## 2.4 | NMJ, Motor Neuron and Muscle Histology

For neuromuscular junction and motor neuron analysis, dis-
sected tissues were fixed 24h in 4% paraformaldehyde, rinsed
in PBS and cut in free-floating sections with a vibratome
(75-µm-thick for muscles, 50-µm-thick for spinal cord; VT-
1000S, Leica Microsystems SAS). For muscle histology and ty-
pology and for spinal cord vascularization analysis, dissected
tissues were individually frozen into -80°C isopentane solution,
and then sectioned in a cryostat (CM3050 S, Leica Microsystems
SAS) into 10-µm-thick sections. The haematoxylin and eosin
(HE) staining of muscle fibres was performed as previously de-
scribed [22]. The detailed methods for immunohistochemistry
analysis and the antibodies used are described in the Tables S1
and S2 of *Supplemental Methods*.

All images were acquired with a Zeiss Z1 AxioObserver mi-
croscope (ZEN 2.3 software) using an ORCA Fusion Camera
(C14440-20UP, Hamamatsu) for fluorescent images and an RGB

AxioCam ICC 1 camera (Zeiss) for colorimetric images. All anal-
yses were done using the ImageJ software (National Institutes of
Health).

2.5 | High-Performance Liquid Chromatography (HPLC)

BIO101 was quantified in plasma collected intracardially the
day of sacrifice after liquid chromatography separation on a
1260 Infinity LC system (Agilent technologies) followed by
quantitative mass spectrometry performed on a QQQ-6420
MS/MS (Agilent Technologies) with electrospray positive ion-
ization (API-ES). Details of these methods are described in the
*Supplemental Methods*.

**2.6 | Protein Extraction and Western Blot Analysis**

Frozen tissues were dissolved in blending buffer using metal
beads (69 989, Qiagen) and a TissueLyser MM300 (Qiagen).
Protein samples (30 µg) were mixed with Laemmli sample buffer
(161-0747, Biorad), denatured at 95°C, and electrophoresed on a
12.5% polyacrylamide gel. Samples were transferred to nitrocel-
lulose membranes using the Trans-Blot Turbo transfer system
(1 704 158 and 1 704 150, Biorad). The blot was blocked in 5% milk
powder in PBS with 0.1% Tween-20 for 1 h, then incubated with
primary antibodies (Table S3) diluted in TBS with 0.1% Tween-20
and 5% bovine serum albumin (24 h, 4°C). After washes, the
blot was incubated with secondary antibodies (Table S3) di-
luted in TBS with 0.1% Tween-20 and 5% bovine serum albu-
min (1 h, RT). Revelation was performed using SuperSignal
West Pico PLUS chemiluminescent substrate (34 577, Thermo
Fisher Scientific) and imaged using ImageQuant LS4000 (GE
Healthcare Bio-Science).

**2.7 | Culture Cell, Treatment and Metabolic Assay**

Human immortalized myoblasts from one type 2 SMA patient
were provided by Institute of Myology (Paris) under a non-
profit-to-non-profit uniform biological material transfer agree-
ment (UBMTA). Cells were cultured on 0.01% gelatin-coated
flasks in a growth medium (GM; Table S3) at a seeding density
of 5 × 10³ cells/cm. For proliferation assay, GM was implemented
with Hoechst 33342 (1 µg/mL, H1399, Thermo Fisher Scientific)
and cells were automatically scored using the ImageXpress Pico
(Molecular Devices) before and after 48 h of treatment with
BIO101 (5 µM), the Mas receptor antagonist A779 (PP-H6705-00,
Bio-Techne; 10 µM) or both.

For differentiation, cells were plated at 40×10³ cells/cm on
Matrigel (356 237, Corning BV) in GM, and when cells reached
~90% confluency, medium was switched to differentiation
medium (DM; Table S3) with BIO101 (5 µM), A779 (10 µM) or
both. Cells were either tested for the metabolic assay at 2 days
of differentiation using Seahorse XFe24 FluxPaks (102 340-
100, Agilent Technologies) and XF Cell Mito Stress Test
(103 015-100, Agilent Technologies) according to manufactur-
er's instructions (Table S5), or fixed (4% paraformaldehyde,
20 min, RT; 10 099 464, VWR) at 2 or 6 days of differentiation,
permeabilized (0.5% Tween-20 in TBS, 30 min, RT) and stained
with anti-MF20 antibody (1:40, DSHB; 0.5% Tween-20 and 8%
donkey serum in TBS, 1 h, 4°C), Hoechst 33342 and donkey anti-
mouse Alexa Fluor 488 antibody (1:400, 715-545-150, Jackson
ImmunoResearch; 0.5% Tween-20 and 4% donkey serum in TBS,
1 h, RT) for assessing myotube diameter and nuclei number per
myotube.

Additional details are presented in the Supplemental Methods.

## 2.8 | Statistical Analysis

Following the instructions from ARRIVE2.0 guideline and the
national ethical recommendation, we determined the minimum
number of animals per group needed to achieve 85% of statistical
power using the G*Power 3.1.9.7 calculator with muscle capacity
as outcome measure for in vivo evaluations, and the myofiber
cross-section-area for ex vivo evaluations. For survival, Kaplan-
Meier survival curves were generated and followed by the
log-rank test for statistical significance. Following recommen-
dations, in vivo data are represented as mean ± standard error of
the mean, while ex vivo data are represented as mean ± standard
deviation. For multiple group comparisons, statistical signifi-
cance was determined using a Kruskal–Wallis test with post hoc
Dunn's test or a two-way ANOVA with post hoc Sidak test or a
mixed model of repeated measures with post hoc Sidak test. For
two group comparisons, statistical significance was determined
with nonparametric Mann–Whitney test or t-test according to
normal distribution of data evaluated by Kolmogorov–Smirnoff
test. Statistical analyses and graphs were performed with the
GraphPad Prism version 8.

## 3 | Results

### 3.1 | BIO101 Chronic Treatment Improves Muscle Function in Severe and Mild Taiwanese SMA Mouse Models

Based on a previous study, the bioavailability of the phar-
maceutic grade preparation of 20-hydroxyecdysone (> 97%),
BIO101, was assessed after a single bolus at 50 mg/kg in post-
natal mice at 10.5 days of age, the late-symptomatic stage in
severe Taiwanese SMA mouse model (Figure 1A). We noted
a progressive decay in its plasmatic concentration until 18 h
post-treatment, confirming the need of daily treatment with
BIO101.

On Taiwanese SMA mouse model, we noted a negligible benefit
on growth, notably from P6 to P10, and a nonsignificant shift
of the final drop beyond P12 in BIO101-treated severe SMA
mice when compared with vehicle-treated severe SMA mice
(Figure 1B). Consistently, we observed a clinically nonrelevant
gain in survival, with average survival increase by 2.5 days in
BIO101- (13 days) compared with vehicle-treated SMA mice
(10.5 days; log-rank *p* = 0.0133; Figure 1C).

However, BIO101 treatment increased moving capacities (open
field) with significant differences at P7 and P9 ($p \le 0.05$), and

even more the resistance to muscular fatigue (grip test) with sig-
nificant differences at P7 and P10 ($p \le 0.05$) in severe SMA mice
(Figure 1D,E), and a marked improvement over P11.

We next determined if the motor-specific effect of BIO101 could
also be observed in the adult mild Taiwanese SMA mouse
model (Smn$^{Δ7/Δ7}$; tgSMN2$^{+/+}$). When treated daily during the
symptomatic phase from 6 to 9 months of age [23, S18], we ob-
served a constant improvement in time of suspension only for
BIO101-treated mild SMA mice (Figure 1F). Collectively, our
results showed that in Taiwanese SMA mouse models, BIO101
treatment can specifically enhance muscular performances on a
wide range of severities and ages, even when administered after
the onset of motor defects.

### 3.2 | BIO101 Protects Motor Neurons and Improves Spinal Cord Vascularization in Severe Taiwanese SMA Mouse Model.

The specific enhancement of motor function with BIO101 treat-
ment in severe and mild Taiwanese SMA mouse models led us to
question the neuroprotection of different populations of motor
neuron (MN). BIO101 treatment significantly limited the loss of
absolute lateral MNs (+19%; $p \le 0.05$; Figure 2A,B) in BIO101-
treated severe SMA mice compared with vehicle-treated severe
SMA mice, with a significant decrease in the percentage of
small MNs ($< 300 \mu m^2$; Figure 2C). No effect of BIO101 treat-
ment was observed on absolute number and size of MNs for con-
trol mice (Figure S1A,B). We further quantified the proportion
of double-positive immunostained cells for choline acetyltrans-
ferase (ChAT) and either oestrogen-related receptor $\beta$ (ERR$\beta$)
or matrix metallopeptidase 9 (MMP9), known to be markers of
slow and fast MNs, respectively [24, S19]. We noted a significant
decrease in the percentage of MMP9+ MNs ($-20%; p \le 0.05$) as-
sociated with a significant increase in the percentage of ERR$\beta$+
MNs (+20%; $p \le 0.05$) in untreated severe SMA mice when com-
pared with control mice, suggesting a fast-specific degeneration
in this model. A complete restoration of the proportion of fast
over slow MNs was observed in BIO101-treated severe SMA
mice (Figure 2D,E) without any change in the total amount of
slow MNs (Figure S1C), suggesting a specific neuroprotection of
fast MN population.

In control mice, if BIO101 treatment did not modify the propor-
tion of ChAT+ MMP9+ (Figure S1D), it induced a significant
decrease in the proportion ($-23%; p \le 0.05$) of ChAT+ ERR$\beta$+
cells (Figure S1E), suggesting an increase of a ChAT+ ERR$\beta$-
MMP9- cells subpopulation after treatment. Importantly, those
neuronal effects of BIO101 are associated with a significant
limitation of the defect in lumbar spinal cord vascularization,
assessed by the surface quantification occupied by vessels in spi-
nal cord section, observed in vehicle-treated severe SMA mice
($+50%; p \le 0.05$; Figure 2F,G), without any effect in control mice
(Figure S1F).

All those results support the hypothesis that BIO101 has a
specific effect on MN and can protect sensitive and fast MN
subpopulations in severe Taiwanese SMA mouse model, pos-
sibly through the improvement of the lumbar spinal cord
vascularization.
<figure><img src="image_2.png" /><figcaption>(B)</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>(C)</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>(D)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>(E)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>(F)</figcaption></figure>

<figure><img src="image_7.png" /></figure>

**FIGURE 1** | Effects of BIO101 on severe and mild Taiwanese SMA mouse phenotypes. (A) Evaluation of BIO101 plasma concentration of control mice after a single oral gavage with 50 mg/kg of BIO101 at P10.5 (n=6 at 1 h; n=8 at 6 h; n=3 at 10 h; n=3 at 18 h). (B) Weight curves of vehicle- or BIO101-treated severe SMA mice (n=25 and 19 mice respectively). (C) Survival curves of vehicle-treated (n=13 mice, median survival of 10 days) or BIO101-treated (n=11 mice, median survival of 10 days) severe SMA mice. Longitudinal recording of the total number of squares crossed (open-field test, to evaluate moving capacity) (D) by vehicle- or BIO101-treated severe SMA mice and of the maximal time of hind-limb suspension (grip-test, muscle fatigue) of vehicle- or BIO101-treated severe (n=25 and 16 mice respectively) (E) and mild (n=18 and 15 mice respectively) (F) SMA mice. Data are represented as mean ± SEM with \*p\<0.05 and \*\*p\<0.01 by (B, D and E) unpaired t-test at each age or (C) log-rank test or (F) mixed model of repeated measures with a Sidak post hoc tests.

## 3.3 | BIO101 Preferentially Improves the Maturation of Fast Muscles and Their NMJ in Severe Taiwanese SMA Mouse Model

We then evaluated the muscle-specific adaptations of the neuromuscular maturation, known to be a critical SMA feature linked to disease progression and functional defects [25, 26, S6, S21]. As previously described, we compare three differently affected muscles of the calf [S22]: the fast-twitch flexor *tibialis*, fast-twitch extensor *plantaris* and slow-twitch extensor *soleus* muscles and evaluated BIO101 effects on

neuromuscular junctions (NMJ) and muscle maturation of these three muscles.

For NMJ, only innervated NMJs were analysed as shown in the *tibialis* of vehicle-treated control mice (CV) compared with vehicle (SV)-or BIO101-treated (SB) severe SMA mice (Figure 3A). BIO101 treatment limited the delay of maturation of NMJs with a significant increase in the proportion of the perforated clustering [+42% (*p* ≤ 0.05) in the *tibialis*, +160% (*p* ≤ 0.05) in the *plantaris*, and +56% (*p* ≤ 0.001) in the *soleus*] and a concomitant decrease in the proportion of uniform clustering (Figure 3A,B)
<figure><img src="image_8.png" /><figcaption>**FIGURE 2** | Effects of BIO101 on spinal cord in severe Taiwanese SMA mouse model. (A) Immunofluorescence directed against choline acetyl-transferase (ChAT; motor neuron marker, white), oestrogen-related receptor β (ERRβ; slow-motor neuron marker, green) and matrix metallopeptidase 9 (MMP9; fast-motor neuron marker, red; scale bar, 50 µm; 200× magnification) on 50-µm-thickness cross-sections of spinal cord from vehicle-treated control compared with vehicle- and BIO101-treated severe SMA mice. (B and C) Quantification of the total number of ChAT+ cells depending on their localization (B), and of the distribution of their soma area (C, *n* = 5 mice in each group). (D and E) Quantification of the percentage of MMP9+ ChAT+ (D, *n* = 5 vehicle-treated control mice, *n* = 6 mice in other groups) or ERRβ+ ChAT+ (E, *n* = 7 BIO101-treated severe SMA mice, *n* = 6 mice in other groups) cells over total ChAT+ cells, in 50-µm-thick slice of lumbar spinal cord of vehicle-treated control mice compared with vehicle- or BIO101-treated severe SMA mice at P10.5. (F) Immunofluorescence of CD31 and quantitative analysis of the capillary density in 10-µm-thick slice of lumbar spinal cord of vehicle-treated control mice (*n* = 4) compared with vehicle- or BIO101-treated severe SMA mice at P10.5 (*n* = 4 and *n* = 5 mice respectively; scale bar, 20 µm; 200× magnification). Data are represented as mean ± SD with *p* < 0.05; **p* < 0.01, ****p* < 0.0001 for a comparison between the two indicated groups or with vehicle-treated control mice by (B, D, E and F) unpaired nonparametric Mann–Whitney tests or (C) two-way ANOVA with a Sidak post hoc tests.</figcaption></figure>

in severe SMA mice. No significant difference was observed in BIO101-treated control mice despite muscle-specific trends (Figure S2A).

For myofibers, we analysed the proportion of embryonic Myosin Heavy Chain isoform (MyHC, embryonic fibres) (Figure 3C,D) in the same muscles. We observed that BIO101 limits the expected delay in maturation of both fast-twitch *tibialis* (–92%; *p* ≤ 0.01) and *plantaris* (–73%; *p* ≤ 0.01) muscles of severe SMA mice (Figure 3C,D), without any modification in

the slow-twitch *soleus* muscle (Figure 3D). In control mice, BIO101 treatment unexpectedly increased the percentage of embryonic MyHC positive fibres only in the fast-twitch *tibialis* and *plantaris* muscles (+32% in the *tibialis* and +17% in the *plantaris*; Figure S2B).

Thus, we noted context-specific effects of BIO101 accelerating the maturation of the NMJ and myofibers in SMA muscles and favouring immature neuromuscular junctions and myofibers in control muscles.
<figure><img src="image_9.png" /><figcaption>**FIGURE 3** | Effects of BIO101 on neuromuscular junction and muscle maturation in severe Taiwanese SMA mouse model. (A and B) Immunofluorescence of neurofilament L (NF-L) and synaptosomal-associated protein 25 (SNAP-25) to label the pre-synaptic motor nerve terminals (green), and with α-bungarotoxin to label post-synaptic side of neuromuscular junction (NMJ; red; scale bar, 20 µm; 200× magnification) (A) and quantification of the percentage of uniform and perforated NMJ over total NMJ (B) in the *tibialis*, *plantaris* and *soleus* muscles of vehicle-treated control mice compared with vehicle- or BIO101-treated severe SMA mice at P10.5 (n = 5 mice in each group). (C and D) Immunofluorescence of embryonic myosin heavy chain (MyHC; scale bar, 20 µm; at 200× magnification) (C) and quantitative analysis of their percentage over total number of myofibers (D) in the *tibialis*, *plantaris* and *soleus* muscles of vehicle-treated control mice compared with vehicle- or BIO101-treated severe SMA mice at P10.5 (n = 5 mice in each group). Data are represented as mean ± SD with \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.0001 for a comparison between the two indicated groups or with vehicle-treated control mice; and \#p < 0.05, \#\#p < 0.01, and \#\#\#p < 0.001 for a comparison with vehicle-treated severe SMA mice by (B) two-way ANOVA with a Sidak post hoc tests or (D) unpaired nonparametric Mann-Whitney tests.</figcaption></figure>

## 3.4 | BIO101 Preferentially Improves Fast-Muscle Phenotype and Vasculature in Severe Taiwanese SMA Mouse Model Through SMN-Independent Mechanism

Then, we investigated whether BIO101-induced improvements in motor neuron survival and neuromuscular maturation could be associated with muscular adaptations.

At P10.5, we observed a significant limitation of muscle atrophy (MA) in all studied muscles of severe SMA mice (Figure S3A and Figure 4A), with a concomitant enhancement of the variation among fibre sizes (standard deviation of fibre size per muscle, VCSA) (Figure S3A and Figure 4B). Interestingly, those effects were more prominent on the fast-twitch tibialis (MA + 20%, VCSA +30%, $p \le 0.01$) and *plantaris* (MA + 33%, VCSA +30%, $p \le 0.01$) muscles than on the slow-twitch *soleus* (MA + 13%, VCSA +15%, $p \le 0.05$) muscle (Figure S3A and Figure 4A,B). Moreover, in control mice, BIO101 treatment induced a hypertrophy and increase in VCSA in the fast-twitch *tibialis* muscle only (Figure S3B,C).

In addition, BIO101 treatment significantly increased the total number of fibres per muscle only in the *tibialis* of severe SMA (+11%; $p \le 0.01$; Figure 4C) and control mice (+20%; $p \le 0.01$;

Figure S3D), but failed in limiting SMA-induced aplasia in the *plantaris* and the *soleus*; Figure 4C).

Furthermore, we looked at the muscle typology adaptations in response to treatment by quantifying the proportion of adult MyHC isoforms. If BIO101 treatment did not restore the altered proportion in slow-twitch type I, intermediate type IIa and fast type IIx/D-IIb positive myofibers observed in vehicle-treated severe SMA mice compared with control mice, the treatment induced fast-twitch muscle-specific adaptations in both control and severe SMA mice (Figure 4D), without any modification of the slow-twitch *soleus* (Figure 4D; Figure S3E).

Our results suggest that muscular BIO101 effects favour growth, through the increase in myofiber diameter and/or number according to the cellular context and facilitate intermediate and fast muscle typology whatever the conditions.

Interestingly, those muscular adaptations with BIO101 treatment were associated with a significant limitation of the vascular alteration, assessed by the number of vessels per myofiber, observed in vehicle-treated severe SMA mice with more prominent effects in the fast-twitch muscles [+29% ($p \le 0.05$) in the *tibialis*, and +58% ($p \le 0.01$) in the *plantaris*] than in
<figure><img src="image_10.png" /><figcaption>(B)</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>(C)</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>(D)</figcaption></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>(E)</figcaption></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>(F)</figcaption></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

**FIGURE 4** | Effects of BIO101 on muscle phenotype in severe Taiwanese SMA mouse model. (A, B and C) Quantitative analysis of the mean of cross-sectional area of fibres (A), their standard deviation (B) and the total number of fibres (C) in the *tibialis*, *plantaris* and *soleus* muscles of vehicle-treated control mice compared with vehicle- or BIO101-treated severe SMA mice at P10.5 (n=5 mice in each group). (D) Immunofluorescence of laminin and adult type 1 (blue), type 2A (red) and type 2X/2B (white) isoforms of Myosin Heavy Chain and quantitative analysis of their percentage over the total number of myofibers in the *tibialis*, *plantaris* and *soleus* muscles of vehicle-treated control mice (CV) compared with vehicle- (SV) or BIO101-treated (SB) severe SMA mice at P10.5 (n=5 mice in each group). (E) Immunofluorescence of CD31 and laminin and quantitative analysis of the number of vessels per muscle fibre in the *tibialis*, *plantaris* and *soleus* muscles of vehicle-treated control mice compared with vehicle- or BIO101-treated severe SMA mice at P10.5 (n=5 mice in each group) (scale bars, 20 µm; 200× magnification). (F) Western Blot analysis and quantification of SMN expression in the hindlimb *tibialis*, *plantaris* and *soleus* muscles of vehicle-treated control mice compared with vehicle- or BIO101-treated severe SMA mice (n=6 for *tibialis* and *plantaris*; n=4 for *soleus*; Lanes were run on the same gel but were noncontiguous). Data are represented as mean±SD with \*p\<0.05 and \*\*p\<0.01 for a comparison between the two indicated groups or with vehicle-treated control mice, and \#p\<0.05 for a comparison with vehicle-treated severe SMA mice by (A, B, C, E and F) unpaired nonparametric Mann-Whitney tests or (D) two-way ANOVA with a Sidak post hoc tests.
<figure><img src="image_20.png" /></figure>

**FIGURE 5** | Effects of BIO101 on SMA patient-derived myoblasts. (A) Quantification of the total number of cells before and 48 h after the treatment with BIO101 ± the specific inhibitor of MAS-receptor (A779) in a culture of myoblasts derived from type 2 SMA patient in proliferation medium. (B) Immunofluorescence directed against the Myosin Heavy Chain common sequence to all isoforms (MF20, green) and DNA staining (Hoechst, blue) and quantitative analysis of myotube diameters and number of nuclei per myotube derived from type 2 SMA patient treated with BIO101 ± A779 at 2 (D2) and 6 days (D6) of differentiation (n = 4; scale bar, 50 µm; 200× magnification). (C and D) ECAR Mito stress test seahorse profiles and the analysis of their area under the curve (C) and OCR/ECAR ratio at basal (0 to 18 min) and maximal respiration (53 to 70 min) (D) in myotubes derived from type 2 SMA patient treated during 2 days of differentiation with BIO101 ± A779 (n = 3). (E) Western Blot analysis and quantification of SMN expression in myotube derived from type 2 SMA patient treated with BIO101 ± A779 at 6 days (D6) of differentiation (Lanes were run on the same gel but were noncontiguous). Data are represented as mean ± SD with *p* < 0.05 and **p** < 0.01 for a comparison with vehicle untreated cells by unpaired nonparametric Mann-Whitney tests (A, B, D and E) at 48 h or (C for AUC) for separated days of differentiation or (C, curves) at each time point.

the slow-twitch *soleus* muscle (+20%; *p* ≤ 0.05; Figure 4E). In control mice, we also noted a specific fast-twitch muscle improvement of the vasculature (*tibialis*: +11%; *plantaris*: +22%; *p* ≤ 0.05; Figure S3F).

We then questioned whether those BIO101-induced benefits could rely on modulation in SMN protein expression. After confirming that the BIO101-specific MAS receptor is well expressed in all studied muscles (Figure S3G) and not altered by chronic treatment (Figure S3H), we did not detect any effect of this chronic treatment on SMN protein expression by western blot in the three studied muscles (Figure 4F), demonstrating that all the muscular benefits observed in our study are independent of SMN protein expression.

## 3.5 | BIO101 Accelerates SMA Patient-Derived Myoblast Differentiation and Favour Anaerobic Metabolism

This led us to consider the potential of BIO101 to positively affect muscular SMA patient cells. Although BIO101 did not affect the proliferation of SMA myoblasts over 48 h of culture (Figure 5A),

we noted a significant increase in both myotube diameter (+14%; *p* ≤ 0.05) and number of nuclei per myotube (+22%; *p* ≤ 0.05) at 2 and 6 days of differentiation when compared with untreated SMA myotubes (Figure 5B). Associated with the decrease in *MyoD* and *Myogenin* mRNA expression, two main myogenic factors, at 2 days of differentiation prior to normalization at 6 days of differentiation (Figure S4A), BIO101 seems to accelerate the human myoblast differentiation through fusion process. Importantly, all BIO101 effects on myoblast differentiation were abolished by the specific MAS receptor antagonist A779, while the antagonist alone did not display any effect on myotube fate (Figure 5B and Figure S4A), confirming that BIO101 exerts its effects through MAS receptor signalling.

We then assessed the changes in energy metabolism of SMA patient-derived myotubes at 2 days of differentiation, using Seahorse MitoStress test. If no change was observed on the oxygen consumption rate (OCR) under different conditions of mitochondrial stress (Figure S4B,C), we noted an increase in the extracellular acidification rate (ECAR) after the inhibition of the respiratory chain, at 30 min (+10%; *p* ≤ 0.05; Figure 5C,D). By reporting the OCR to the ECAR, our results support an enhancement of anaerobic catabolism of glucose in SMA myotubes
under BIO101 treatment (Figure 5D), which was not observed in A779 (Figure 5C,D), supporting a MAS-specific effect of BIO101 in favour of fast-twitch myotube energy metabolism.

Finally, as observed in severe SMA-like mice, BIO101-induced benefits were independent of SMN protein expression in

human myotubes after 6 days of differentiation (Figure 5E). Interestingly, the inhibition of MAS receptor with A779 induced a significant decrease in SMN expression when combined with BIO101 and a trend to decrease alone, suggesting a potent role of basal MAS receptor activity on SMN (Figure 5E).

<figure><img src="image_21.png" /><figcaption>**FIGURE 6** | Effects of BIO101 in combination with ASO-10-27 on severe Taiwanese SMA mouse phenotype. (A) Survival curves of severe SMA mice treated with the ASO-mismatch (n = 15 mice, median survival of 13 days), the ASO-10-27 (n = 14 mice, median survival of 18 days), or the ASO-10-27 combined with BIO101 (n = 14 mice, median survival of 22.5 days). (B, C and D) Weight curves (B), and quantification of the maximal time of hind-limb suspension score (grip-test, muscle fatigue) (C), and of the total number of squares crossed (open-field, moving capacity) (D) of severe SMA mice treated with the ASO-mismatch (n = 15 mice), the ASO-10-27 (n = 16 mice) or the ASO-10-27 combined with BIO101 (n = 15 mice). (E and F) Western Blot analysis and quantification of phosphorylated form of AKT (pAKT), total form of AKT (AKT) and SMN expression in the lumbar spinal cord (E) and in a pool of hindlimb *tibialis, plantaris* and *soleus* muscles (F) of severe SMA mice treated with the ASO-mismatch, the ASO-10-27 or the ASO-10-27 combined with BIO101 (n = 2 ASO-mismatch-treated mice; n = 4 mice treated with ASO-10-27 ± BIO101; lanes were run on the same gel but were noncontiguous). (B–D) Data are represented as mean ± SEM while (E and F) data are represented as mean ± SD with \*p < 0.05 and \*\*p < 0.01 for a comparison between the two indicated groups or with vehicle-treated control mice by (A) log-rank test or (B, C and D) unpaired *t*-test at each age or (E and F) unpaired nonparametric Mann–Whitney test.</figcaption></figure>
## 3.6 | BIO101 Potentiates the Benefits of ASO-10-27 Therapy on Motor Function in Severe Taiwanese SMA Mouse Model

Our results showed that BIO101 alone can positively affect the entire motor unit in severe SMA mice leading to specific motor function benefits whatever the age and the severity, and which is also relevant on SMA-patient cells in an SMN-independent manner. This led us to consider BIO101 as a potent combinatorial treatment with SMN-based therapies known for their marked survival benefits with persistent muscular defects [11, S6]. The combination of a single intracerebroventricular (ICV) injection of 8µg of ASO-10-27 at birth with the daily oral administration of BIO101 did not affect the survival gain observed in ASO-10-27-treated severe SMA mice (log-rank ***p=0.0002; +38% in median survival) compared with mismatch-treated severe SMA mice (median survival of 13 days; Figure 6A). A moderate weight gain was observed between P15 and P25 in co-treated severe SMA mice compared with severe SMA mice treated with ASO-10-27 alone (Figure 6B). However, for moving capacities, we observed a significant increase in the total number of crossings at P15 (Figure 6C) followed by a higher plateau beyond P19 for the co-treated severe SMA mice (Figure 6C). Most importantly, with the grip-test, we noted a marked and significant increase of the time of suspension at P23 and P25 for co-treated severe SMA mice compared with severe SMA mice treated with ASO-10-27 alone (Figure 6D), with an impressive maintenance in performance until 47 post-natal days (Figure 6D). Thus, BIO101 specifically improves resistance to muscular fatigue in combination with SMN-based therapy. Finally, despite the increase of SMN protein induced by ASO-10-27 injection and the proper triggering of the AKT intracellular pathway (Figure 6E,F), BIO101 treatment failed in increasing SMN protein expression at P10.5 in both spinal cord (Figure 6E) and hind limb muscles (Figure 6F) of co-treated severe SMA mice compared with severe SMA mice treated with ASO-10-27 alone. These results highlight the promising ability of BIO101 to benefit muscular function in combination with ASO-10-27.

## 4 | Discussion

The advances in SMN-based therapy for SMA have completely changed disease progression, with the emergence of new symptoms, leading to consider complementary treatments targeting the remaining alteration of neuromuscular function and/or the non-responding patients. We demonstrated that a daily oral administration of BIO101 can specifically improve muscular function in severe (infanthood) and mild (adulthood) Taiwanese SMA mouse models. The effects on severe SMA mice occurred through preferential protection of all the components of fast motor units. We demonstrated that BIO101 can also positively impact human myoblasts from type II SMA patients, favouring the differentiation, as previously demonstrated in mice or human control myoblasts. Importantly, all BIO101-induced benefits are independent of SMN expression. Thus, when combined with the SMN-based therapy ASO-10-27, BIO101 potentializes motor function improvement, highlighting the therapeutic potential of BIO101 for all severities of SMA.

Considering the crucial role of muscle function on the quality of life of SMA patients, the most striking result of our study relies on the ability of BIO101 to improve muscle performance in Taiwanese SMA mouse models (Figures 1E,F and 6D), administered alone or in combination with the ASO-10-27, whatever the considered severity and age. This specific functional effect of BIO101, without relevant effects on the survival of severe SMA mice, could rely on the limitation of muscular atrophy and the protection of lumbar motor neurons through direct or indirect effects. Indeed, the acceleration of the maturation of the neuromuscular structures could have an effect on cross-section area of myofibers which both are known to favour muscular performance by enhancing muscle force and functional effectiveness [27, S23]. Those gains improve the ability of mice to move freely (open-field) and to sustain longer muscular contraction for equivalent weight (grip-test) due to lower involvement regarding maximal force, both parameters of muscular function with important clinical relevance for SMA patients. In addition, the impact of muscle state on motor neuron function and survival in SMA is well known [28]. Thus, by improving muscle phenotype in severe SMA mice, BIO101 could indirectly limit motor neuron defects through specific myokines release and retrograde factor production, both known to protect motor neurons even in SMA [29]. Ultimately, this muscle-induced improvement of motor neuron state can positively loop to enhance in turn neuromuscular activity and therefore limit muscle atrophy [22, 30]. Moreover, BIO101 improved spinal cord vascularization, which could also participate in neuroprotection by favouring nutrient delivery and metabolism function [31].

We noted a poor amount of motor neuron degeneration, affecting preferentially the lateral subpopulation, a specific phenotype for our Taiwanese severe SMA mouse model when compared with previous studies on the same model [22, 32, S22, S24] and contrasting with a recent study showing no degeneration in this model [33]. This discrepancy confirms the constant evolution of the mouse model of SMA over time and generations, reinforcing the idea that SMA is a systemic disease leading to the alteration of the neuromuscular function through metabolic defects and synaptopathy. By its multi-tissular effects, BIO101 seems to match perfectly to disease specificities and heterogeneity.

The BIO101-induced limitation of muscular atrophy in severe SMA mice (Figure 4A) could be linked to the BIO101/MAS receptor intracellular signalling activation favouring an increase of proteosynthesis through the regulation of AKT/mTOR pathway and/or to the partial inhibition of myostatin gene expression [17, S15], already demonstrate as beneficial on animal models of SMA [34, 35, S9, S25]. Moreover, the ASO-10-27 treatment [34] is known to improve myostatin expression which could potentialize the effects of its inhibition through BIO101 treatment and explain the more pronounced functional benefits when both treatments are combined. However, the exact mechanisms by which BIO101 treatment exerts its effects need further investigation.

Another major aspect of BIO101 effects relies on the highest benefits observed in the fast-twitch *tibialis* and *plantaris* muscles compared with the slow-twitch *soleus* muscle, (Figures 3 and 4). These specific effects could be explained by muscle-specific intracellular adaptations regarding their types, and notably to the
Ca²⁺ homeostasis known to be highly altered in SMA [32, 36] and differentially regulated in slow- and fast-twitch muscles [37, S26]. We know that MAS receptor allows transient Ca²⁺ release from the endoplasmic reticulum [S15]. Thus, imposing an equiv- alent influx of Ca²⁺ in a slow- versus fast-twitch muscle with BIO101 treatment may not result in the same intracellular ad-aptations, notably through Ca2+-sensitive signalling pathways like CAMKII, known to be altered in SMA [38]. Interestingly, the BIO101 effects on muscular vascularization also present this fast-twitch preference, which could partly explain muscular im- provement through metabolic benefits.

In addition, BIO101 seems to modulate the entire motor unit fate
during development in favour of fast/intermediate motor unit in
control and severe SMA mice, with a change in the proportion
of MN+ ERRβ+ and MN+ MMP9+ (Figure S1E; Figure 2D,E),
associated with a faster muscle typology with BIO101 treatment
(Figure S3E; Figure 4D). This effect is also supported by the
improvement of anaerobic performance of type II SMA myo-
tubes (Figure 5C,D). This BIO101-induced adaptation pushes
energetic capacities and lowers the system requirements in
mitochondrial function and vascularization, making it a drug
candidate well adapted to known SMA alterations and condition
[39]. Extending the study to SMA Type I and Type III myoblasts
will also provide critical insights into different disease severity
and potential specific adaptations.

Interestingly, all the beneficial effects of BIO101 are indepen-
dent of SMN protein expression (Figures 4F, 5E, 6E and 6F),
which reinforces its potent for combinatorial treatment and for
mild and adult forms of the disease.

Finally, despite clear evidence of multi-tissular and multi-level
benefits of BIO101 leading to muscular function improvement,
the treatment presented negligible effects on survival in severe
SMA mice, as monotherapy and in combination with ASO10-27,
when compared with other approaches like physical exercise,
pharmacological activation of the NMDA receptor or genetic
invalidation of the IGF-1 receptor [22, 30, 38]. This peculiarity
could be explained by a limited improvement in cardiac func-
tion [40, S27] and/or respiratory muscle function. However, even
if the effects of BIO101 on cardiac tissue have not been investi-
gated in this study and deserve further analysis, the molecule
20E, and by extension BIO101, is known to improve cardio-
vascular as well as respiratory function, even in disease context
[S17, S28]. Thus, these contrasting results could rely either on a
specific tissular and cellular status of SMA cardio-respiratory
system, which could limit the effect of BIO101, or on the cellular-
specific effect of BIO101, which can evolve along maturation
and age, being more pronounced on cardio-respiratory system
at adulthood. This peculiarity needs to be extensively explored
and described with specific research programs. However, this
limitation observed on severe mouse model has a completely dif-
ferent interest for SMA patient clinical care. Patients can benefit
from adapted clinical care with respiratory and nutritional aids,
while motor function remains orphan of specific care, for pa-
tients treated or not with SMN-based therapies.

To conclude, BIO101 appears as a new promising therapeutic
option for SMA patients in combination with SMN-dependent
therapies such as ASO therapy, or as monotherapy for milder

forms regarding its safety, as shown in clinical trials in adults
or in non-clinical studies in juvenile animals, and oral admin-
istration. An optimization of the BIO101 administration fre-
quency could be necessary to enhance the beneficial effect of the
molecule in SMA patients but its capacity to mediate different
pharmacological effects according to the cellular context and to
induce benefits on a wide range of severities and ages, makes it
well-adapted to the clinical heterogeneity of SMA.

Acknowledgements

C.Be. received a PhD scholarship from the *Association nationale de la recherche et de la technologie (ANRT)*. This work was essentially supported by Biophytis funding. This work was also supported by INSERM and Université Paris Cité fundings. Human immortalized myoblasts from type 2 SMA patients were provided by Institute of Myology (Paris) under non-profit-to-non-profit uniform biological material transfer agreement (UBMTA). All authors wish to thank the entire team of the animal facility, UMS BioMedTech facilities CNRS UMS 2009, for animal care.

Conflicts of Interest

C.Be., S.C., P.N.H., C.Ba., R.L., S.V., P.D. and M.L. are, or were, employ-
ees of Biophytis and own equity in the company. C.Be., R.L., S.V., P.D.,
M.L., F.C. and O.B. are inventors or contributors on patent(s) concern-
ing the use of BIO101 in SMA. They declare, however, that the compa-
ny's potential commercial interests had no impact on the conduct of this
research work.

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## Supporting Information

Additional supporting information can be found online in the Supporting Information section. **Figure S1:** Effects of BIO101 on spinal cord in control mice. Quantification of the total number of ChAT+ cells depending on their localization (medial and lateral) (A) and of the distribution of their soma area (B) per 50-µm-thick slice of lumbar spinal cord of vehicle- compared with BIO101-treated control mice at P10.5 (n=5 mice in each group). (C) Quantification of the total number ERRβ+ ChAT+ (n=7 BIO101-treated SMA-like mice, n=6 in other groups) cells over total ChAT+ cells, in 50-µm-thick slice of lumbar spinal cord of vehicle-treated control mice compared with vehicle- or BIO101-treated SMA-like mice at P10.5. (D and E) Quantification of the proportion of double positives MMP9-ChAT (D) or ERRβ-ChAT (E) cells over total ChAT+ cells in 50-µm-thick slice of lumbar spinal cord of vehicle- (n=5 for the double positives MMP9-ChAT; n=6 for the double positives ERRβ-ChAT) compared with BIO101-treated control mice (n=6) at P10.5. (F) Quantitative analysis of the capillary density
in the ventral horn of lumbar spinal cord of vehicle- compared with BIO101-treated control mice at P10.5 (n=4 mice in each group). Data are represented as mean±SD with *p<0.05 for a comparison between the two indicated groups by (A, C, D, E and F) unpaired nonparametric Mann-Whitney tests or (B) two-way ANOVA with a Sidak post hoc tests.

**Figure S2:** Effects of BIO101 on neuromuscular junction and muscle maturation in control mice. (A) Quantitative analyses of the percentage of uniform and perforated NMJ over total NMJ in the *tibialis, plantaris* and *soleus* muscles of vehicle- compared with BIO101-treated control mice at P10.5 (n=5 mice in each group). (B) Quantitative analysis of the percentage of myofibers expressing embryonic Myosin Heavy Chain isoform over total number of myofibers in the *tibialis, plantaris* and *soleus* muscles of vehicle- compared with BIO101-treated control mice at P10.5 (n=5 mice in each group). Data are represented as mean±SD with *p<0.05 for a comparison between the two indicated groups by (A) Kruskal-Wallis test with a Sidak post hoc tests or (B) unpaired nonparametric Mann-Whitney tests.

**Figure S3:** Effects of BIO101 on muscle phenotype in control mice. (A) Images of haematoxylin-eosin staining on *tibialis* muscles of vehicle-treated control mice compared with vehicle- or BIO101-treated SMA mice at P10.5 (scale bar: 25 µm) (B-D) Quantitative analysis of the mean cross-sectional area of fibres (B), their standard deviation (C) and the total number of fibres (D) in the *tibialis, plantaris* and *soleus* muscles of vehicle- compared with BIO101-treated control mice at P10.5 (n=5 mice in each group). (E) Quantitative analysis of the percentage of myofibers expressing adult type 1, type 2A and type 2X/2B Myosin Heavy Chain isoforms over total number of myofibers in the *tibialis, plantaris* and *soleus* muscles of vehicle- (CV) compared with BIO101-treated (CB) control mice at P10.5 (n=5 mice in each group). (F) Quantitative analysis of the number of vessels per muscle fibre in the *tibialis, plantaris* and *soleus* muscle of vehicle- compared with BIO101-treated control mice at P10.5 (n=5 mice in each group). (G and H) Quantification of the relative mRNA levels of Mas in the *tibialis, plantaris* and *soleus* muscles of vehicle-treated control mice (n=5 in each group) (G) and in vehicle-treated control mice compared with BIO101-treated control mice and vehicle- or BIO101-treated SMA-like mice at P10.5 for the *tibialis, plantaris* and *soleus* muscles (n=5 mice in each group) (H). Data are represented as mean±SD with *p<0.05 and **p<0.01 for a comparison between the two indicated groups by (B, C, D and F) unpaired nonparametric Mann-Whitney tests or (E) two-way ANOVA with a Sidak post hoc tests or (G and H) Kruskal-Wallis test with a Sidak post hoc tests.

**Figure S4:** Effects of BIO101 on SMA patient-derived myoblast differentiation and metabolism. (A) Quantification of the relative mRNA levels of MyoD and Myogenin in myotubes derived from type 2 SMA patient treated during 2 (D2) and 6 (D6) days of differentiation with BIO101 (BIO)±A779 (BIO+A779 or A779) compared with untreated (UT) myotubes (n=3). (B and C) OCR Mito Stress test Seahorse profiles (B) and quantification of metabolic parameters (C) (BR, basal respiration; PL, proton leak; ATP, ATP production; MR, mitochondrial respiration; SRC, spare respiratory capacity; NMOC, non-mitochondrial oxygen consumption), in myotubes derived from type 2 SMA patient treated during 2 days of differentiation with BIO101±A779 (n=3). Data are represented as mean±SD with *p<0.05 for a comparison with UT condition (A) Kruskal-Wallis test with a Sidak post hoc tests. **Table S1:** Supplementary Information. **Table S2:** Supplementary Information. **Table S3:** Supplementary Information. **Table S4:** Supplementary Information. **Table S5:** Supplementary Information. **Table S6:** Supplementary Information.