Treatment for Myopalladin-related Congenital Myopathy – TreatMYPN

To achieve our objectives, we combine:

• An MKO mouse model

The constitutive myopalladin‐deficient mouse (MKO) is followed longitudinally up to 12 months. At each time point (1.5, 3, 6, 9, and 12 months), body weight is recorded and muscle strength assessed via the grip test to quantify functional weakness. Muscles harvested at these ages undergo standard histological analyses (H&E, NADH, Gomori, COX, SDH) combined with fiber typing by immunofluorescence to determine whether the histopathological phenotypes observed in mice mirror those seen in patients. In parallel, myofiber ultrastructure is examined by electron microscopy. Finally, biomechanics are studied at two levels: whole‐muscle stretch to assess global mechanical response and isolated‐fiber traction‐relaxation tests from which Young’s modulus and relaxation time are extracted to characterize muscle fiber elasticity and viscoelasticity.

• A 3D muscle model (3DMM) for in vitro studies

To model MYPN‐CM in vitro, we culture WT and MKO mouse satellite cells on aligned hydrogels, which after several days yield highly differentiated, mature, contractile myofibers. The quality of these 3DMMs is validated by Z‐line striation and nuclear localization; in MKO constructs, Z‐line dissolution and centralized nuclei serve as key pathological markers. Following transduction of MKO 3DMMs with the AAV‐MYPN vector, these structural parameters will be re‐evaluated to determine the extent of rescue, and the contractile function of the 3DMMs will be measured to confirm a direct functional benefit from MYPN re‐expression.

• Transcriptomic analyses

To elucidate the molecular mechanisms underlying MYPN‐CM, we will perform single‐nuclei RNA sequencing (snRNA‐seq) on MKO and WT muscle, focusing on pathways related to sarcomeric architecture and muscle homeostasis. Differentially expressed genes will be validated by qPCR and immunoblotting to quantify precisely the affected transcripts and proteins. To ensure translational relevance, we will compare these transcriptomic and proteomic profiles with data from MYPN‐CM patient and control biopsies, thereby identifying robust biomarkers applicable in a clinical context.

• AAV vector development

The human MYPN gene will be cloned under a dMCK promoter into an AAV plasmid developed by Généthon to optimize muscle‐specific expression. Cloning and viral production will be carried out in partnership with Généthon. We will first validate transduction efficiency in vitro on 3DMMs, then confirm MYPN re‐expression by western blot and immunofluorescence. Next, the vector will be administered intramuscularly to MKO mice to verify local MYPN expression, followed by intravenous delivery to assess tissue distribution (muscle versus off‐target organs) and immunogenicity. Finally, the previously defined molecular, histopathological, and functional readouts will be used to measure phenotypic restoration.

During this first year, we’ve made decisive strides in understanding and tracking the muscle disease caused by loss of myopalladin. To start, we established reliable molecular, tissue-based, and functional markers using both our MKO mouse model and lab‐grown 3D muscle cultures.

By just three months of age, both male and female MKO mice already show clear muscle weakness: their grip strength is about 20% lower in both front and hind limbs, and their body and muscle weights, especially in key muscles like the tibialis anterior (TA), gastrocnemius, and quadriceps, have declined. This tells us that some muscles are particularly vulnerable to myopalladin loss.

Under the microscope, tissue stains and electron microscopy reveal atrophy of muscle fibers and internalized nuclei. We also see “ring fibers” in the TA and distinct “cap” structures, well-defined peripheral zones packed with thin filaments and Z-line fragments, exactly like those found in biopsies from MYPN-CM patients. These findings highlight a deeply disrupted sarcomere architecture.

On the biomechanical side, isolated MKO fibers contract less forcefully, are stiffer (less able to deform), and relax more quickly after being stretched, evidence of altered elasticity and viscoelasticity.

In our 3D cultures, MKO myofibers lose their α-actinin striations and show central nuclei, underlining that without myopalladin, muscle structure and function are altered.

Together, these combined markers, reduced strength, tissue abnormalities, and mechanical dysfunction, give us a powerful toolkit to measure the success of our upcoming gene therapies and pave the way for the next phases of in vitro and in vivo validation.

In the coming months, we will complete the full phenotyping of 120 mice (6 mice × 2 sexes × 5 ages) to refine our functional and histopathological readouts, reduce statistical variability, and strengthen the robustness of our biomarkers. Simultaneously, we will enter the most advanced preclinical phase of the TreatMYPN project. For the three objectives focused on the production and administration of AAV-MYPN vectors, we have nearly finalized cloning the MYPN transgene under the dMCK promoter into a Généthon-optimized AAV backbone. Viral production will begin shortly, and by the end of the year we will conduct both initial in vitro transduction assays on 3DMM and intramuscular and systemic injections in MKO mice. These steps will allow us to validate MYPN expression, assess vector biodistribution and tolerability, and then measure its impact on our established readouts, thereby preparing the preclinical dossier for rapid advancement to clinical trials.

Congenital myopathies (CM) are severe monogenic muscle diseases affecting humans from birth or early infancy and having a strong impact on patient survival and quality of life leading to muscle weakness provoking in some cases gait loss. Nemaline Myopathy (NEM) and Cap myopathy, are two of the most common CM with an estimated frequency of 1 in 50,000 live births. They are characterized by the presence of protein inclusions with a rod shape (rods) and cap in the muscle biopsy. In 2017 we and others have reported a CM caused by biallelic mutations in the myopalladin gene MYPN, leading to loss of function of MYPN, with rods and caps in the myofibers. MYPN-CM are rare, they have an important impact on patients’ quality of life resulting from early onset muscle weakness possibly leading to gait loss, and have no cure. The monogenic nature of MYPN-CM, and the size of MYPN gene made MYPN-CM suitable for AAV mediated therapeutic approaches. Nevertheless, their rarity precludes appeal for pharmaceutical companies. Recent studies in a murine MYPN knock out model (MKO) demonstrated that MYPN promotes skeletal muscle growth through activation of the SRF pathway. Our MKO muscles myopathologic analysis showed myofibers atrophy, increased nuclear internalizations, and ring-shape fibers, reminiscent of caps, never reported before. An innovative technique developed in our lab that consists in creating a model of 3D mature myofibers derived from MKO muscle satellite cells (MuSCs) cultured in vitro on innovative hydrodynamic gels (3DMM) showed that MKO MuSCs exhibited a-actinin striation, dissolution at Z-line levels, a-actinin accumulation in some myofibers, and prominent centrally placed nuclei. With the goal of developing a therapeutic strategy for MYPN-CM, we will employ a translational approach using complementary preclinical models, 3DMM and MKO with these specific aims: 1. Definition of molecular (sNuclei RNA seq), myopathologic, and clinical readouts; 2. AAV-based gene therapy strategy development and in vitro validation on 3DMM; 3. Evaluation of the therapeutic benefit in vivo in MKO. The clinico-myopathologic expertise of the coordinator, the presence of a clear myopathologic phenotype (ring fibers etc.) in MKO and 3DMM, the utilization or cutting edge RNASeq techniques perfectly mastered in our lab, together with the know-how on AAV gene rescuing strategies for myopathies (Généthon, collaboration) will allow to accelerate the gene therapy development and to comprehend the molecular mechanisms underlying MYPN-CM. The full academic setting of this project will serve as template to develop the therapy of other rare CMs.