The t(4;11) translocation, which drives KMT2A::AFF1 fusion gene expression, is associated with poor prognosis in pediatric and adult B-cell Acute Lymphoblastic Leukemia (B-ALL). KMT2A::AFF1 fusion circular RNAs (f-circRNAs) have been identified in B-ALL, though their contribution to leukemia has not yet been outlined. We identified a novel recurrent KMT2A::AFF1-derived back-splicing junction joining AFF1 exon 7 to KMT2A exon 3 (AK_7_3) in SEM, RS4;11, and ALL-PO B-ALL cell lines, all of which carry the t(4;11)(q24;q23)/KMT2A::AFF1 translocation. This sequence was observed in RT-PCR products encompassing two splicing variants of KMT2A exon 4, which are also present in the linear KMT2A::AFF1 chimera. AK_7_3 was also found in 12/18 pediatric and 17/24 adult t(4;11)-positive B-ALL patients, including three paired diagnosis/relapse samples, whereas it was absent in 23 t(4;11)-negative B-ALL patients. Whole-transcriptome analysis of AK_7_3-knockdown SEM cells identified upregulated genes involved in oxidative stress response and regulation of apoptosis. Cell death analysis confirmed the pro-apoptotic impact of AK_7_3 silencing. Confocal analyses, Seahorse bioenergetic profiling, and electron microscopy revealed an overproduction of reactive oxygen species, increased mitochondrial membrane potential, mitochondrial respiration, and dysmorphic mitochondria after AK_7_3 knockdown in KMT2A::AFF1 cell lines. In summary, we identified AK_7_3 as a novel f-circRNA back-splicing junction recurrent in t(4;11)-positive B-ALL patients at both diagnosis and relapse. Our data provide evidence for a functional role of this molecule as an anti-apoptotic agent that affects mitochondrial function, suggesting its potential involvement in KMT2A::AFF1-driven B-ALL leukemogenesis.

Chloride channels are involved in many cellular processes, including cell volume regulation, modulation of cell excitability, and electrolyte and water secretion. Mutations of these proteins are associated with heterogeneous diseases such as myotonia, cystic fibrosis, epilepsy, deafness, lysosomal storage disease, and various kinds of renal and ophthalmic dysfunctions, also known as channelopathies. Thus, drugs targeting chloride channels may have important therapeutic applications. In this context, fenamates, commonly used for their anti-inflammatory properties, have been explored for drug repurposing in chloride channelopathies thanks to their ability to modulate multiple chloride channels. This narrative review resumes the effects of niflumic acid (NFA), flufenamic acid (FFA), mefenamic acid (MFA), meclofenamic acid (MCFA), and tolfenamic acid (TFA) on different types of chloride channel. It emerges that fenamates have a wide spectrum of activities on these channels that vary depending on multiple factors like channel isoforms, extracellular and intracellular conditions, and cell and tissue types. They may also exhibit both activating and inhibitory effects depending on their concentration. Therefore, thanks to their variegated modulatory activity on chloride channels, fenamates might be considered promising lead compounds for the development of new drug candidates that can target these altered channels involved in channelopathies. Trial Registration: EudraCT number: 2021-000708-39; ClinicalTrials.gov identifier: NCT029930005 and NCT02429570.

Cationic ion channels are transmembrane proteins that regulate the flux of cations (potassium, sodium, and calcium) across cell membrane, playing a pivotal role in many cellular functions. Disruptions of their activity can lead to the so-called genetic or acquired channelopathies, a heterogeneous group of diseases that affect multiple human systems. Fenamates, a class of nonsteroidal anti-inflammatory drugs, has recently emerged as modulators of cationic ion channels highlighting the possibility of their repurposing for the treatment ion channel-related disorders, such as channelopathies, chronic pain, epilepsy, cardiac arrhythmias, and cancers. In this review, the ability of fenamates (i.e. niflumic, flufenamic, mefenamic, meclofenamic, and tolfenamic acids) to differentially modulate the activity of cationic ion channels is described. Overall, preclinical and clinical studies suggest that fenamates represent a promising class of compounds for drug repurposing and for the development of new molecules, offering novel therapeutic opportunities for patients affected by ion channel-related disorders.

Objectives:Non-dystrophic myotonias (NDM) are rare diseases due to mutations in the voltage-gated sodium (Nav1.4) and chloride (ClC-1) channels expressed in skeletal muscle fibers. We provide an up-to-date review of pharmacological treatments available for NDM patients and experimental studies aimed at identifying alternative treatments and at better understanding the mechanisms of actions. Methods:Literature research was performed using PubMed and ClinicalTrial.gov. Results:Today, the sodium channel blocker mexiletine is the drug of choice for treatment of NDM. Alternative drugs include other sodium channel blockers and the carbonic anhydrase inhibitor acetazolamide. Preclinical studies suggest that activators of ClC-1 channels or voltage-gated potassium channels may have antimyotonic potential. Conclusions:An increasing number of antimyotonic drugs would help to design a precision therapy to address personalized treatment of myotonic individuals.

The small Rho GTP-binding proteins are important cell morphology, function, and apoptosis regulators. Unlike other Rho proteins, RhoB can be subjected to either geranylgeranylation (RhoB-GG) or farnesylation (RhoB-F), making that the only target of the farnesyltransferase inhibitor (FTI). Fluorescence resonance energy transfer experiments revealed that RhoB is activated by hyperosmolarity. By contrast, hyposmolarity did not affect RhoB activity. Interestingly, treatment with farnesyltransferase inhibitor-277 (FTI-277) decreased the cell size. To evaluate whether RhoB plays a role in volume reduction, renal collecting duct MCD4 cells and Human Kidney, HK-2 were transiently transfected with RhoB-wildtype-Enhance Green Fluorescence Protein (RhoB-wt-EGFP) and RhoB-CLLL-EGFP which cannot undergo farnesylation. A calcein-based fluorescent assay revealed that hyperosmolarity caused a significant reduction of cell volume in mock and RhoB-wt-EGFP-expressing cells. By contrast, cells treated with FTI-277 or expressing the RhoB-CLLL-EGFP mutant did not properly respond to hyperosmolarity with respect to mock and RhoB-wt-EGFP expressing cells. These findings were further confirmed by 3D-LSCM showing that RhoB-CLLL-EGFP cells displayed a significant reduction in cell size compared to cells expressing RhoB-wt-EGFP. Moreover, flow cytometry analysis revealed that RhoB-CLLL-EGFP expressing cells as well as FTI-277-treated cells showed a significant increase in cell apoptosis. Together, these data suggested that: (i) RhoB is sensitive to hyperosmolarity and not to hyposmolarity; (ii) inhibition of RhoB farnesylation associates with an increase in cell apoptosis, likely suggesting that RhoB might be a paramount player controlling apoptosis by interfering with responses to cell volume change.

Multiple myeloma (MM) progression is closely dependent on cells in the bone marrow (BM) microenvironment, including fibroblasts (FBs) and immune cells. In their BM niche, MM cells adhere to FBs sustaining immune evasion, drug resistance and the undetectable endurance of tumor cells known as minimal residual disease (MRD). Here, we describe the novel bi-specific designed ankyrin repeat protein (DARPin) α-FAPx4-1BB (MP0310) with FAP-dependent 4-1BB agonistic activity. The α-FAPx4-1BB DARPin simultaneously binds to FAP and 4-1BB overexpressed by activated FBs and immune cells, respectively. Although flow cytometry analysis showed that T and NK cells from MM patients were not activated and did not express 4-1BB, stimulation with daratumumab or elotuzumab, monoclonal antibodies (mAbs) currently used for the treatment of MM, significantly upregulated 4-1BB both in vitro and in MM patients following mAb-based therapy. The mAb-induced 4-1BB overexpression allowed the engagement of α-FAPx4-1BB that acted as a bridge between FAP+FBs and 4-1BB+NK cells. Therefore, α-FAPx4-1BB enhanced both the adhesion of daratumumab-treated NK cells on FBs as well as their activation by improving release of CD107a and perforin, hence MM cell killing via antibody-mediated cell cytotoxicity (ADCC). Interestingly, α-FAPx4-1BB significantly potentiated daratumumab-mediated ADCC in the presence of FBs, suggesting that it may overcome the BM FBs' immunosuppressive effect. Overall, we speculate that treatment with α-FAPx4-1BB may represent a valuable strategy to improve mAb-induced NK cell activity fostering MRD negativity in MM patients through the eradication of latent MRD cells.

Background: The nondystrophic myotonias are rare muscle hyperexcitability disorders caused by gain-of-function mutations in the SCN4A gene or loss-of-function mutations in the CLCN1 gene. Clinically, they are characterized by myotonia, defined as delayed muscle relaxation after voluntary contraction, which leads to symptoms of muscle stiffness, pain, fatigue, and weakness. Diagnosis is based on history and examination findings, the presence of electrical myotonia on electromyography, and genetic confirmation. Methods: Next-generation sequencing including the CLCN1 and SCN4A genes was performed in patients with clinical neuromuscular disorders. Electromyography, Short Exercise Test, in vivo and in vitro electrophysiology, site-directed mutagenesis and heterologous expression were collected. Results: A heterozygous point mutation (c.1775C > T, p.Thr592Ile) of muscle voltage-gated sodium channel α subunit gene (SCN4A) has been identified in five female patients over three generations, in a family with non-dystrophic myotonia. The muscle stiffness and myotonia involve mainly the face and hands, but also affect walking and running, appearing early after birth and presenting a clear cold sensitivity. Very hot temperatures, menstruation and pregnancy also exacerbate the symptoms; muscle pain and a warm-up phenomenon are variable features. Neither paralytic attacks nor post-exercise weakness has been reported. Muscle hypertrophy with cramp-like pain and increased stiffness developed during pregnancy. The symptoms were controlled with both mexiletine and acetazolamide. The Short Exercise Test after muscle cooling revealed two different patterns, with moderate absolute changes of compound muscle action potential amplitude. Conclusions: The p.Thr592Ile mutation in the SCN4A gene identified in this Sardinian family was responsible of clinical phenotype of myotonia.

Immunoglobulin (Ig) replacement therapy (IgRT) consists of the administration of low-dose human polyclonal Igs for the treatment of primary and secondary hypogammaglobulinemia that are associated with recurrent infections and immune dysfunction. IgRT restores physiological antibody levels and induces an immunomodulatory effect by strengthening immune effector cells, thus reducing infections. Here, we describe the pharmacology of different Ig formulations with a particular focus on their mechanism of action as low-dose IgRT, including the direct anti-microbial effect and the immunomodulatory function. In addition, we describe the use of therapeutic Igs for the management of multiple myeloma (MM), a hematologic malignancy characterized by severe secondary hypogammaglobulinemia associated with poor patient outcome. In MM settings, IgRT prevents life-threatening and recurrent infections showing promising results regarding patient survival and quality of life. Nevertheless, the clinical benefits of IgRT are still controversial. A deeper understanding of the immune-mediated effects of low-dose IgRT will provide the basis for novel combined therapeutic options and personalized therapy in MM and other conditions characterized by hypogammaglobulinemia.

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.

Extracellular vesicles (EVs) have emerged as important players in cell-to-cell communication within the bone marrow (BM) of multiple myeloma (MM) patients, where they mediate several tumor-associated processes. Here, we investigate the contribution of fibroblasts-derived EVs (FBEVs) in supporting BM angiogenesis. We demonstrate that FBEVs' cargo contains several angiogenic cytokines (i.e., VEGF, HGF, and ANG-1) that promote an early over-angiogenic effect independent from EVs uptake. Interestingly, co-culture of endothelial cells from MM patients (MMECs) with FBEVs for 1 or 6 h activates the VEGF/VEGFR2, HGF/HGFR, and ANG-1/Tie2 axis, as well as the mTORC2 and Wnt/β-catenin pathways, suggesting that the early over-angiogenic effect is a cytokine-mediated process. FBEVs internalization occurs after longer exposure of MMECs to FBEVs (24 h) and induces a late over-angiogenic effect by increasing MMECs migration, chemotaxis, metalloproteases release, and capillarogenesis. FBEVs uptake activates mTORC1, MAPK, SRC, and STAT pathways that promote the release of pro-angiogenic cytokines, further supporting the pro-angiogenic milieu. Overall, our results demonstrate that FBEVs foster MM angiogenesis through dual time-related uptake-independent and uptake-dependent mechanisms that activate different intracellular pathways and transcriptional programs, providing the rationale for designing novel anti-angiogenic strategies.

Non-dystrophic myotonias include several entities with possible clinical overlap, i.e. myotonia congenita caused by CLCN1 gene mutations, as well as paramyotonia congenita and sodium channel myotonia caused by SCN4A gene mutations. Herein, we describe the clinical features of five relatives affected by clinical and neurophysi-ological myotonia, with an aspecific and mixed phenotype. Next-generation sequencing identified the novel p. K1302R variant in SCN4A and the p.H838P variant in CLCN1. Segregation of the two mutations with the disease was confirmed by genotyping affected and non-affected family members. Patch-clamp experiments showed that sodium currents generated by p.K1302R and WT hNav1.4 were very similar. Mutant channel showed a small negative shift (5 mV) in the voltage-dependence of activation, which increased the likelihood of the channel to open at more negative voltages. The p.H838P mutation caused a reduction in chloride current density and a small voltage-dependence shift towards less negative potentials, in agreement with its position into the CBS2 domain of the C-terminus. Our results demonstrated that the mild functional alterations induced by p.K1302R and p.H838P in combination may be responsible for the mixed myotonic phenotypes. The K1302R mutant was sensitive to mexiletine and lamotrigine, suggesting that both drugs might be useful for the K1302R carriers.

Invasive and painful procedures, which often induce feelings of anxiety, are necessary components of pediatric cancer treatment, and adequate pain and anxiety management during these treatments is of pivotal importance. In this context, it is widely recognized that a holistic approach, including pharmacological and non-pharmacological modalities, such as distraction techniques, should be the standard of care. Recent evidence suggested the use of virtual reality (VR) as an effective non-pharmacological intervention in pediatrics. Therefore, this systematic review aims to analyze previously published studies on the effectiveness of VR for the management of pain and/or anxiety in children and adolescents with hematological or solid cancer. Medline, SCOPUS, Web of Science, ProQuest, CINAHL, and The Cochrane Central Register of Controlled Trials were used to search for relevant studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses checklist. Randomized controlled trial, crossover trial, cluster randomized trial, and quasi-experimental studies were included. Thirteen studies, published between 1999 and 2022, that fulfilled the inclusion criteria were included. Regarding the primary outcomes measured, pain was considered in five studies, anxiety in three studies, and the remaining five studies analyzed the effectiveness of VR for both pain and anxiety reduction. Our findings suggested a beneficial effect of VR during painful vascular access procedures. Limited data are available on the reduction of anxiety in children with cancer.

Angiogenesis represents a pivotal hallmark of multiple myeloma (MM) that correlates to patients' prognosis, overall survival, and drug resistance. Hence, several anti-angiogenic drugs that directly target angiogenic cytokines (i.e., monoclonal antibodies, recombinant molecules) or their cognate receptors (i.e., tyrosine kinase inhibitors) have been developed. Additionally, many standard antimyeloma drugs currently used in clinical practice (i.e., immunomodulatory drugs, bisphosphonates, proteasome inhibitors, alkylating agents, glucocorticoids) show anti-angiogenic effects further supporting the importance of inhibiting angiogenesis from potentiating the antimyeloma activity. Here, we review the most important anti-angiogenic therapies used for the management of MM patients with a particular focus on their pharmacological profile and on their anti-angiogenic effect in vitro and in vivo. Despite the promising perspective, the direct targeting of angiogenic cytokines/receptors did not show a great efficacy in MM patients, suggesting the need to a deeper knowledge of the BM angiogenic niche for the design of novel multi-targeting anti-angiogenic therapies.

Several commercially available and newly synthesized riluzole analogs were evaluated in vitro as voltage-gated skeletal muscle sodium-channel blockers. Data obtained from the patch-clamp technique demonstrated that potency is well correlated with lipophilicity and the introduction of a protonatable amino function in the benzothiazole 2-position enhances the use-dependent behavior. The most interesting compound, the 2-piperazine analog of riluzole (14), although slightly less potent than the parent compound in the patch-clamp assay as well as in an in vitro model of myotonia, showed greater use-dependent Nav1.4 blocking activity. Docking studies allowed the identification of the key interactions that 14 makes with the amino acids of the local anesthetic binding site within the pore of the channel. The reported results pave the way for the identification of novel compounds useful in the treatment of cell excitability disorders.

Intracellular pH (pHi) regulation is a challenge for the exocrine pancreas, where the luminal secretion of bicarbonate-rich fluid is accompanied by interstitial flows of acid. This acid-base transport requires a plethora of ion transporters, including bicarbonate transporters and the Na+/H+ exchanger isoform 1 (NHE1), which are dysregulated in Pancreatic Ductal Adenocarcinoma (PDAC). PDAC progression is favored by a Collagen-I rich extracellular matrix (ECM) which exacerbates the physiological interstitial acidosis. In organotypic cultures of normal human pancreatic cells (HPDE), parenchymal cancer cells (CPCs) and cancer stem cells (CSCs) growing on matrices reproducing ECM changes during progression, we studied resting pHi, the pHi response to fluxes of NaHCO3 and acidosis and the role of NHE1 in pHi regulation. Our findings show that: (i) on the physiological ECM, HPDE cells have the most alkaline pHi, followed by CSCs and CPCs, while a Collagen I-rich ECM reverses the acid-base balance in cancer cells compared to normal cells; (ii) both resting pHi and pHi recovery from an acid load are reduced by extracellular NaHCO3, especially in HPDE cells on a normal ECM; (iii) cancer cell NHE1 activity is less affected by NaHCO3. We conclude that ECM composition and the fluctuations of pHe cooperate to predispose pHi homeostasis towards the presence of NaHCO3 gradients similar to that expected in the tumor.

Angiogenesis, the formation of new capillaries from preexisting blood vessels, plays an important role in cancer progression. When the tumor mass expands, the balance is shifted toward a pro-angiogenic milieu to maintain sustainable angiogenic processes. In this context, there is an up-regulation of several pro-angiogenic factors, including vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), placental growth factor (PlGF), platelet derived endothelial cell growth factor (PD-ECGF), angiopoietins (Angs), transforming growth factors (TGFs) -α and -β, and epidermal cell growth factor (EGF), which collectively activate the proliferation of circulating endothelial progenitor cells (EPCs) able to enter in the peripheral blood circulation, migrating to sites of angiogenesis. Hence, the number of antiangiogenic agents developed for cancer treatment has risen over the past years. To date, the most common approaches to the inhibition of the VEGF axis include the blockade of VEGF receptors (VEGFRs) or ligands by neutralizing antibodies, as well as the tyrosine kinase inhibitors (TKIs), immunomodulatory drugs (IMiDs), and monoclonal antibodies (mAbs). Here, we focus on the role of circulating EPCs, which mediate the cross-talk between cancer angiogenesis and neoplastic clone, as potential novel targets for antiangiogenic drugs with particular relevance for hematological malignancies.

Multiple myeloma (MM) is the second most common hematological malignancy, and despite the introduction of innovative therapies, remains an incurable disease. Identifying early and minimally or non-invasive biomarkers for predicting clinical outcomes and therapeutic responses is an active field of investigation. Malignant plasma cells (PCs) reside in the bone marrow (BM) microenvironment (BMME) which comprises cells (e.g., tumour, immune, stromal cells), components of the extracellular matrix (ECM) and vesicular and non-vesicular (soluble) molecules, all factors that support PCs’ survival and proliferation. The interaction between PCs and BM stromal cells (BMSCs), a hallmark of MM progression, is based not only on intercellular interactions but also on autocrine and paracrine circuits mediated by soluble or vesicular components. In fact, PCs and BMSCs secrete various cytokines, including angiogenic cytokines, essential for the formation of specialized niches called “osteoblastic and vascular niches”, thus supporting neovascularization and bone disease, vital processes that modulate the pathophysiological PCs–BMME interactions, and ultimately promoting disease progression. Here, we aim to discuss the roles of cytokines and growth factors in pathogenetic pathways in MM and as prognostic and predictive biomarkers. We also discuss the potential of targeted drugs that simultaneously block PCs’ proliferation and survival, PCs–BMSCs interactions and BMSCs activity, which may represent the future goal of MM therapy.

Ion channels are pore-forming proteins that allow ions to flow across plasma membranes and intracellular organelles in both excitable and non-excitable cells. They are involved in the regulation of several biological processes (i.e., proliferation, cell volume and shape, differentiation, migration, and apoptosis). Recently, the aberrant expression of ion channels has emerged as an important step of malignant transformation, tumor progression, and drug resistance, leading to the idea of "onco-channelopathy". Here, we review the contribution of ion channels and transporters in multiple myeloma (MM), a hematological neoplasia characterized by the expansion of tumor plasma cells (MM cells) in the bone marrow (BM). Deregulation of ion channels sustains MM progression by modulating intracellular pathways that promote MM cells' survival, proliferation, and drug resistance. Finally, we focus on the promising role of ion channels as therapeutic targets for the treatment of MM patients in a combination strategy with currently used anti-MM drugs to improve their cytotoxic activity and reduce adverse effects.

Skeletal muscle ion channelopathies are rare genetic diseases mainly characterized by myotonia (muscle stiffness) or periodic paralysis (muscle weakness). Here, we reviewed the available therapeutic options in non-dystrophic myotonias (NDM) and periodic paralyses (PP), which consists essentially in drug repositioning to address stiffness or weakness attacks. Empirical use followed by successful randomized clinical trials eventually led to the orphan drug designation and marketing authorization granting of mexiletine for NDM and dichlorphenamide for PP. Yet, these treatments neither consider the genetic cause of the diseases nor address the individual variability in drug response. Thus, ongoing research aims at the identification of repurposed drugs alternative to mexiletine and dichlorphenamide to allow personalization of treatment. This review highlights how drug repurposing may represent an efficient strategy in rare diseases, allowing reduction of drug development time and costs in a context in which the return on investment may be particularly challenging.

Myotonia congenita (MC) is an inherited rare disease characterized by impaired muscle relaxation after contraction, resulting in muscle stiffness. It is caused by loss-of-function mutations in the skeletal muscle chloride channel ClC-1, important for the stabilization of resting membrane potential and for the repolarization phase of action potentials. Thanks to in vitro functional studies, the molecular mechanisms by which ClC-1 mutations alter chloride ion influx into the cell have been in part clarified, classifying them in "gating-defective" or "expression-defective" mutations. To date, the treatment of MC is only palliative because no direct ClC-1 activator is available. An ideal drug should be one which is able to correct biophysical defects of ClC-1 in the case of gating-defective mutations or a drug capable to recover ClC-1 protein expression on the plasma membrane for trafficking-defective ones. In this study, we tested the ability of niflumic acid (NFA), a commercial nonsteroidal anti-inflammatory drug, to act as a pharmacological chaperone on trafficking-defective MC mutants (A531V, V947E). Wild-type (WT) or MC mutant ClC-1 channels were expressed in HEK293 cells and whole-cell chloride currents were recorded with the patch-clamp technique before and after NFA incubation. Membrane biotinylation assays and western blot were performed to support electrophysiological results. A531V and V947E mutations caused a decrease in chloride current density due to a reduction of ClC-1 total protein level and channel expression on the plasma membrane. The treatment of A531V and V947E-transfected cells with 50 µM NFA restored chloride currents, reaching levels similar to those of WT. Furthermore, no significant difference was observed in voltage dependence, suggesting that NFA increased protein membrane expression without altering the function of ClC-1. Indeed, biochemical experiments confirmed that V947E total protein expression and its plasma membrane distribution were recovered after NFA incubation, reaching protein levels similar to WT. Thus, the use of NFA as a pharmacological chaperone in trafficking defective ClC-1 channel mutations could represent a good strategy in the treatment of MC. Because of the favorable safety profile of this drug, our study may easily open the way for confirmatory human pilot studies aimed at verifying the antimyotonic activity of NFA in selected patients carrying specific ClC-1 channel mutations.