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Background: Adult patients with adrenoleukodystrophy have a poor prognosis owing to development of adrenomyeloneuropathy. Additionally, a large proportion of patients with adrenomyeloneuropathy develop life-threatening progressive cerebral adrenoleukodystrophy. Leriglitazone is a novel selective peroxisome proliferator-activated receptor gamma agonist that regulates expression of key genes that contribute to neuroinflammatory and neurodegenerative processes implicated in adrenoleukodystrophy disease progression. We aimed to assess the effect of leriglitazone on clinical, imaging, and biochemical markers of disease progression in adults with adrenomyeloneuropathy. Methods: ADVANCE was a 96-week, randomised, double-blind, placebo-controlled, phase 2–3 trial done at ten hospitals in France, Germany, Hungary, Italy, the Netherlands, Spain, the UK, and the USA. Ambulatory men aged 18–65 years with adrenomyeloneuropathy without gadolinium enhancing lesions suggestive of progressive cerebral adrenoleukodystrophy were randomly assigned (2:1 without stratification) to receive daily oral suspensions of leriglitazone (150 mg starting dose; between baseline and week 12, doses were increased or decreased to achieve plasma concentrations of 200 μg·h/mL [SD 20%]) or placebo by means of an interactive response system and a computer-generated sequence. Investigators and patients were masked to group assignment. The primary efficacy endpoint was change from baseline in the Six-Minute Walk Test distance at week 96, analysed in the full-analysis set by means of a mixed model for repeated measures with restricted maximum likelihood and baseline value as a covariate. Adverse events were also assessed in the full-analysis set. This study was registered with ClinicalTrials.gov, NCT03231878; the primary study is complete; patients had the option to continue treatment in an open-label extension, which is ongoing. Findings: Between Dec 8, 2017, and Oct 16, 2018, of 136 patients screened, 116 were randomly assigned; 62 [81%] of 77 patients receiving leriglitazone and 34 [87%] of 39 receiving placebo completed treatment. There was no between-group difference in the primary endpoint (mean [SD] change from baseline leriglitazone: –27·7 [41·4] m; placebo: –30·3 [60·5] m; least-squares mean difference –1·2 m; 95% CI –22·6 to 20·2; p=0·91). The most common treatment emergent adverse events in both the leriglitazone and placebo groups were weight gain (54 [70%] of 77 vs nine [23%] of 39 patients, respectively) and peripheral oedema (49 [64%] of 77 vs seven [18%] of 39). There were no deaths. Serious treatment-emergent adverse events occurred in 14 (18%) of 77 patients receiving leriglitazone and ten (26%) of 39 patients receiving placebo. The most common serious treatment emergent adverse event, clinically progressive cerebral adrenoleukodystrophy, occurred in six [5%] of 116 patients, all of whom were in the placebo group. Interpretation: The primary endpoint was not met, but leriglitazone was generally well tolerated and rates of adverse events were in line with the expected safety profile for this drug class. The finding that cerebral adrenoleukodystrophy, a life-threatening event for patients with adrenomyeloneuropathy, occurred only in patients in the placebo group supports further investigation of whether leriglitazone might slow the progression of cerebral adrenoleukodystrophy. Funding: Minoryx Therapeutics.

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy, and increased incidence in diabetes. The underlying pathophysiological mechanism of FRDA, driven by a significantly decreased expression of frataxin (FXN), involves increased oxidative stress, reduced activity of enzymes containing iron‑sulfur clus-ters (ISC), defective energy production, calcium dyshomeostasis, and impaired mitochondrial biogenesis, leading to mitochondrial dysfunction. The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcriptional factor playing a key role in mitochondrial function and biogenesis, fatty acid storage, energy metabolism, and antioxidant defence. It has been previously shown that the PPARγ/PPARγ coactivator 1 alpha (PGC-1α) pathway is dysregulated when there is frataxin deficiency, thus contributing to FRDA pathogenesis and supporting the PPARγ pathway as a potential therapeutic target. Here we assess whether MIN-102 (INN: leriglitazone), a novel brain penetrant and orally bioavailable PPARγ agonist with an improved profile for central nervous system (CNS) diseases, rescues phenotypic features in cellular and an-imal models of FRDA. In frataxin-deficient dorsal root ganglia (DRG) neurons, leriglitazone increased frataxin protein levels, reduced neurite degeneration and α-fodrin cleavage mediated by calpain and caspase 3, and increased survival. Leriglitazone also restored mitochondrial membrane potential and partially reversed decreased levels of mitochondrial Na+/Ca2+exchanger (NCLX), resulting in an improvement of mitochon-drial functions and calcium homeostasis. In frataxin-deficient primary neonatal cardiomyocytes, leriglitazone prevented lipid droplet accumulation without increases in frataxin levels. Furthermore, leriglitazone improved motor function deficit in YG8sR mice, a FRDA mouse model. In agreement with the role of PPARγ in mitochondrial biogenesis, leriglitazone significantly increased markers of mitochondrial biogenesis in FRDA patient cells. Overall, these results suggest that targeting the PPARγ pathway by leriglitazone may provide an efficacious therapy for FRDA increasing the mitochondrial function and biogenesis that could increase fra-taxin levels in compromised frataxin-deficient DRG neurons. Alternately, leriglitazone improved the energy metabolism by increasing the fatty acid β-oxidation in frataxin-deficient cardiomyocytes without elevation of frataxin levels. This could be linked to a lack of significant mitochondrial biogenesis and cardiac hypertrophy. This work was supported by Retos-Colaboraci ́on 2017 (RTC-2017- 5867-1), ENISA Jovenes Emprendedores 2012, Torres Quevedo 2017 (PTQ-17-09233) and Region Wallonne (SPW-EER/DRDT/DPjR/DEMO/ ML/D ́ef-7939).

X-linked adrenoleukodystrophy (X-ALD), a potentially fatal neurometabolic disorder with no effective pharmacological treatment, is characterized by clinical manifestations ranging from progressive spinal cord axonopathy [adrenomyeloneuropathy (AMN)] to severe demyelination and neuroinflammation (cerebral ALD-cALD), for which molecular mechanisms are not well known. Leriglitazone is a recently developed brain penetrant full PPARγ agonist that could modulate multiple biological pathways relevant for neuroinflammatory and neurodegenerative diseases, and particularly for X-ALD. We found that leriglitazone decreased oxidative stress, increased adenosine 5'-triphosphate concentration, and exerted neuroprotective effects in primary rodent neurons and astrocytes after very long chain fatty acid-induced toxicity simulating X-ALD. In addition, leriglitazone improved motor function; restored markers of oxidative stress, mitochondrial function, and inflammation in spinal cord tissues from AMN mouse models; and decreased the neurological disability in the EAE neuroinflammatory mouse model. X-ALD monocyte-derived patient macrophages treated with leriglitazone were less skewed toward an inflammatory phenotype, and the adhesion of human X-ALD monocytes to brain endothelial cells decreased after treatment, suggesting the potential of leriglitazone to prevent the progression to pathologically disrupted blood-brain barrier. Leriglitazone increased myelin debris clearance in vitro and increased myelination and oligodendrocyte survival in demyelination-remyelination in vivo models, thus promoting remyelination. Last, leriglitazone was clinically tested in a phase 1 study showing central nervous system target engagement (adiponectin increase) and changes on inflammatory biomarkers in plasma and cerebrospinal fluid. The results of our study support the use of leriglitazone in X-ALD and, more generally, in other neuroinflammatory and neurodegenerative conditions.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal disorder characterised by the accumulation of a complex pattern of lipids in the lysosomal-late endosomal system. More than 300 disease-causing mutations have been identified so far in the NPC1 and NPC2 genes, including indel, missense, nonsense and splicing mutations. Only one genomic deletion, of more than 23 kb, has been previously reported. We describe two larger structural variants, encompassing NPC1 and flanking genes, as a cause of the disease. QMPSF, SNP inheritance and CytoScan® HD Array were used to confirm and further characterise the presence of hemizygous deletions in two patients. One of the patients (NPC-57) bore a previously described missense mutation (p.T1066N) and an inherited deletion that included NPC1, C18orf8 and part of ANKRD29 gene. The second patient (NPC-G1) had a 1-bp deletion (c.852delT; p.F284Lfs*26) and a deletion encompassing the promoter region and exons 1-10 of NPC1 and the adjacent ANKRD29 and LAMA3. This study characterised two novel chromosomal microdeletions at 18q11-q12 that cause NPC disease and provide insight into missing NPC1 mutant alleles.

The number of mutations identified deep in introns which activate or create novel splice sites resulting in pathogenic pseudoexon inclusion in mRNA continues to grow for inherited metabolic disease (IMD) and other human genetic diseases. A common characteristic is that the native splice sites remain intact thus retaining the potential for normal splicing. Antisense oligonucleotides (AO) have been shown to modulate the splicing pattern by steric hindrance of the recognition and binding of the splicing apparatus to the selected sequences. In the case of pseudoexons, AO force the use of the natural splice sites, recovering normally spliced transcripts encoding functional protein. This review summarizes the present knowledge of antisense splicing modulation as a molecular therapy approach for pseudoexon-activating mutations, with a focus in IMD. Although the feasibility of treatment for patients with IMD has yet to be proven, it appears to be clinically promising, as positive results have been reported in cellular and animal models of disease, and antisense therapy for splicing modulation is currently in the clinical trials phase for Duchenne muscular dystrophy patients. Here, we review the most recent advances in AO stability, targeting and delivery, and other issues to be considered for an effective treatment in the clinical setting. Although the number of patients who can be potentially treated is low for each IMD, it represents an excellent therapeutical option as a type of personalized molecular medicine which is especially relevant for diseases for which there is, to date, no efficient treatment.

Niemann-Pick type C disease is an autosomal recessive disorder caused by mutations in either the NPC1 or NPC2 gene. While most of the mutations are missense, a few splicing mutations have also been described. We identified and characterized a novel point mutation c.1554-1009G>A located in intron 9 of the NPC1 gene in a Spanish patient. Sequencing of the cDNA from the patient showed that this intronic mutation creates a cryptic donor splice site resulting in the incorporation of 194 bp of intron 9 as a new exon (pseudoexon) in the mRNA. This new transcript bears a premature termination codon and is degraded by the nonsense-mediated mRNA decay mechanism. Experimental confirmation that the point mutation generates the inclusion of a pseudoexon in the mRNA was obtained using a minigene. A specific antisense morpholino oligonucleotide targeted to the cryptic splice site was designed and transfected into fibroblasts from the patient. Using this approach, normal splicing was restored. These results demonstrate the importance of screening deep intronic regions and support the efficacy of antisense therapeutics for the treatment of diseases caused by pseudoexon-generating mutations.

Pharmacological chaperone therapy (PCT) is a rather new approach consisting in targeting incorrectly folded proteins by small molecules, thus, facilitating the correct folding of the protein and inducing a recovery of its functionality. Many diseases result from mutations on specific genes; this patent review focuses on those pathologies where PCT has a potential application for enzymatic enhancement. Rare diseases are the main area where PCT has been applied and the most advanced compounds are aiming to cure lysosomal storage disorders such as Fabry, Pompe or Gaucher. Until now, most compounds used as pharmacological chaperones were based on substrate-like chemical structures but recently new nonsubstrate-like and non-inhibitory compounds have been disclosed for Gaucher and Pompe diseases. This initiates a new era for pharmacological chaperones with more diverse chemical structures and binding modes. This review covers the patents relating to enzyme enhancement on pharmacological chaperone therapy. Only an update is presented for Gaucher disease, where PCT is highly applied and recently reviewed.

Niemann–Pick type C (NPC) disease is a fatal autosomal recessive neurodegenerative disorder caused, most commonly, by mutations in the NPC1 gene. At the cellular level, the disease is characterized by the storage of multiple lipids in the endosomal–lysosomal system, including free cholesterol, glycosphingolipids, sphingomyelin and the catabolic product of sphingolipids, sphingosine. Therapeutic options for NPC disease are relatively limited. One drawback for the development of novel therapies is the lack of suitable human neuronal cell models. In this work, a stable SH-SY5Y cell model for NPC disease was generated using short hairpin RNAs. An inhibition of the NPC1 expression of around 90% was obtained at the RNA level. The NPC1 knockdown was confirmed at the protein level. To characterize the stable cell line generated, cholesterol levels were analyzed in the NPC1-knockdown SH-SY5Y cells by filipin staining and gas chromatography–mass spectrometry. A characteristic NPC pattern and a twofold increase of the free cholesterol levels, related to intact SH-SY5Y cells, were found. Moreover, sphingolipids were analyzed by liquid chromatography–mass spectrometry and an increase in ganglioside GM2 levels was observed. The stable NPC1-knockdown SH-SY5Y cell line generated in the present study provides a human neuronal cell model for this lethal disease that could be a valuable tool for the study of future therapeutic approaches.

Mutations in the NPC1 or NPC2 gene are responsible for Niemann-Pick type C (NPC) disease (OMIM #257220), an autosomal recessive neurodegenerative lysosomal storage disorder caused by an incorrect regulation of intracellular lipid trafficking. A molecular analysis carried out in 30 unrelated patients identified 43 distinct mutations in the NPC1 gene, 12 of which had not been previously described. The novel NPC1 alleles were four amino acid substitutions (p.F995L, p.F1079S, p.L1106P and p.G1209E), a nonsense mutation (p.E1089X), a 1-bp insertion (p.L1117PfsX4), an in-frame deletion (p.N916del), four intronic changes (c.58-3280C>G, c.882-28A>T, c.2604+5G>A and c.3591+5G>A) that affect the splicing mechanism, and the first deletion including the whole gene described in NPC disease. In all the splice site mutations, the formation of abnormal spliced transcripts was confirmed by cDNA analysis, and mRNA degradation by the nonsense-mediated mRNA decay process was also assessed. As it has been previously reported in this disease, genotype-phenotype correlations are limited due to the large number of private mutations. We describe for the first time one homozygous patient for p.I1061T mutation, who presented the severe infantile clinical onset, and another patient with the variant biochemical phenotype, whose clinical presentation was the neonatal form of the disease.

Niemann-Pick disease (NPD) types A/B are both caused by a deficiency of lysosomal acid sphingomyelinase and display autosomal recessive inheritance. These two types of the disease were described according to the presence (type A) or absence (type B) of neurological symptoms. We present a molecular analysis of 19 Spanish NPD A/B patients and two from Maghreb. Eight of the patients had type A and 13 had type B NPD. All mutant SMPD1 alleles were identified, including 17 different mutations, 10 of which were novel. The only frequent mutations in the 21 NPD patients were c.1823_1825delGCC (p.R608del) (38%) and c.1445C>A (p.A482E) (9%). Genotype-phenotype correlations were established for most of the mutations and, in particular, the p.R608del-type B association was confirmed. This mutation accounts for 61.5% of the mutant alleles in the type B subgroup of patients. Expression studies performed on six of the identified mutations confirmed them to be disease-causing due to their low enzyme activity. An allele with a mutation affecting a non-canonical donor splice site produced only aberrant mRNAs corresponding to previously reported non-functional SMPD1 minor transcripts. This study is the first exhaustive mutational analysis of Spanish Niemann-Pick A/B disease patients.