Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene1-3. In FCMD, the SVA insertion occurs in the 3′-untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant mRNA splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in FCMD patients. This region spans part of the 3′ end of the fukutin coding region, proximal part of the 3′ UTR, and the SVA insertion. Correspondingly, fukutin mRNA transcripts in FCMD patients and SVA knock-in (KI) model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin C-terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer, and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in FCMD patient cells and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of α-dystroglycan (α-DG) and laminin binding by α-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia4, neutral lipid storage disease5) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known6-8, we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical clinical treatment for FCMD and other SVA-mediated diseases.

Background Hereditary transthyretin (ATTRv) amyloidosis is a rare, inherited, progressive disease caused by mutations in the transthyretin (TTR) gene. We aimed to assess the efficacy and safety of long-term treatment with patisiran, an RNA interference therapeutic that inhibits TTR production, in patients with ATTRv amyloidosis with polyneuropathy. MethodsThis multi-country, multi-centre, open-label extension (OLE) trial enrolled patients at 43 sites in 19 countries as of 24 September 2018. Patients were eligible if they had completed the phase 3 APOLLO (randomised, double-blind, placebo-controlled [2:1], 18-month study) or phase 2 OLE (single-arm, 24-month study) parent studies and tolerated the study drug. Eligible patients from APOLLO (APOLLO-patisiran [received patisiran during APOLLO] and APOLLO-placebo [received placebo during APOLLO] groups) and the phase 2 OLE (phase 2 OLE patisiran group) studies enrolled in this Global OLE trial and receive patisiran 0•3 mg/kg by intravenous infusion every 3 weeks for up to 5 years. Efficacy assessments include measures of polyneuropathy (modified Neuropathy Impairment Score +7 [mNIS+7]), quality of life, autonomic symptoms, nutritional status, disability, ambulation status, motor function, and cardiac stress. Patients included in the current efficacy analyses are those who had completed 12-month efficacy assessments as of the data cut-off. Safety analyses included all patients who received ≥1 dose of patisiran up to the data cut-off. The Global OLE is ongoing with no new enrolment, and current findings are based on the 12-month interim analysis. The study is registered with ClinicalTrials.gov, NCT02510261.

Background Lipoarabinomannan (LAM) is a major antigen of Mycobacterium tuberculosis (MTB). In this report, we evaluated the ability of a novel immunoassay to measure concentrations of LAM in sputum as a biomarker of bacterial load prior to and during treatment in pulmonary tuberculosis (TB) patients. Methods and findings Phage display technology was used to isolate monoclonal antibodies binding to epitopes unique in LAM from MTB and slow-growing nontuberculous mycobacteria (NTM). Using these antibodies, a sandwich enzyme-linked immunosorbent assay (LAM-ELISA) was developed to quantitate LAM concentration. The LAM-ELISA had a lower limit of quantification of 15 pg/mL LAM, corresponding to 121 colony-forming units (CFUs)/mL of MTB strain H37Rv. It detected slow-growing NTMs but without cross-reacting to common oral bacteria. Two clinical studies were performed between the years 2013 and 2016 in Manila, Philippines, in patients without known human immunodeficiency virus (HIV) coinfection. In a case-control cohort diagnostic study, sputum specimens were collected from 308 patients (aged 17-69 years; 62% male) diagnosed as having pulmonary TB diseases or non-TB diseases, but who could expectorate sputum, and were then evaluated by smear microscopy, BACTEC MGIT 960 Mycobacterial Detection System (MGIT) and Lowenstein-Jensen (LJ) culture, and LAM-ELISA. Some sputum specimens were also examined by Xpert MTB/RIF. The LAM-ELISA detected all smear- and MTB-culture–positive samples ( n = 70) and 50% ( n = 29) of smear-negative but culture-positive samples ( n = 58) (versus 79.3%; 46 positive cases by the Xpert MTB/RIF), but none from non-TB patients ( n = 56). Among both LAM and MGIT MTB-culture-positive samples, log 10 -transformed LAM concentration and MGIT time to detection (TTD) showed a good inverse relationship (r = −0.803, p < 0.0001). In a prospective longitudinal cohort study, 40 drug-susceptible pulmonary TB patients (aged 18-69 years; 60% male) were enrolled during the first 56 days of the standard 4-drug therapy. Declines in sputum LAM concentrations correlated with increases of MGIT TTD in individual patients. There was a 1.29 log 10 decrease of sputum LAM concentration, corresponding to an increase of 221 hours for MGIT TTD during the first 14 days of treatment, a treatment duration often used in early bactericidal activity (EBA) trials. Major limitations of this study include a relatively small number of patients, treatment duration up to only 56 days, lack of quantitative sputum culture CFU count data, and no examination of the correlation of sputum LAM to clinical cure. Conclusions These results indicate that the LAM-ELISA can determine LAM concentration in sputum, and sputum LAM measured by the assay may be used as a bi...

Immune responses to parasitic pathogens are affected by the host physiological condition. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) are transporters of lipids between the liver and peripheral tissues, and modulate pro-inflammatory immune responses. Pathogenic mycobacteria are parasitic intracellular bacteria that can survive within macrophages for a long period. Macrophage function is thus key for host defense against mycobacteria. These basic facts suggest possible effects of HDL and LDL on mycobacterial diseases, which have not been elucidated so far. In this study, we found that HDL and not LDL enhanced mycobacterial infections in human macrophages. Nevertheless, we observed that HDL remarkably suppressed production of tumor necrosis factor alpha (TNF-α) upon mycobacterial infections. TNF-α is a critical host-protective cytokine against mycobacterial diseases. We proved that toll-like receptor (TLR)-2 is responsible for TNF-α production by human macrophages infected with mycobacteria. Subsequent analysis showed that HDL downregulates TLR2 expression and suppresses its intracellular signaling pathways. This report demonstrates for the first time the substantial action of HDL in mycobacterial infections to human macrophages.

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