Effect of Adding a Six-Week Course of Doxycycline to Intensive Hygiene-Based Care for Improving Lymphedema in a Rural Setting of Mali: A Double-Blind, Randomized Controlled 24-Month Trial.

Lymphedema (LE) is one the most disfiguring chronic manifestations of lymphatic filariasis. Its management relies primarily on limb hygiene and local care. A previous study in Ghana demonstrating a beneficial effect of doxycycline on LE led to the current multicenter trial on the efficacy of doxycycline in filarial LE. A randomized placebo-controlled trial was initiated in two rural health districts in Mali. Patients with LE stages 1-3 were randomized to receive either doxycycline (200 mg/day) or placebo over a 6-week monitored treatment period and were then followed every 6 months for 2 years. Both groups received materials for limb hygiene that was carried out daily for the entire 2-year study. The primary endpoint was lack of progression in LE stage at 24 months. One hundred patients were enrolled in each study arm. The baseline sociodemographic characteristics of each group were largely similar. There was no significant difference at month 24 after treatment initiation in the number of subjects showing progression in LE stage between the two treatment arms (P = 0.5921). Importantly, however, the number of attacks of acute adenolymphangitis (ADLA) was reduced in both arms, but there was no significant difference between the two groups at any follow-up time point (all P >0.23). Doxycycline was well tolerated in those receiving the drug. When added to daily self-administered limb hygiene, a 6-week course of doxycycline (200 mg) was not superior to placebo in increasing the improvement associated with hygiene alone in LE volume, stage, or frequency of ADLA attacks over a 24-month period.

LEDoxy-SL: A Placebo-Controlled, Double-Blind, Randomized, 24-Month Trial of Six Weeks of Daily Doxycycline Plus Hygiene-Based Essential Care for Reducing Progression of Filarial Lymphedema in Sri Lanka.

Morbidity management of filarial lymphedema remains a challenge even during the post-lymphatic filariasis elimination era in Sri Lanka despite provision of the predominantly hygiene-based WHO Essential Package of Care. Because prior studies have suggested that 6 weeks of doxycycline may reduce progression of limb lymphedema, we conducted a randomized, placebo-controlled, superiority study to evaluate this possibility in Sri Lanka. Patients aged 14 to 65 years with lymphedema in one or both legs received either 200 mg of doxycycline daily for 6 weeks or matching placebo. The primary efficacy endpoint was improvement or lack of progression in lymphedema stage at 24 months postenrollment. Secondary endpoints included change in lymphedema stage at 12 and 24 months, frequency of acute adenolymphangitis episodes, and perceived disability measured by the WHO Disability Assessment Schedule 2.0 (WHODAS 2.0). Training and supplies for limb hygiene were provided throughout the study. Two hundred participants (100 in each arm) with lymphedema of Dreyer stages 1 to 3 were enrolled. By the end of the 2-year study, 29% of the doxycycline patients and 34% of those on placebo showed improvement (i.e., a decrease in lymphedema stage), whereas 11% and 15% of the two groups showed worsening of the lymphedema. Adenolymphangitis rates were comparable in the two groups (43 doxycycline and 38 placebo recipients), although attacks lasted slightly longer in placebo patients (6.5 days versus 5.2 days). In both groups, perceived disability improved initially, with partial rebound in the second year. Only 34 adverse events affecting 24 patients (11%) occurred during the 6-week treatment period. Although doxycycline did not significantly impact lymphedema progression in this study, the results clearly indicate that clinical and personal benefits can be obtained from intensive hygiene management alone.

Transcriptome- and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes.

Spinal muscular atrophy (SMA) genes, SMN1 and SMN2 (hereinafter referred to as SMN1/2), produce multiple circular RNAs (circRNAs), including C2A-2B-3-4 that encompasses early exons 2A, 2B, 3 and 4. C2A-2B-3-4 is a universally and abundantly expressed circRNA of SMN1/2. Here we report the transcriptome- and proteome-wide effects of overexpression of C2A-2B-3-4 in inducible HEK293 cells. Our RNA-Seq analysis revealed altered expression of ~ 15% genes (4172 genes) by C2A-2B-3-4. About half of the affected genes by C2A-2B-3-4 remained unaffected by L2A-2B-3-4, a linear transcript encompassing exons 2A, 2B, 3 and 4 of SMN1/2. These findings underscore the unique role of the structural context of C2A-2B-3-4 in gene regulation. A surprisingly high number of upregulated genes by C2A-2B-3-4 were located on chromosomes 4 and 7, whereas many of the downregulated genes were located on chromosomes 10 and X. Supporting a cross-regulation of SMN1/2 transcripts, C2A-2B-3-4 and L2A-2B-3-4 upregulated and downregulated SMN1/2 mRNAs, respectively. Proteome analysis revealed 61 upregulated and 57 downregulated proteins by C2A-2B-3-4 with very limited overlap with those affected by L2A-2B-3-4. Independent validations confirmed the effect of C2A-2B-3-4 on expression of genes associated with chromatin remodeling, transcription, spliceosome function, ribosome biogenesis, lipid metabolism, cytoskeletal formation, cell proliferation and neuromuscular junction formation. Our findings reveal a broad role of C2A-2B-3-4, and expands our understanding of functions of SMN1/2 genes.

Transcriptome- and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes.

Spinal muscular atrophy (SMA) genes, SMN1 and SMN2, produce multiple circular RNAs (circRNAs), including C2A-2B-3-4 that encompasses early exons 2A, 2B, 3 and 4. Here we report the transcriptome- and proteome-wide effects of overexpression of C2A-2B-3-4 in inducible HEK293 cells. Our RNA-Seq analysis revealed altered expression of ~ 15% genes (4,172 genes) by C2A-2B-3-4. About half of the affected genes by C2A-2B-3-4 remained unaffected by L2A-2B-3-4, a linear transcript encompassing exons 2A, 2B, 3 and 4 of SMN1/SMN2. These fifindings underscore the unique role of the structural context of C2A-2B-3-4 in gene regulation. A surprisingly high number of upregulated genes by C2A-2B-3-4 were located on chromosomes 4 and 7, whereas many of the downregulated genes were located on chromosomes 10 and X. Supporting a cross-regulation of SMN1/SMN2 transcripts, C2A-2B-3-4 and L2A-2B-3-4 upregulated and downregulated SMN1/SMN2 mRNAs, respectively. Proteome analysis revealed 61 upregulated and 57 downregulated proteins by C2A-2B-3-4 with very limited overlap with those affected by L2A-2B-3-4. Independent validations confirmed the effect of C2A-2B-3-4 on expression of genes associated with chromatin remodeling, transcription, spliceosome function, ribosome biogenesis, lipid metabolism, cytoskeletal formation, cell proliferation and neuromuscular junction formation. Our findings reveal a broad role of C2A-2B-3-4, a universally expressed circRNA produced by SMN1/SMN2.

Synergistic Effect of an Antisense Oligonucleotide and Small Molecule on Splicing Correction of the Spinal Muscular Atrophy Gene.

Spinal muscular atrophy (SMA) is treated by increasing the level of Survival Motor Neuron (SMN) protein through correction of SMN2 exon 7 skipping or exogenous expression of SMN through gene therapy. Currently available therapies have multiple shortcomings, including poor body-wide distribution, invasive delivery, and potential negative consequences due to high doses needed for clinical efficacy. Here we test the effects of a combination treatment of a splice-correcting antisense oligonucleotide (ASO) Anti-N1 with the small compounds risdiplam and branaplam. We show that a low-dose treatment of Anti-N1 with either compound produces a synergistic effect on the inclusion of SMN2 exon 7 in SMA patient fibroblasts. Using RNA-Seq, we characterize the transcriptomes of cells treated with each compound as well as in combination. Although high doses of each individual treatment trigger widespread perturbations of the transcriptome, combination treatment of Anti-N1 with risdiplam and branaplam results in minimal disruption of gene expression. For individual genes targeted by the 3 compounds, we observe little to no additive effects of combination treatment. Overall, we conclude that the combination treatment of a splice-correcting ASO with small compounds represents a promising strategy for achieving a high level of SMN expression while minimizing the risk of off-target effects.

A super minigene with a short promoter and truncated introns recapitulates essential features of transcription and splicing regulation of the SMN1 and SMN2 genes.

Here we report a Survival Motor Neuron 2 (SMN2) super minigene, SMN2Sup, encompassing its own promoter, all exons, their flanking intronic sequences and the entire 3'-untranslated region. We confirm that the pre-mRNA generated from SMN2Sup undergoes splicing to produce a translation-competent mRNA. We demonstrate that mRNA generated from SMN2Sup produces more SMN than an identical mRNA generated from a cDNA clone. We uncover that overexpression of SMN triggers skipping of exon 3 of SMN1/SMN2. We define the minimal promoter and regulatory elements associated with the initiation and elongation of transcription of SMN2. The shortened introns within SMN2Sup preserved the ability of camptothecin, a transcription elongation inhibitor, to induce skipping of exons 3 and 7 of SMN2. We show that intron 1-retained transcripts undergo nonsense-mediated decay. We demonstrate that splicing factor SRSF3 and DNA/RNA helicase DHX9 regulate splicing of multiple exons in the context of both SMN2Sup and endogenous SMN1/SMN2. Prevention of SMN2 exon 7 skipping has implications for the treatment of spinal muscular atrophy (SMA). We validate the utility of the super minigene in monitoring SMN levels upon splicing correction. Finally, we demonstrate how the super minigene could be employed to capture the cell type-specific effects of a pathogenic SMN1 mutation.

Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.

BACKGROUND: Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay. CONCLUSIONS/SIGNIFICANCE: Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.

Diverse targets of SMN2-directed splicing-modulating small molecule therapeutics for spinal muscular atrophy.

Designing an RNA-interacting molecule that displays high therapeutic efficacy while retaining specificity within a broad concentration range remains a challenging task. Risdiplam is an FDA-approved small molecule for the treatment of spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. Branaplam is another small molecule which has undergone clinical trials. The therapeutic merit of both compounds is based on their ability to restore body-wide inclusion of Survival Motor Neuron 2 (SMN2) exon 7 upon oral administration. Here we compare the transcriptome-wide off-target effects of these compounds in SMA patient cells. We captured concentration-dependent compound-specific changes, including aberrant expression of genes associated with DNA replication, cell cycle, RNA metabolism, cell signaling and metabolic pathways. Both compounds triggered massive perturbations of splicing events, inducing off-target exon inclusion, exon skipping, intron retention, intron removal and alternative splice site usage. Our results of minigenes expressed in HeLa cells provide mechanistic insights into how these molecules targeted towards a single gene produce different off-target effects. We show the advantages of combined treatments with low doses of risdiplam and branaplam. Our findings are instructive for devising better dosing regimens as well as for developing the next generation of small molecule therapeutics aimed at splicing modulation.

A refined and updated health impact assessment of the Global Programme to Eliminate Lymphatic Filariasis (2000-2020).

BACKGROUND: Lymphatic filariasis (LF) is a neglected tropical disease (NTD). In 2000 the World Health Organization (WHO) established the Global Programme to Eliminate Lymphatic Filariasis (GPELF). A key component of this programme is mass drug administration (MDA). Between 2000 and 2020, the GPELF has delivered over 8.6 billion treatments to at-risk populations. The last impact assessment of the programme evaluated the treatments provided between 2000-2014. The goal of this analysis is to provide an updated health impact assessment of the programme, based on the numbers treated between 2000-2020. METHODS: We updated and refined a previously established model that estimates the number of clinical manifestations and disability-adjusted life years (DALYs) averted by the treatments provided by the GPELF. The model comprises three different population cohorts that can benefit from MDA provided (those protected from acquiring infection, those with subclinical morbidity prevented from progressing and those with clinical disease alleviated). The treatment numbers were updated for all participating countries using data from the WHO. In addition, data relating to the estimated number of individuals initially at risk of LF infection were updated where possible. Finally, the DALY calculations were refined to use updated disability weights. RESULTS: Using the updated model and corresponding treatment data, we projected that the total benefit cohort of the GPELF (2000-2020) would consist of approximately 58.5 million individuals and the programme would avert 44.3 million chronic LF cases. Over the lifetime of the benefit cohorts, this corresponded to 244 million DALYs being averted. CONCLUSION: This study indicates that substantial health benefits have resulted from the first 20 years of the GPELF. It is important to note that the GPELF would have both additional benefits not quantified by the DALY burden metric as well as benefits on other co-endemic diseases (such as soil-transmitted helminths, onchocerciasis and scabies)-making the total health benefit underestimated. As with the past impact assessments, these results further justify the value and importance of continued investment in the GPELF.

Control and elimination of lymphatic filariasis in Oceania: Prevalence, geographical distribution, mass drug administration, and surveillance in Samoa, 1998-2017.

Lymphatic filariasis (LF) is a major public health problem globally and in the Pacific Region. The Global Programme to Eliminate LF has made great progress but LF is persistent and resurgent in some Pacific countries and territories. Samoa remains endemic for LF despite elimination efforts through multiple two-drug mass drug administrations (MDA) since 1965, including renewed elimination efforts started in 1999 under the Pacific Programme for Elimination of LF (PacELF). Despite eight rounds of national and two rounds of subnational MDA under PacELF, Samoa failed transmission assessment surveys (TAS) in all three evaluation units in 2017. In 2018, Samoa was the first to distribute countrywide triple-drug MDA using ivermectin, diethylcarbamazine (DEC), and albendazole. This paper provides a review of MDAs and historical survey results from 1998 to 2017 in Samoa and highlights lessons learnt from LF elimination efforts, including challenges and potential ways to overcome them to successfully achieve elimination.

High Concentration of an ISS-N1-Targeting Antisense Oligonucleotide Causes Massive Perturbation of the Transcriptome.

Intronic splicing silencer N1 (ISS-N1) located within Survival Motor Neuron 2 (SMN2) intron 7 is the target of a therapeutic antisense oligonucleotide (ASO), nusinersen (Spinraza), which is currently being used for the treatment of spinal muscular atrophy (SMA), a leading genetic disease associated with infant mortality. The discovery of ISS-N1 as a promising therapeutic target was enabled in part by Anti-N1, a 20-mer ASO that restored SMN2 exon 7 inclusion by annealing to ISS-N1. Here, we analyzed the transcriptome of SMA patient cells treated with 100 nM of Anti-N1 for 30 h. Such concentrations are routinely used to demonstrate the efficacy of an ASO. While 100 nM of Anti-N1 substantially stimulated SMN2 exon 7 inclusion, it also caused massive perturbations in the transcriptome and triggered widespread aberrant splicing, affecting expression of essential genes associated with multiple cellular processes such as transcription, splicing, translation, cell signaling, cell cycle, macromolecular trafficking, cytoskeletal dynamics, and innate immunity. We validated our findings with quantitative and semiquantitative PCR of 39 candidate genes associated with diverse pathways. We also showed a substantial reduction in off-target effects with shorter ISS-N1-targeting ASOs. Our findings are significant for implementing better ASO design and dosing regimens of ASO-based drugs.

Baseline Mapping of Neglected Tropical Diseases in Africa: The Accelerated WHO/AFRO Mapping Project.

Mapping is a prerequisite for effective implementation of interventions against neglected tropical diseases (NTDs). Before the accelerated World Health Organization (WHO)/Regional Office for Africa (AFRO) NTD Mapping Project was initiated in 2014, mapping efforts in many countries were frequently carried out in an ad hoc and nonstandardized fashion. In 2013, there were at least 2,200 different districts (of the 4,851 districts in the WHO African region) that still required mapping, and in many of these districts, more than one disease needed to be mapped. During its 3-year duration from January 2014 through the end of 2016, the project carried out mapping surveys for one or more NTDs in at least 2,500 districts in 37 African countries. At the end of 2016, most (90%) of the 4,851 districts had completed the WHO-required mapping surveys for the five targeted Preventive Chemotherapy (PC)-NTDs, and the impact of this accelerated WHO/AFRO NTD Mapping Project proved to be much greater than just the detailed mapping results themselves. Indeed, the AFRO Mapping Project dramatically energized and empowered national NTD programs, attracted donor support for expanding these programs, and developed both a robust NTD mapping database and data portal. By clarifying the prevalence and burden of NTDs, the project provided not only the metrics and technical framework for guiding and tracking program implementation and success but also the research opportunities for developing improved diagnostic and epidemiologic sampling tools for all 5 PC-NTDs-lymphatic filariasis, onchocerciasis, schistosomiasis, soil-transmitted helminthiasis, and trachoma.

Medicine donation programmes supporting the global drive to end the burden of neglected tropical diseases.

Neglected tropical diseases (NTDs) are targeted for global control or elimination. Recognising that the populations most in need of medicines to target NTDs are those least able to support and sustain them financially, the pharmaceutical industry created mechanisms for donating medicines and expertise to affected countries through partnerships with the WHO, development agencies, non-governmental organisations and philanthropic donors. In the last 30 y, companies have established programmes to donate 17 different medicines to overcome the burden of NTDs. Billions of tablets, capsules, intravenous and oral solutions have been donated, along with the manufacturing, supply chains and research necessary to support these efforts. Industry engagement has stimulated other donors to support NTDs with funds and oversight so that the 'heath benefit' return on investment in these programmes is truly a 'best value in public health'. Many current donations are 'open-ended', promising support as long as necessary to achieve defined health targets. Extraordinary global health advances have been made in filariasis, onchocerciasis, trachoma, trypanosomiasis, leishmaniasis, schistosomiasis, intestinal parasites and others; and these advances are taking place in the context of strengthening health systems and meeting the global development goals espoused by the WHO. The pharmaceutical manufacturers, already strong collaborators in initiating or supporting these disease-targeted programmes, have committed to continuing their partnership roles in striving to meet the targets of the WHO's new NTD roadmap to 2030.

Towards a comprehensive research and development plan to support the control, elimination and eradication of neglected tropical diseases.

To maximise the likelihood of success, global health programmes need repeated, honest appraisal of their own weaknesses, with research undertaken to address any identified gaps. There is still much to be learned to optimise work against neglected tropical diseases. To facilitate that learning, a comprehensive research and development plan is required. Here, we discuss how such a plan might be developed.

Setting the stage for a Global Programme to Eliminate Lymphatic Filariasis: the first 125 years (1875-2000).

The development of the World Health Organization's Global Programme to Eliminate Lymphatic Filariasis (GPELF) can be interpreted through many different lenses-e.g. one focusing on the health or economic plight of affected individuals and populations, another tracking the individuals and organizations responsible for building the programme or, as in this review, one identifying each of the critical requirements and specific hurdles that need to be addressed in order to successfully construct the programme. For almost 75 y after the life cycle of LF was first described, the principal tool for countering it was vector control. Discovery that diethylcarbamazine (and later ivermectin and albendazole) could effectively treat affected and at-risk populations, along with the availability of a simple, field-based diagnostic test to monitor programme progress, provided the essential tools for LF elimination. Recognition of this potential by the global health community (including the World Health Assembly) led two pharmaceutical companies (GlaxoSmithKline and Merck) to make enormous, unprecedented donations of albendazole and ivermectin to achieve this goal. Additional resource support from the public and private sectors and from health ministries in the 80 LF-endemic countries led to the creation of a Global Alliance to Eliminate LF, which launched the GPELF in 2000, just 125 y after the LF life cycle was first described.

Evolution of the monitoring and evaluation strategies to support the World Health Organization's Global Programme to Eliminate Lymphatic Filariasis.

The Global Programme to Eliminate Lymphatic Filariasis (GPELF) was established with the ambitious goal of eliminating LF as a public health problem. The remarkable success of the GPELF over the past 2 decades in carrying out its principal strategy of scaling up and scaling down mass drug administration has relied first on the development of a rigorous monitoring and evaluation (M&E) framework and then the willingness of the World Health Organization and its community of partners to modify this framework in response to the practical experiences of national programmes. This flexibility was facilitated by the strong partnership that developed among researchers, LF programme managers and donors willing to support the necessary research agenda. This brief review summarizes the historical evolution of the GPELF M&E strategies and highlights current research needed to achieve the elimination goal.

The First Orally Deliverable Small Molecule for the Treatment of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is 1 of the leading causes of infant mortality. SMA is mostly caused by low levels of Survival Motor Neuron (SMN) protein due to deletion of or mutation in the SMN1 gene. Its nearly identical copy, SMN2, fails to compensate for the loss of SMN1 due to predominant skipping of exon 7. Correction of SMN2 exon 7 splicing by an antisense oligonucleotide (ASO), nusinersen (Spinraza™), that targets the intronic splicing silencer N1 (ISS-N1) became the first approved therapy for SMA. Restoration of SMN levels using gene therapy was the next. Very recently, an orally deliverable small molecule, risdiplam (Evrysdi™), became the third approved therapy for SMA. Here we discuss how these therapies are positioned to meet the needs of the broad phenotypic spectrum of SMA patients.

Characteristics of circular RNAs generated by human Survival Motor Neuron genes.

Circular RNAs (circRNAs) belong to a diverse class of stable RNAs expressed in all cell types. Their proposed functions include sponging of microRNAs (miRNAs), sequestration and trafficking of proteins, assembly of multimeric complexes, production of peptides, and regulation of transcription. Backsplicing due to RNA structures formed by an exceptionally high number of Alu repeats lead to the production of a vast repertoire of circRNAs by human Survival Motor Neuron genes, SMN1 and SMN2, that code for SMN, an essential multifunctional protein. Low levels of SMN due to deletion or mutation of SMN1 result in spinal muscular atrophy (SMA), a major genetic disease of infants and children. Mild SMA is also recorded in adult population, expanding the spectrum of the disease. Here we review SMN circRNAs with respect to their biogenesis, sequence features, and potential functions. We also discuss how SMN circRNAs could be exploited for diagnostic and therapeutic purposes.