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.

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.

Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs.

Circular RNAs (circRNAs) perform diverse functions, including the regulation of transcription, translation, peptide synthesis, macromolecular sequestration and trafficking. Inverted Alu repeats capable of forming RNA:RNA duplexes that bring splice sites together for backsplicing are known to facilitate circRNA generation. However, higher limits of circRNAs produced by a single Alu-rich gene are currently not predictable due to limitations of amplification and analyses. Here, using a tailored approach, we report a surprising diversity of exon-containing circRNAs generated by the Alu-rich Survival Motor Neuron (SMN) genes that code for SMN, an essential multifunctional protein in humans. We show that expression of the vast repertoire of SMN circRNAs is universal. Several of the identified circRNAs harbor novel exons derived from both intronic and intergenic sequences. A comparison with mouse Smn circRNAs underscored a clear impact of primate-specific Alu elements on shaping the overall repertoire of human SMN circRNAs. We show the role of DHX9, an RNA helicase, in splicing regulation of several SMN exons that are preferentially incorporated into circRNAs. Our results suggest self- and cross-regulation of biogenesis of various SMN circRNAs. These findings bring a novel perspective towards a better understanding of SMN gene function.

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.

The Health and Economic Burdens of Lymphatic Filariasis Prior to Mass Drug Administration Programs.

BACKGROUND: The Global Programme to Eliminate Lymphatic Filariasis (GPELF) was launched in 2000 with the goal of eliminating lymphatic filariasis (LF) as a public health problem by 2020. Despite considerable progress, the current prevalence is around 60% of the 2000 figure, with the deadline looming a year away. Consequently, there is a continued need for investment in both the mass drug administration (MDA) and morbidity management programs, and this paper aims to demonstrate that need by estimating the health and economic burdens of LF prior to MDA programs starting in GPELF areas. METHODS: A previously developed model was used to estimate the numbers of individuals infected and individuals with symptomatic disease, along with the attributable number of disability-adjusted life years (DALYs). The economic burden was calculated by quantifying the costs incurred by the health-care system in managing clinical cases, the patients' out-of-pocket costs, and their productivity costs. RESULTS: Prior to the MDA program, approximately 129 million people were infected with LF, of which 43 million had clinical disease, corresponding to a DALY burden of 5.25 million. The average annual economic burden per chronic case was US $115, the majority of which resulted from productivity costs. The total economic burden of LF was estimated at US $5.8 billion annually. CONCLUSIONS: These results demonstrate the magnitude of the LF burden and highlight the continued need to support the GPELF. Patients with clinical disease bore the majority of the economic burden, but will not benefit much from the current MDA program, which is aimed at reducing transmission. This assessment further highlights the need to scale up morbidity management programs.

The Elimination of Neglected Tropical Diseases: A Case Study Exemplifying How Foreign Assistance Funding Can Be Catalytic in Reducing the Burden of Major Global Health Conditions.

In 2006, following direct advocacy and published rationale, the US Agency for International Development (USAID) established a neglected tropical diseases (NTDs) program to support the scale-up of integrated platforms to target the elimination and control of 5 NTDs-lymphatic filariasis, trachoma, onchocerciasis, schistosomiasis, and soil-transmitted helminthiasis. By 2017, more than 2.3 billion NTD treatments had been delivered to at-risk populations in 25 countries, leveraging $19 billion in donated drugs-approximately $26 dollars in donated medicine per $1 spent by USAID. As a result, most of the supported countries are on track to achieve their elimination goals (for lymphatic filariasis and trachoma) by 2020 or 2021 and their control goals soon thereafter. Though "small" when compared to other global health initiatives, this investment proved to be catalytic, and indeed highlights how foreign assistance funding can be transformative, in reducing the burden of major global health conditions such as NTDs.

High-affinity RNA targets of the Survival Motor Neuron protein reveal diverse preferences for sequence and structural motifs.

The Survival Motor Neuron (SMN) protein is essential for survival of all animal cells. SMN harbors a nucleic acid-binding domain and plays an important role in RNA metabolism. However, the RNA-binding property of SMN is poorly understood. Here we employ iterative in vitro selection and chemical structure probing to identify sequence and structural motif(s) critical for RNA-SMN interactions. Our results reveal that motifs that drive RNA-SMN interactions are diverse and suggest that tight RNA-SMN interaction requires presence of multiple contact sites on the RNA molecule. We performed UV crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to identify cellular RNA targets of SMN in neuronal SH-SY5Y cells. Results of HITS-CLIP identified a wide variety of targets, including mRNAs coding for ribosome biogenesis and cytoskeleton dynamics. We show critical determinants of ANXA2 mRNA for a direct SMN interaction in vitro. Our data confirms the ability of SMN to discriminate among close RNA sequences, and represent the first validation of a direct interaction of SMN with a cellular RNA target. Our findings suggest direct RNA-SMN interaction as a novel mechanism to initiate the cascade of events leading to the execution of SMN-specific functions.

Activation of a cryptic 5' splice site reverses the impact of pathogenic splice site mutations in the spinal muscular atrophy gene.

Spinal muscular atrophy (SMA) is caused by deletions or mutations of the Survival Motor Neuron 1 (SMN1) gene coupled with predominant skipping of SMN2 exon 7. The only approved SMA treatment is an antisense oligonucleotide that targets the intronic splicing silencer N1 (ISS-N1), located downstream of the 5' splice site (5'ss) of exon 7. Here, we describe a novel approach to exon 7 splicing modulation through activation of a cryptic 5'ss (Cr1). We discovered the activation of Cr1 in transcripts derived from SMN1 that carries a pathogenic G-to-C mutation at the first position (G1C) of intron 7. We show that Cr1-activating engineered U1 snRNAs (eU1s) have the unique ability to reprogram pre-mRNA splicing and restore exon 7 inclusion in SMN1 carrying a broad spectrum of pathogenic mutations at both the 3'ss and 5'ss of the exon 7. Employing a splicing-coupled translation reporter, we demonstrate that mRNAs generated by an eU1-induced activation of Cr1 produce full-length SMN. Our findings underscore a wider role for U1 snRNP in splicing regulation and reveal a novel approach for the restoration of SMN exon 7 inclusion for a potential therapy of SMA.

Gender-Specific Amelioration of SMA Phenotype upon Disruption of a Deep Intronic Structure by an Oligonucleotide.

Spinal muscular atrophy (SMA), the leading genetic disease of children, is caused by low levels of survival motor neuron (SMN) protein. Here, we employ A15/283, an antisense oligonucleotide targeting a deep intronic sequence/structure, to examine the impact of restoration of SMN in a mild SMA mouse model. We show gender-specific amelioration of tail necrosis upon subcutaneous administrations of A15/283 into SMA mice at postnatal days 1 and 3. We also demonstrate that a modest increase in SMN due to early administrations of A15/283 dramatically improves testicular development and spermatogenesis. Our results reveal near total correction of expression of several genes in adult testis upon temporary increase in SMN during early postnatal development. This is the first demonstration of in vivo efficacy of an antisense oligonucleotide targeting a deep intronic sequence/structure. This is also the first report of gender-specific amelioration of SMA pathology upon a modest peripheral increase of SMN.

An intronic structure enabled by a long-distance interaction serves as a novel target for splicing correction in spinal muscular atrophy.

Here, we report a long-distance interaction (LDI) as a critical regulator of alternative splicing of Survival Motor Neuron 2 (SMN2) exon 7, skipping of which is linked to spinal muscular atrophy (SMA), a leading genetic disease of children and infants. We show that this LDI is linked to a unique intra-intronic structure that we term internal stem through LDI-1 (ISTL1). We used site-specific mutations and Selective 2'-Hydroxyl Acylation analyzed by Primer Extension to confirm the formation and functional significance of ISTL1. We demonstrate that the inhibitory effect of ISTL1 is independent of hnRNP A1/A2B1 and PTB1 previously implicated in SMN2 exon 7 splicing. We show that an antisense oligonucleotide-mediated sequestration of the 3' strand of ISTL1 fully corrects SMN2 exon 7 splicing and restores high levels of SMN and Gemin2, a SMN-interacting protein, in SMA patient cells. Our results also reveal that the 3' strand of ISTL1 and upstream sequences constitute an inhibitory region that we term intronic splicing silencer N2 (ISS-N2). This is the first report to demonstrate a critical role of a structure-associated LDI in splicing regulation of an essential gene linked to a genetic disease. Our findings expand the repertoire of potential targets for an antisense oligonucleotide-mediated therapy of SMA.

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.

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.

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.

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.

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.

A forward genetic screen to explore chloroplast protein import in vivo identifies Moco sulfurase, pivotal for ABA and IAA biosynthesis and purine turnover.

A genetic screen in Arabidopsis was developed to explore the regulation of chloroplast protein import in vivo using two independent reporters representing housekeeping and photosynthetic pre-proteins. We first used 5-enolpyruvylshikimate 3-phosphate synthase (EPSP synthase*), a key enzyme in the shikimic acid pathway, with a mutation that confers tolerance to the herbicide glyphosate. Because the EPSP synthase* pre-protein must be imported for its function, the loss of glyphosate tolerance provided an initial indication of an import deficiency. Second, the fate of GFP fused to a ferredoxin transit peptide (FD5-GFP) was determined. A class of altered chloroplast import (aci) mutants showed both glyphosate sensitivity and FD5-GFP mislocalized to nuclei. aci2-1 was selected for further study. Yellow fluorescent protein (YFP) fused to the transit peptide of EPSP synthase* or the small subunit of Rubisco was not imported into chloroplasts, but also localized to nuclei during protoplast transient expression. Isolated aci2-1 chloroplasts showed a 50% reduction in pre-protein import efficiency in an in vitro assay. Mutants did not grow photoautotrophically on media without sucrose and were small and dark green in soil. aci2-1 and two alleles code for Moco-sulfurase, which activates the aldehyde oxidases required for the biosynthesis of the plant hormones abscisic acid (ABA) and indole-acetic acid (IAA) and controls purine nucleotide (ATP and GTP) turnover and nitrogen recycling via xanthine dehydrogenase. These enzyme activities were not detected in aci2-1. ABA, IAA and/or purine turnover may play previously unrecognized roles in the regulation of chloroplast protein import in response to developmental, metabolic and environmental cues.

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.

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.