Viral protein-based nanoparticles (part 2): Pharmaceutical applications

Viral protein nanoparticles (ViP NPs) such as virus-like particles and virosomes are structures halfway between viruses and synthetic nanoparticles. The biological nature of ViP NPs endows them with the biocompatibility, biodegradability, and functional properties that many synthetic nanoparticles lack. At the same time, the absence of a viral genome avoids the safety concerns of viruses. Such characteristics of ViP NPs offer a myriad of opportunities for theirapplication at several points across disease development: from prophylaxis to diagnosis and treatment. ViP NPs present remarkable immunostimulant properties, and thus the vaccination field has benefited the most from these platforms capable of overcoming the limitations of both traditional and subunit vaccines. This was reflected in the marketing authorization of several VLP- and virosome-based vaccines. Besides, ViP NPs inherit the ability of viruses to deliver their cargo to target cells. Because of that, ViP NPs are promising candidates as vectors for drug and gene delivery, and for diagnostic applications. In this review, we analyze the pharmaceutical applications of ViP NPs, describing the products that are commercially available or under clinical evaluation, but also the advances that scientists are making toward the implementation of ViP NPs in other areas of major pharmaceutical interestThis publication is part of the following grants: PID2021–124986OB-I00 (Project “IMMMA”) funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”; AC21_2/00046 (Project “RAIN”) funded by Health Institute Carlos III/MCIN/AEI/ 10.13039/501100011033 and by “European Union NextGenerationEU/EURONANOMED 3; PID2021–126510NB-I00 funded by MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”; and ED431G 2019/02 grant funded by Xunta de Galicia. RMC is a recipient of a predoctoral grant from the Ministerio de Educación y Formación Profesional (FPU20/01525)S

Co-occurrence of mutations in NF1 and other susceptibility genes in pheochromocytoma and paraganglioma

Co-occurrent mutations; Germline mutation; PheochromocytomaMutaciones concurrentes; Mutación de la línea germinal; FeocromocitomaMutacions concurrents; Mutació de la línia germinal; FeocromocitomaIntroduction: The percentage of patients diagnosed with pheochromocytoma and paraganglioma (altogether PPGL) carrying known germline mutations in one of the over fifteen susceptibility genes identified to date has dramatically increased during the last two decades, accounting for up to 35-40% of PPGL patients. Moreover, the application of NGS to the diagnosis of PPGL detects unexpected co-occurrences of pathogenic allelic variants in different susceptibility genes. Methods: Herein we uncover several cases with dual mutations in NF1 and other PPGL genes by targeted sequencing. We studied the molecular characteristics of the tumours with co-occurrent mutations, using omic tools to gain insight into the role of these events in tumour development. Results: Amongst 23 patients carrying germline NF1 mutations, targeted sequencing revealed additional pathogenic germline variants in DLST (n=1) and MDH2 (n=2), and two somatic mutations in H3-3A and PRKAR1A. Three additional patients, with somatic mutations in NF1 were found carrying germline pathogenic mutations in SDHB or DLST, and a somatic truncating mutation in ATRX. Two of the cases with dual germline mutations showed multiple pheochromocytomas or extra-adrenal paragangliomas - an extremely rare clinical finding in NF1 patients. Transcriptional and methylation profiling and metabolite assessment showed an “intermediate signature” to suggest that both variants had a pathological role in tumour development. Discussion: In conclusion, mutations affecting genes involved in different pathways (pseudohypoxic and receptor tyrosine kinase signalling) co-occurring in the same patient could provide a selective advantage for the development of PPGL, and explain the variable expressivity and incomplete penetrance observed in some patients.This work was supported by the Instituto de Salud Carlos III (ISCIII), through the “Acción Estratégica en Salud” (AES) (projects PI18/00454 to AC and PI20/01169 to MR), cofounded by the European Regional Development Fund (ERDF). SM was supported by the Spanish Ministry of Science, Innovation and Universities “Formación del Profesorado Universitario— FPU” fellowship with ID number FPU19/04940

Novel DNMT3A Germline Variant in a Patient with Multiple Paragangliomas and Papillary Thyroid Carcinoma

Over the past few years, next generation technologies have been applied to unravel the genetics of rare inherited diseases, facilitating the discovery of new susceptibility genes. We recently found germline DNMT3A gain-of-function variants in two patients with head and neck paragangliomas causing a characteristic hypermethylated DNA profile. Here, whole-exome sequencing identifies a novel germline DNMT3A variant (p.Gly332Arg) in a patient with bilateral carotid paragangliomas, papillary thyroid carcinoma and idiopathic intellectual disability. The variant, located in the Pro-Trp-Trp-Pro (PWWP) domain of the protein involved in chromatin targeting, affects a residue mutated in papillary thyroid tumors and located between the two residues found mutated in microcephalic dwarfism patients. Structural modelling of the variant in the DNMT3A PWWP domain predicts that the interaction with H3K36me3 will be altered. An increased methylation of DNMT3A target genes, compatible with a gain-of-function effect of the alteration, was observed in saliva DNA from the proband and in one independent acute myeloid leukemia sample carrying the same p.Gly332Arg variant. Although further studies are needed to support a causal role of DNMT3A variants in paraganglioma, the description of a new DNMT3A alteration in a patient with multiple clinical features suggests a heterogeneous phenotypic spectrum related to DNMT3A germline variants

Co-occurrence of mutations in NF1 and other susceptibility genes in pheochromocytoma and paraganglioma

IntroductionThe percentage of patients diagnosed with pheochromocytoma and paraganglioma (altogether PPGL) carrying known germline mutations in one of the over fifteen susceptibility genes identified to date has dramatically increased during the last two decades, accounting for up to 35-40% of PPGL patients. Moreover, the application of NGS to the diagnosis of PPGL detects unexpected co-occurrences of pathogenic allelic variants in different susceptibility genes.MethodsHerein we uncover several cases with dual mutations in NF1 and other PPGL genes by targeted sequencing. We studied the molecular characteristics of the tumours with co-occurrent mutations, using omic tools to gain insight into the role of these events in tumour development.ResultsAmongst 23 patients carrying germline NF1 mutations, targeted sequencing revealed additional pathogenic germline variants in DLST (n=1) and MDH2 (n=2), and two somatic mutations in H3-3A and PRKAR1A. Three additional patients, with somatic mutations in NF1 were found carrying germline pathogenic mutations in SDHB or DLST, and a somatic truncating mutation in ATRX. Two of the cases with dual germline mutations showed multiple pheochromocytomas or extra-adrenal paragangliomas - an extremely rare clinical finding in NF1 patients. Transcriptional and methylation profiling and metabolite assessment showed an “intermediate signature” to suggest that both variants had a pathological role in tumour development.DiscussionIn conclusion, mutations affecting genes involved in different pathways (pseudohypoxic and receptor tyrosine kinase signalling) co-occurring in the same patient could provide a selective advantage for the development of PPGL, and explain the variable expressivity and incomplete penetrance observed in some patients

Delineating the motor phenotype of SGCE-myoclonus dystonia syndrome

Objective To perform phenotype and genotype characterization in myoclonus-dystonia patients and to validate clinical rating tools. Method Two movement disorders experts rated patients with the Burke-Fahn-Marsden and Unified-Myoclonus rating scales using a video-recording protocol. Clinimetric analysis was performed. SGCE mutations were screened by Sanger sequencing and multiplex ligation-dependent probe amplification. Results 48 patients were included and 43/48 rated. Mean age at assessment was 12.9±10.5 years (range 3–51) and 88% were ≤18 years of age. Myoclonus was a universal sign with a rostro-caudal severity-gradient. Myoclonus increased in severity and spread to lower limbs during action tests. Stimulus-evoked myoclonus was observed in 86.8% cases. Dystonia was common but mild. It had a focal distribution and was action-induced, causing writer's cramp (69%) and gait dystonia (34%). The severity of both myoclonus and dystonia had a strong impact on hand writing and walking difficulties. The Unified Myoclonus Rating scale showed the best clinimetric properties for the questionnaire, action myoclonus and functional subscales, and exceeded the Burke-Fahn-Marsden scale in its utility in assessing functional impairment in MDS patients. Twenty-one different SGCE mutations were identified in 45/48 patients, eleven being novel (most prevalent p. Val187*, founder mutation in Canary Islands). Conclusion This study quantifies the severity of the motor phenotype in SGCE-myoclonus dystonia syndrome, with a special focus on children, and identifies disabilities in gross and fine motor tasks that are essential for childhood development. Our results contribute to the knowledge of SGCE-related MDS in the early stage of evolution, where disease-modifying therapies could be initiated in order to prevent long-term social and physical burdens.This work was supported by a Grant from Myoclonus Dystonia Syndrome Spanish association (ALUDME) (grant PIC/124/16), grant FPU16/07203 from Ministerio de Educación y formación profesional, and grant PI15/00287 and PI18/01319 from the Plan Nacional de I + D + I

Analysis of Telomere Maintenance Related Genes Reveals NOP10 as a New Metastatic-Risk Marker in Pheochromocytoma/Paraganglioma

One of the main problems we face with PPGL is the lack of molecular markers capable of predicting the development of metastases in patients. Telomere-related genes, such as TERT and ATRX, have been recently described in PPGL, supporting the association between the activation of immortalization mechanisms and disease progression. However, the contribution of other genes involving telomere preservation machinery has not been previously investigated. In this work, we aimed to analyze the prognostic value of a comprehensive set of genes involved in telomere maintenance. For this study, we collected 165 PPGL samples (97 non-metastatic/63 metastatic), genetically characterized, in which the expression of 29 genes of interest was studied by NGS. Three of the 29 genes studied, TERT, ATRX and NOP10, showed differential expression between metastatic and non-metastatic cases, and alterations in these genes were associated with a shorter time to progression, independent of SDHB-status. We studied telomere length by Q-FISH in patient samples and in an in vitro model. NOP10 overexpressing tumors displayed an intermediate-length telomere phenotype without ALT, and in vitro results suggest that NOP10 has a role in telomerase-dependent telomere maintenance. We also propose the implementation of NOP10 IHC to better stratify PPGL patients