Gene replacement therapy for spinal muscular atrophy: safety and preliminary efficacy in a Brazilian cohort.

Spinal muscular atrophy (SMA) is a motor neuron disease associated with progressive muscle weakness, ventilatory failure, and reduced survival. Onasemnogene abeparvovec is the first gene replacement therapy (GT) approved to treat this condition. An observational retrospective study was conducted to assess adverse events and efficacy of GT in SMA patients. Forty-one patients with SMA (58.5% females and 80.1% SMA type 1) were included. The mean age at GT dosing was 18 (±6.4) months. Thirty-six patients (87.8%) were under previous treatment with nusinersen, and 10 (24.4%) continued nusinersen after GT. Mean CHOP-INTEND increased 13 points after 6 months and this finding did not differ between groups according to nusinersen maintenance after GT (p = 0.949). Among SMA type 1 patients, 14 (46.6%) reached the ability to sit alone. Liver transaminases elevation at least two times higher than the upper limit of normal value occurred in 29 (70.7%) patients. Thrombocytopenia occurred in 13 (31.7%) patients, and one presented thrombotic microangiopathy. Older age (>2 years) was associated with more prolonged use of corticosteroids (p = 0.021). GT is effective in SMA patients, combined nusinersen after GT did not appear to add gain in motor function and older age is associated with prolonged corticosteroid use.

Real-life experience with inotersen at CEPARM, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro.

BACKGROUND: Hereditary transthyretin amyloidosis (ATTRv) is an inherited, progressive, and fatal disease still largely underdiagnosed. Mutations in the transthyretin (TTR) gene cause the TTR protein to destabilize, misfold, aggregate, and deposit in body tissues, which makes ATTRv a disease with heterogeneous clinical phenotype. OBJECTIVE: To describe the long-term efficacy and safety of inotersen therapy in patients with ATTRv peripheral neuropathy (ATTRv-PN). METHODS: Patients who completed the NEURO-TTR pivotal study and the NEURO-TTR OLE open-label extension study migrated to the present study and were followed-up for at least 18 more months to an average of 67 months and up to 76 months since day 1 of the inotersen therapy (D1-first dose of inotersen). Disease progression was evaluated by standard measures. RESULTS: Ten ATTRv-PN patients with Val30Met mutation were included. The mean disease duration on D1 was of 3 years, and the mean age of the patients was of 46.8 years. During an additional 18-month follow up, neurological function, based on the Neuropathy Impairment Score and the Polyneuropathy Disability Score, functionality aspects (Karnofsky Performance Status), and nutritional and cardiac aspects were maintained. No new safety signs have been noted. CONCLUSION: The treatment with inotersen was effective and well tolerated for the average of 67 months and up to 76 months. Our results are consistent with those of larger phase-III trials.

ANTECEDENTES: Amiloidose hereditária por transtirretina (ATTRv) é uma doença hereditária, progressiva e fatal ainda largamente subdiagnosticada. Mutações no gene transtirretina (TTR) promovem desestabilização, desdobramento, agregação e depósito da proteína TTR em tecidos do corpo, o que faz da ATTRv uma doença de fenótipo clínico heterogêneo. OBJETIVO: Descrever a eficácia e segurança da terapia com inotersena no longo prazo em pacientes com neuropatia periférica ATTRv (ATTRv-PN). MéTODOS: Pacientes que completaram o estudo pivotal NEURO-TTR e o estudo de extensão aberta NEURO-TTR OLE migraram para este estudo e foram acompanhados por no mínimo 18 meses adicionais, em média por 67 meses, e por até 76 meses, desde o dia 1 da terapia com inotersena (D1­primeira dose de inotersena). A progressão da doença foi avaliada por medidas padronizadas. RESULTADOS: Dez pacientes com ATTRv-PN com mutação Val30Met foram incluídos. A duração média da doença no D1 era de 3 anos, e a média de idade dos pacientes era de 46,8 anos. Durante o período de acompanhamento adicional de 18 meses, a função neurológica, baseada no Neuropathy Impairment Score e no Polyneuropathy Disability Score, os aspectos de funcionalidade (Karnofsky Performance Status), nutricional e cardíacos estavam mantidos. Não se observou nenhum novo sinal de segurança. CONCLUSãO: O tratamento com inotersena foi eficaz e bem tolerado por 67 meses em média, e por até 76 meses. Nossos resultados são consistentes com os de estudos maiores de fase III.

Hereditary Transthyretin Amyloidosis and the Impact of Classic and New Treatments on Kidney Function: A Review.

Hereditary transthyretin amyloidosis (ATTRv) is a rare, progressive, and life-threatening disease caused by misfolded transthyretin (TTR) proteins that aggregate as abnormal amyloid fibrils and accumulate throughout the body. The kidney is one of the main organs affected in amyloid light chain (AL) amyloidosis and ATTRv amyloidosis. The most common clinical presentation is proteinuria, which consists mainly of albumin; this is the first step in the natural history of ATTRv nephropathy. Not all TTR mutations are equal in terms of ATTRv kidney involvement. Kidney involvement in ATTRv itself is difficult to define, given the numerous associated confounding factors. There are several treatments available to treat ATTRv, including orthotopic liver transplant (OLT), which is the classic treatment for ATTRv. However, we should be careful regarding the use of calcineurin inhibitors in the setting of OLT because these can be nephrotoxic. New treatments for amyloidosis may have an impact on kidney function, including drugs that target specific pathways involved in the disease. Tafamidis and diflunisal, which are TTR stabilizers, patisiran (RNA interference agent), and inotersen (antisense oligonucleotide inhibitor) have been shown to reduce TTR amyloid. Tafamidis and patisiran are medications that have reduced the progression of kidney disease in amyloidosis, but inotersen and diflunisal may damage kidney function.

Circ-ABCA1 promotes oxidized low-density lipoprotein-induced inflammation and phenotypic switch in vascular smooth muscle cells.

OBJECTIVE: Atherosclerosis (AS) is a chronic inflammatory disease of the arterial wall, in which Human Vascular Smooth Muscle Cells (HVSMCs) are involved. Nevertheless, the functions and mechanisms of circRNAs in oxidized Low-Density Lipoprotein (ox-LDL)-induced vascular smooth muscle cells remain unclear. METHODS: Circ-ABCA1 expression was measured in the models of AS. Then, in the vitro model, oligonucleotide transfection was performed, followed by an analysis of VSMC proliferation, migration, inflammation, and phenotypic switch. Also, in the in vivo model, mice were injected with shRNA lentivirus, followed by histological examination of aortic tissues. Finally, the interaction of circ-ABCA1, miR-885-5p, and ROCK2 was identified. RESULTS: Circ-ABCA1, was confirmed to be overexpressed in ox-LDL-induced VSMCs and mouse models of AS. Functionally, silencing circ-ABCA1 via oligonucleotide transfection suppressed VSMC proliferation, migration, inflammation, and phenotypic switch in vitro and prevented AS development in mice in vivo. Mechanistically, circ-ABCA1 absorbed miR-885-5p, which targeted ROCK2. CONCLUSION: Taken together, the data from this study suggest that circ-ABCA1 mediates cellular inflammation and phenotype switching through the miR-885-5p/ROCK2 axis in ox-LDL-induced VSMCs, and the circ-ABCA1/miR-885-5p/ROCK2 axis is a new potential biomarker for the treatment of AS.

Aptamer-Based Recognition of Breast Tumor Cells: A New Era for Breast Cancer Diagnosis.

Breast cancer is one of the leading causes of death among women worldwide and can be classified into four major distinct molecular subtypes based on the expression of specific receptors. Despite significant advances, the lack of biomarkers for detailed diagnosis and prognosis remains a major challenge in the field of oncology. This study aimed to identify short single-stranded oligonucleotides known as aptamers to improve breast cancer diagnosis. The Cell-SELEX technique was used to select aptamers specific to the MDA-MB-231 tumor cell line. After selection, five aptamers demonstrated specific recognition for tumor breast cell lines and no binding to non-tumor breast cells. Validation of aptamer specificity revealed recognition of primary and metastatic tumors of all subtypes. In particular, AptaB4 and AptaB5 showed greater recognition of primary tumors and metastatic tissue, respectively. Finally, a computational biology approach was used to identify potential aptamer targets, which indicated that CSKP could interact with AptaB4. These results suggest that aptamers are promising in breast cancer diagnosis and treatment due to their specificity and selectivity.

Cost-Effectiveness of Onasemnogene Abeparvovec Compared With Nusinersen and Risdiplam in Patients With Spinal Muscular Atrophy Type 1 in Brazil: Custo-Efetividade do Onasemnogeno Abeparvoveque (AVXS-101) em Comparação ao Nusinersena e Risdiplam em Pacientes com Atrofia Muscular Espinhal Tipo 1 no Brasil.

OBJECTIVES: This study aimed to evaluate the cost-effectiveness of the onasemnogene abeparvovec in relation to nusinersen and risdiplam in the treatment of spinal muscular atrophy type 1 from the perspective of the Brazilian Unified Health System. METHODS: A Markov model was built on a lifetime horizon. Short-term data were obtained from clinical trials of the technologies and from published cohort survival curves (long term). Costs were measured in current 2022 local currency (R$) values and benefits in quality-adjusted life-years (QALYs). Utility values were derived from type 1 spinal muscular atrophy literature, whereas costs related to technologies and maintenance care in each health state were obtained from official sources of reimbursement in Brazil. Deterministic and probabilistic, as well as scenario, sensitivity analyses were performed. RESULTS: Compared with the less costly strategy (nusinersen), the use of onasemnogene abeparvovec resulted in an incremental cost of R$2.468.448,06 ($975 671.169 - purchasing power parity [PPP]) and a 3-QALY increment and incremental cost-effectiveness ratio of R$742.890,92 ($293 632.774 - PPP)/QALY. Risdiplam had an extended dominance from other strategies, resulting in an incremental cost-effectiveness ratio of R$926.586,22 ($366 239.612 - PPP)/QALY compared with nusinersen. Sensitivity analysis showed a significant impact of the follow-up time of the cohort and the cost of acquiring onasemnogene abeparvovec. CONCLUSIONS: Over a lifetime horizon, onasemnogene abeparvovec seems to be a potentially more effective option than nusinersen and risdiplam, albeit with an incremental cost. Such a trade-off should be weighed in efficiency criteria during decision making and outcome monitoring from the perspective of the Brazilian Unified Health System.

New Allele-Specific Oligonucleotide (ASO) amplifications for Toxoplasma gondii rop18 allele typing: Analysis of 86 human congenital infections in Brazil.

This study aimed to detect and differentiate Toxoplasma gondii by the allele typing of its polymorphic rop18 gene. For this purpose, a novel genotyping system using allele-specific oligonucleotides (ASOs) was designed, consisting of three ASO pairs. The first and third pairs specifically amplify rop18 allele I and allele III, while the second pair amplify both allele I and II. Genomic DNA from 86 congenital infections was analyzed by ASO-PCRs, successfully typing 82 (95.35%) samples. The remaining 4 samples (4.65%) required sequencing and single nucleotide polymorphism (SNP) analysis of the amplification products. The distribution of samples according to rop18 alleles was: 39.5% of allele III, 38.4% of allele II, 19.8% of mixed rop18 alleles (I/III or II/III), and 2.3% of allele I. The six severely compromised infants exhibited the highest parasite load levels and were infected during the first and early second trimesters of pregnancy. Among these cases, two were associated with rop18 allele I parasites, two with mixed rop18 alleles (I/III), one with allele II, and one with allele III parasites. In conclusion, all severe cases of congenital toxoplasmosis were infected during early pregnancy, but they were not exclusively associated with rop18 allele I parasites, as observed in murine toxoplasmosis. Furthermore, nearly one-fifth of parasites were non-archetypal, exhibiting more than one rop18 allele, indicating a higher genetic diversity of Toxoplasma gondii in this South American sample. Overall, a robust T. gondii rop18 allele typing was developed and suggested that congenital toxoplasmosis in humans involves complex mechanisms beyond the parasite genotype.

Monitoramento do horizonte tecnológico: Nusinersena para Atrofia Muscular Espinhal 5q tipo 1 / Technological horizon monitoring: Nusinersen for Spinal Muscular Atrophy 5q type 1

OBJETIVO: Monitorar a implementação, utilização, custo do tratamento e impacto orçamentário do nusinersena para atrofia muscular espinhal (AME) 5q tipo I no SUS. HISTÓRICO DA INCORPORAÇÃO E IMPLEMENTAÇÃO: -Demanda de incorporação de origem externa, solicitada pela Biogen. -Incorporação em abril de 2019, condicionada à reavaliação em três anos após a implementação da tecnologia no SUS (Relatório de recomendação da Conitec nº 449 e Portaria de incorporação SCTIE/MS nº 24, de 24 de abril de 2019). -Protocolo Clínico e Diretrizes Terapêuticas publicado pela Portaria Conjunta SCTIE/SAES/MS nº 15, de 22 de outubro de 2019. -Inclusão do procedimento no Sistema de Gerenciamento da Tabela de Procedimentos, Medicamentos, Órteses/Próteses e Materiais Especiais do SUS pela Portaria SAES/MS nº 1.232, de 22 de outubro de 2019. -Primeiro registro de dispensação da tecnologia no SUS em dezembro de 2019. MÉTODO: Estudo de coorte aberta retrospectiva, realizado com dados de mundo real de dispensação nacionais, referentes ao período de dezembro de 2019 (início da dispensação do medicamento) a dezembro de 2022. Os dados de utilização foram extraídos da Sala Aberta de Inteligência em Saúde (Sabeis), que é originado do Sistema de Informações Ambulatoriais do SUS (SIA/SUS). Os preços unitários dos medicamentos foram consultados na base do Sistema Integrado de Administração de Serviços Gerais (SIASG). UTILIZAÇÃO DO MEDICAMENTO NO SUS: Do primeiro registro, em dezembro de 2019, até dezembro de 2022 foram atendidos 306 usuários. Desses, 72% estavam ativos no tratamento no último ano observado. Na primeira dispensação, os indivíduos eram mais frequentemente do sexo masculino (55%), com menos de dois anos de idade (70%) e residiam na região nordeste (36%). PREÇO DE AQUISIÇÃO OBSERVADO: Foram identificados três registros de compras federais do nusinersena no período avaliado. A primeira aquisição com preço unitário de R$ 159 mil e a segunda e terceira por R$ 160 mil. CUSTO ANUAL DO TRATAMENTO: Considerando o preço de aquisição observado e a posologia do medicamento, o custo esperado para o primeiro ano de tratamento, por usuário, foi R$ 960 mil, incluindo as doses de ataque (0, 14, 28 dias + intervalo de 30 dias) e as doses de manutenção (quadrimestrais). Já no segundo ano de tratamento, só com as doses de manutenção (quadrimestrais), o custo anual esperado, por usuário, foi R$ 480 mil. IMPACTO ORÇAMENTÁRIO OBSERVADO: Estimou-se que o impacto orçamentário observado para o medicamento nusinersena para AME 5q tipo 1 no SUS foi de R$ 80,7 milhões no primeiro ano (12/2019 a 11/2020) para atender 132 usuários com 505 frascos-ampola, R$ 94,9 milhões no segundo ano (12/2020 a 11/2021) para atender 183 usuários com 593 frascos-ampola e R$ 101,8 milhões no terceiro ano (12/2021 a 11/2022) para atender 216 usuários com 636 frascos-ampola. Os valores observados foram inferiores aos estimados no relatório de recomendação, incluindo quantidade de usuários, de frascos-ampola e do preço do medicamento.(AU)

Aptamer-Based Immunotheranostic Strategies.

The escape from immune surveillance is a hallmark of cancer progression. The classic immune checkpoint molecules PD-1, PD-L1, CTLA-4, LAG-3, TIM-3 novel ones are part of a sophisticated system of up- and downmodulation of the immune system, which is unregulated in cancer. In recent years, there have been remarkable advances in the development of targeting strategies, focused principally on immunotherapies aiming at blocking those molecules involved in the evasion of the immune system. However, there are still challenges to predicting their efficacy due to the wide heterogeneity of clinical responses. Thus, there is a need to develop new strategies, and theranostics has much to contribute in this field. Besides that, aptamers have emerged as promising molecules with the potential to generate a huge impact in the immunotheranostic field. They are single-stranded oligonucleotides with a unique self-folding tridimensional structure, with high affinity and specificity for the target. In particular, their small size and physicochemical characteristics make them a versatile tool for designing theranostic strategies. Here, we review the progress in theranostic strategies based on aptamers against immune checkpoints, and highlight the potential of those approaches.

Trypanosoma cruzi antigen detection in blood to assess treatment efficacy and cure in mice models of Chagas disease.

Introduction: Chagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi. Although endemic mainly in Latin America, CD has become a global public health problem due to migration of infected individuals to non-endemic regions. Despite progress made in drug development, preclinical assays for drug discovery are required to accelerate the development of new drugs with reduced side effects, which are much needed for human treatment. Methods: We used a cure model of infected mice treated with Fexinidazole (FZ) to further validate a novel Enzyme Linked Aptamer (ELA) assay that detects parasite biomarkers circulating in the blood of infected animals. Results: The ELA assay showed cure by FZ in ~71% and ~77% of mice infected with the VL-10 and Colombiana strains of T. cruzi, respectively. The ELA assay also revealed superior treatment efficacy of FZ compared to Benznidazole prior to immunosuppression treatment. Discussion: Our study supports the use of ELA assay as an alternative to traditional serology or blood PCR to assess the efficacy of antichagasic drugs during their preclinical phase of development. Further, the combination of high sensitivity and ease of use make this parasite antigen detection assay an attractive new tool to facilitate the development of much needed new therapies for CD.

Development of a Molecular Aptamer Beacon Applied to Magnetic-Assisted RNA Extraction for Detection of Dengue and Zika Viruses Using Clinical Samples.

Limitations in the detection of cocirculating flaviviruses such as Dengue and Zika lead us to propose the use of aptameric capture of the viral RNA in combination with RT-PCR (APTA-RT-PCR). Aptamers were obtained via SELEX and next-generation sequencing, followed by colorimetric and fluorescent characterizations. An APTA-RT-PCR assay was developed, optimized, and tested against the viral RNAs in 108 serum samples. After selection, sequence APTAZC10 was designed as a bifunctional molecular beacon (APTAZC10-MB), exhibiting affinity for the viral targets. APTA-RT-PCR was able to detect Dengue and Zika RNA in 43% and 8% of samples, respectively. Our results indicate that APTAZC10-MB and APTA-RT-PCR will be useful to improve the detection of Dengue and Zika viruses in a fast molecular assay for the improvement of infectious disease surveillance.

Aptamers as Theragnostic Tools in Prostate Cancer.

Despite of the capacity that several drugs have for specific inhibition of the androgen receptor (AR), in most cases, PCa progresses to an androgen-independent stage. In this context, the development of new targeted therapies for prostate cancer (PCa) has remained as a challenge. To overcome this issue, new tools, based on nucleic acids technology, have been developed. Aptamers are small oligonucleotides with a three-dimensional structure capable of interacting with practically any desired target, even large targets such as mammalian cells or viruses. Recently, aptamers have been studied for treatment and detection of many diseases including cancer. In PCa, numerous works have reported their use in the development of new approaches in diagnostics and treatment strategies. Aptamers have been joined with drugs or other specific molecules such as silencing RNAs (aptamer-siRNA chimeras) to specifically reduce the expression of oncogenes in PCa cells. Even though these studies have shown good results in the early stages, more research is still needed to demonstrate the clinical value of aptamers in PCa. The aim of this review was to compile the existing scientific literature regarding the use of aptamers in PCa in both diagnosis and treatment studies. Since Prostate-Specific Membrane Antigen (PSMA) aptamers are the most studied type of aptamers in this field, special emphasis was given to these aptamers.

Determination of association equilibrium constant from single molecule fluorescence localization microscopy.

Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants. In this work, we use as a test system two complementary homo-oligonucleotides, one strand marked with Cyanine 3.5 and the other with Alexa Fluor 647. Hybridization is controlled by the amount of each strand, temperature, and the ionic force, and measured in steady state emission. The same samples are examined in Stochastic Optical Reconstruction Microscopy (STORM) experiments with split-field simultaneous two-colour detection. The effect of multiblinking, labelling-detection efficiency, and determination of the critical distance for association are discussed. We consistently determine values in STORM coincident with those of the bulk experiment.

The interaction of dengue virus capsid protein with negatively charged interfaces drives the in vitro assembly of nucleocapsid-like particles.

Dengue virus (DENV) causes a major arthropod-borne viral disease, with 2.5 billion people living in risk areas. DENV consists in a 50 nm-diameter enveloped particle in which the surface proteins are arranged with icosahedral symmetry, while information about nucleocapsid (NC) structural organization is lacking. DENV NC is composed of the viral genome, a positive-sense single-stranded RNA, packaged by the capsid (C) protein. Here, we established the conditions for a reproducible in vitro assembly of DENV nucleocapsid-like particles (NCLPs) using recombinant DENVC. We analyzed NCLP formation in the absence or presence of oligonucleotides in solution using small angle X-ray scattering, Rayleigh light scattering as well as fluorescence anisotropy, and characterized particle structural properties using atomic force and transmission electron microscopy imaging. The experiments in solution comparing 2-, 5- and 25-mer oligonucleotides established that 2-mer is too small and 5-mer is sufficient for the formation of NCLPs. The assembly process was concentration-dependent and showed a saturation profile, with a stoichiometry of 1:1 (DENVC:oligonucleotide) molar ratio, suggesting an equilibrium involving DENVC dimer and an organized structure compatible with NCLPs. Imaging methods proved that the decrease in concentration to sub-nanomolar concentrations of DENVC allows the formation of regular spherical NCLPs after protein deposition on mica or carbon surfaces, in the presence as well as in the absence of oligonucleotides, in this latter case being surface driven. Altogether, the results suggest that in vitro assembly of DENV NCLPs depends on DENVC charge neutralization, which must be a very coordinated process to avoid unspecific aggregation. Our hypothesis is that a specific highly positive spot in DENVC α4-α4' is the main DENVC-RNA binding site, which is required to be firstly neutralized to allow NC formation.

Mipomersen in Familial Hypercholesterolemia: An Update on Health-Related Quality of Life and Patient-Reported Outcomes.

Familial hypercholesterolemia (FH) is an autosomal dominant condition that leads to significantly elevated low-density lipoprotein cholesterol (LDL-C) levels and an elevated risk for cardiovascular disease. Mipomersen is an antisense oligonucleotide inhibitor targeted to apolipoprotein B-100 (apoB-100) mRNA that is administered via subcutaneous injection. Once administered, mipomersen causes selective degradation of the apoB-100 mRNA and inhibition of protein translation. This ultimately results in substantial reductions in LDL-C and other lipoprotein levels. Mipomersen is approved for the treatment of homozygous FH. In this review, we discuss its mechanism, current evidence, limitations of use including adverse events, and impact on health-related quality of life.

Residue interactions affecting the deprotonation of internal guanine moieties in oligodeoxyribonucleotides, calculated by FMO methods.

The effect of vicinal molecular groups on the intrinsic acidity of a central guanine residue in short single-stranded DNA models and the potentials exerted by the backbone and the nucleobases on the leaving proton were determined by the fragment molecular orbital (FMO) method, in terms of quantum descriptors (QDs) and pair interaction interfragment decomposition analysis (PIEDA). The acidity of the central guanine moiety decreased with increasing oligonucleotide length, in response to changes by less than 1 eV in the ionization potential, global softness, electrophilicity index, and electronegativity descriptors. The differences in these descriptors were majorly interpreted in terms of the electrostatic influence of the negative charges residing on the backbone of the molecule. Additionally, this electric-field effect was determined explicitly for the displacement of the test hydronium ion to a distance of 250 Å from its original position, resulting in good agreement with calculations of the variation in Gibbs free energies, obtained from physical experiments conducted on the identical oligonucleotide sequences. The reported results are useful for biophysical applications of deoxyriboligonucleotides containing guanine residues in order to induce local negative charges at specific positions in the DNA chain.

Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.

Patisiran e Inotersen en amiloidosis familiar mediada por transtiretina / Patisiran and Inotersen in familial amyloidosis transthyretin-mediated

CONTEXTO: La amiloidosis constituye un amplio espectro de enfermedades que se caracterizan por el depósito extracelular de un material fibrilar, generando depósitos insolubles y tóxicos en diferentes tejidos en forma de haces con conformación anómala de láminas beta cruzada, conocida como amiloide. El amiloide acumulado puede provocar daño celular y deterioro de la función de los órganos. Se sabe que al menos 30 proteínas humanas forman fibrillas amiloides. Una de sus formas es, la amiloidosis por transtiretina (ATTR), una enfermedad multisistémica, progresiva, que resulta del mal plegamiento, agregación y depósito de la proteína transportadora transtiretina (TTR), en varios tejidos del cuerpo. La Sociedad Internacional de Amiloidosis ha definido dos formas principales de este tipo de amiloidosis: la amiloidosis ATTR adquirida, denominada amiloidosis ATTRwt (wt por por sus siglas en inglés, wild-type) y la amiloidosis ATTR hereditaria, denominada hATTR (de sus siglas en inglés hereditary transthyretin amyloidosis), que se desarrolla como resultado de mutaciones en el gen TTR; que en consecuencia desestabiliza la proteína. Se estima que su prevalencia global es de 0,87 a 1,1 por millón de personas. Aproximadamente, 50000 personas padecen esta enfermedad en todo el mundo. Su presentación suele darse entre, la tercera y quinta década de la vida y de no tratarse, los síntomas de la ATTR son progresivos pudiendo llegar a causar la muerte, la que suele producirse entre tres y 15 años después de la presentación. TECNOLOGÍA: Los inhibidores de los precursores de amiloide, son fármacos conocidos como oligonucleótidos, que actúan en el interior de la célula a nivel del ARN. Tienen la capacidad de degradar o "silenciar" el ARN mensajero (ARNm), de esta forma, inhiben la síntesis hepática de la proteína TTR por interferência del ARNm. Por consiguiente, se bloquea la expresión de la proteína TTR, reduciendo la síntesis de la proteína precursora del amiloide. OBJETIVO: El objetivo del presente informe es evaluar la evidencia disponible acerca de la eficacia, seguridad y aspectos relacionados a las políticas de cobertura del uso de patisiran e inotersen en pacientes con amiloidosis familiar mediada por transtiretina. MÉTODOS: Se realizó una búsqueda en las principales bases de datos bibliográficas, en buscadores genéricos de internet, y financiadores de salud. Se priorizó la inclusión de revisiones sistemáticas (RS), ensayos clínicos controlados aleatorizados (ECAs), evaluaciones de tecnologías sanitarias (ETS), evaluaciones económicas, guías de práctica clínica (GPC) y políticas de cobertura de diferentes sistemas de salud. RESULTADOS: Se incluyeron tres ECAs, dos RS, tres GPC,siete evaluaciones económicas, y ocho informes de políticas de cobertura de patisirán e inotersen para amiloidosis familiar mediada por transtiretina. CONCLUSIONES: Evidencia de moderada calidad muestra que el uso de patisiran en pacientes con polineuropatia (estadio 1 y estadio 2) por amiloidosis familiar hereditaria mediada por transtiretina, probablemente presente una mejoría considerable de la polineuropatía y calidad de vida con respecto al cuidado usual. Si bien el patisirán presentó efectos adversos leves o moderados, estos fueron bien tolerados. No se observaron diferencias significativas en la mortalidad entre el patisiran y el cuidado usual. Evidencia de moderada calidad muestra que el uso de inotersen en pacientes con polineuropatia (estadio 1 y estadio 2) por amiloidosis familiar hereditaria mediada por trasntiretina, probablemente presente una mejoría considerable de la polineuropatía y calidad de vida con respecto a no usarlo. El uso de inotersen en comparación con el cuidado usual más placebo presentó efectos adversos importantes, tales como a glomerulonefritis y plaquetopenia. No se ha encontrado evidencia que compare en forma directa el patisiran con el inotersen, ni con otras drogas activas para el tratamiento de la amiloidosis familiar hereditaria mediada por transtiretina. Es por ello que la interpretación de la magnitud del beneficio neto de cada una fue realizada de manera indirecta y a través del juicio de valores del equipo de investigación. Por lo que estos efectos "comparativos" deben ser evaluados con cautela debido a las importantes diferencias entre los estudios, en cuanto a su población, tiempo de tratamiento y mínimas diferencias en las escalas utilizadas para la evaluación de la severidad de la polineuropatía. Esta evidencia sugeriría que probablemente el uso de patisiran podría favorecer a una reducción en la progresión de la polineuropatía y una mejoría en la calidad de vida en comparación con el inotersen. Hasta la fecha, no hay estudios que evalúen la seguridad y la eficacia de estas drogas en el subgrupo de pacientes con amiloidosis cardiaca. Solamente existe información basada en análisis de subgrupos de los ensayos clínicos disponibles, de los cuales no se obtuvieron resultados concluyentes. El patisiran e inotersen no se encuentran aprobadas aún en Argentina, por lo que su uso es compassivo o en el contexto de estudios clínicos. Ambas drogas se encuentran aprobadas por la Agencia Europea de Medicamentos y la Administración de Alimentos y Medicamentos de los Estados Unidos. Existe un consenso entre las guías de práctica clínica relevadas en recomendar el uso de patisiran o inotersen para el tratamiento de pacientes con polineuropatía (estadio 1 y estadio 2) en amiloidosis familiar hereditaria medida por transtiretina, considerando que la enfermedad es devastadora tanto para el paciente como para los familiares y que los resultados de los estudios, si bien, no son a largo plazo, fueron significativos para evitar la progresión de la polineuropatía. No se dispone de información económica respecto a estas drogas en Argentina, los datos son relevados de principalmente de sistemas de salud internacionales, tales como el sistema de salud del Reino Unido, que previo a un acuerdo comercial confidencial -por lo que se desconoce el precio convenido- , consideraron que ambas drogas resultaron costo efectivas para el umbral de pago de ese sistema de salud para el caso de enfermedades severas ultrararas, en las que contemplan umbrales de costo-efectividad hasta diez veces superiores al estándar. Sin embargo, no es posible realizar esa asunción para el sistema de salud de Argentina.

Complete Mesoscopic Parameterization of Single LNA Modifications in DNA Applied to Oncogene Probe Design.

The use of mesoscopic models to describe the thermodynamic properties of locked nucleic acid (LNA)-modified nucleotides can provide useful insights into their properties, such as hydrogen-bonding and stacking interactions. In addition, the mesoscopic parameters can be used to optimize LNA insertion in probes, to achieve accurate melting temperature predictions, and to obtain duplex opening profiles at the base-pair level. Here, we applied this type of model to parameterize a large set of melting temperatures for LNA-modified sequences, from published sources, covering all possible nearest-neighbor configurations. We have found a very large increase in Morse potentials, which indicates very strong hydrogen bonding as the main cause of improved LNA thermodynamic stability. LNA-modified adenine-thymine (AT) was found to have similar hydrogen bonding to unmodified cytosine-guanine (CG) base pairs, while for LNA CG, we found exceptionally large hydrogen bonding. In contrast, stacking interactions, which were thought to be behind the stability of LNA, were similar to unmodified DNA in most cases. We applied the new LNA parameters to the design of BRAF, KRAS, and EGFR oncogene variants by testing all possible LNA modifications. Selected sequences were then synthesized and had their hybridization temperatures measured, achieving a prediction accuracy within 1 °C. We performed a detailed base-pair opening analysis to discuss specific aspects of these probe hybridizations that may be relevant for probe design.