Nuevos tratamientos farmacológicos para la miocardiopatía hipertrófica: foco en inhibidores de miosina / Hypertrophic Cardiomiopathy: new pharmacologic therapies focused on myosin inhibitors

La miocardiopatía hipertrófica (MCH), definida clínicamente como una hipertrofia ventricular izquierda no explicada por sobrecarga hemodinámica ni por infiltración, es la enfermedad miocárdica hereditaria más común. El tratamiento actual se basa en terapias de reducción septal en casos de Miocardiopatía hipertrófica obstructiva (MCHo), prevención de muerte súbita cardíaca mediante desfibrilador automático implantable y en el uso de fármacos para disminuir la obstrucción dinámica del tracto de salida del ventrículo izquierdo. Recientemente se han desarrollado diversos agentes que actúan sobre los mecanismos que, a nivel celular, llevan a la aparición de hipertrofia ventricular. Este artículo se centra en formas de tratamiento farmacológico desarrolladas en los últimos años, en especial con inhibidores de miosina. Se detallan los mecanismos de acción como también los resultados de diversos estudios clínicos de evaluaciones recientes.

Current clinical management of Hypertrophic Cardiomyopathy is mainly based on modifying physiologic mechanisms to decrease left ventricular outflow tract obstruction. Also important are agents that prevent and treat ventricular arrythmias. This review article is focused on the cellular mechanisms that underlie the development of ventricular hypertrophy and the effect of new myosin inhibitors such as mavcamten and aficamten. Promising results of recent clinical trials evaluating the effect of these agents are analyzed. The potential benefits of genetic therapy are also discussed.

Aplicaciones clínicas de la herramienta CRISPR-Cas / Clinical Applications of the CRISPR-Cas Tool

Resumen El desarrollo de tecnologías para la edición del genoma ha abierto la posibilidad de apuntar directamente y modificar secuencias genómicas en casi todo tipo de células eucariotas. La edición del genoma ha ampliado nuestra capacidad para dilucidar la contribución de la genética a las enfermedades al promover la creación de modelos celulares y animales más precisos de procesos patológicos y ha comenzado a mostrar su potencial en una variedad de campos, que van desde la investigación básica hasta la biotecnología aplicada y biomédica. Entre estas tecnologías, el uso de las repeticiones palindrómicas cortas agrupadas regularmente espaciadas ha acelerado, en gran medida, el progreso de la edición de genes desde el concepto hasta la práctica clínica, generando, además, interés debido, no solo a su precisión y eficiencia, sino también a la rapidez y a los costos necesarios para su implementación en comparación con otras tecnologías de edición genómica. En esta revisión se presenta información recabada de publicaciones indexadas en la base de datos PubMed que se encontraron mediante el uso de palabras claves asociadas con la tecnología y que se filtraron para retener solo aquellas con evidencias de avances clínicamente relevantes y que permiten demostrar algunas de las aplicaciones que tiene esta tecnología en la investigación, pronóstico y tratamiento de enfermedades genéticas, cardiovasculares, virales, entre otras; esto con el objetivo de dar a conocer la situación actual de los avances en aplicaciones clínicas de la herramienta CRISPR-Cas y fomentar aún más la investigación en esta tecnología, la cual, tal como se evidencia a lo largo de esta revisión, posee una gran versatilidad y un amplio rango de aplicaciones, lo que ofrece una enorme oportunidad en el campo de la medicina genómica, pero que, a su vez, requiere un mayor fomento en su investigación para mejorar la tecnología y acercarla aún más a consolidar aplicaciones clínicas de uso seguro, confiable y consistente.

Abstract The development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all types of eukaryotic cells. Genome editing has expanded our ability to elucidate the contribution of genetics to disease by promoting the creation of more precise cellular and animal models of disease processes and has begun to show its potential in a variety of fields, ranging from basic research to applied and biomedical biotechnology. Among these technologies, the use of clustered regularly spaced short palindromic repeats have greatly accelerated the progress of gene editing from concept to clinical practice, further generating interest due not only to its precision and efficiency, but also to the speed and costs required for its implementation compared to other genomic editing methods. This review presents information collected from indexed publications in the PubMed database that were found by using keywords associated with the technology and filtered to retain only those with evidence of clinically relevant advances that demonstrate some of the applications that this technology has in research, prognosis, and treatment of genetic, cardiovascular, and viral diseases, among others; this with the aim of show the current situation of advances in clinical applications of the CRISPR-Cas tool and further encourage research in this technology, which, as evidenced throughout this review, has a great versatility and a wide range of applications, which offers an enormous opportunity in the field of genomic medicine but which, in turn, requires greater support in its research to improve the technology and bring it even closer to consolidating clinical applications of safe, reliable and consistent use.

Terapia genética de injeção intravítrea de rAAV2-ND4 para neuropatia óptica hereditária de Leber: uma revisão sistemática / rAAV2-ND4 intravitreal injection gene therapy for Leber's hereditary optic neuropathy: a systematic review

RESUMO A neuropatia óptica hereditária de Leber é uma doença mitocondrial hereditária neurodegenerativa. A taxa potencial de recuperação espontânea é controversa na literatura. A terapia genética tem sido estudada como suporte aos pacientes. O objetivo desta revisão foi avaliar qualitativamente a segurança, os efeitos adversos e a eficácia da terapia gênica disponível. Trata-se de uma revisão sistemática de artigos indexados nas bases de dados PubMed®, Biblioteca Virtual em Saúde, SciELO, Cochrane, ScienceDirect, Scopus e Lilacs no primeiro semestre de 2021. Os critérios de inclusão e filtros foram: artigos relacionados ao tema, estudos randomizados, ensaios clínicos, trabalhos em humanos, últimos 5 anos, nas línguas portuguesa, inglesa e espanhola e texto completo disponível gratuitamente. Os parâmetros de exclusão foram: artigos duplicados, fuga ao tema, artigos de revisão, trabalhos não disponíveis e que fugiam aos critérios de inclusão. O coeficiente de kappa foi 0,812. A terapia não apresentou efeitos adversos sérios em nenhum dos artigos selecionados, e os efeitos menores sofreram 100% de remissão espontânea após o tratamento. Apesar de NAbs terem sido encontrados no soro de alguns pacientes, não houve associação entre a resposta imune adaptativa e a injeção do vetor viral. O tratamento foi eficaz na melhora da acuidade e campo visual. Vários estudos confirmaram a eficácia da terapia gênica, em doses baixas e médias, na melhora da acuidade visual e também sobre os efeitos adversos comuns relacionados à altas doses. A resposta imune humoral e a variação dos NAbs no soro foi citada em mais de um artigo. A terapia foi eficaz na melhora da acuidade visual e os efeitos adversos que surgiram foram tratados facilmente. No entanto, a resposta imune humoral ainda precisa ser estudada.

ABSTRACT Leber's Hereditary Optic Neuropathy (LHON) is an inherited neurodegenerative mitochondrial disease. The potential rate of spontaneous recovery is controversial in the literature. Gene therapy has been studied to support patients. The objective of this review was to qualitatively assess the safety, adverse effects, and efficacy of available gene therapy. This is a systematic review of articles indexed in PubMed®, VHL, SciELO, Cochrane, ScienceDirect, Scopus, and Lilacs databases, in the first half of 2021. Inclusion criteria and filters were: articles related to the topic, randomized studies, clinical trials, work in humans, last 5 years, in Portuguese, English, and Spanish and full text available for free. The exclusion parameters were: duplicate articles, not related to the topic, review articles, not available works, and works that did not meet the inclusion criteria. The kappa coefficient was 0.812. The therapy had no serious adverse effects in any of the selected articles, and minor effects experienced 100% spontaneous remission after treatment. Although NAbs were found in the serum of some patients, there was no association between the adaptive immune response and the injection of the viral vector. The treatment was effective in improving acuity and visual field. Several studies have confirmed the effectiveness of gene therapy, at low and medium doses, in improving visual acuity and also on common adverse effects related to high doses. The humoral immune response and the variation in serum NAbs was cited in more than one article. The therapy was effective in improving visual acuity and the adverse effects that arose were easily treated. However, the humoral immune response still needs to be studied.

Antibióticos innovadores contra la farmacorresistencia bacteriana obtenido por tecnología de edición genética CRISPR/Cas9 / Innovative antibiotics against bacterial drug resistance obtained by CRISPR/cas9 gene editing technology

Introducción: El aumento de la resistencia a los antimicrobianos constituye actualmente una peligrosa amenaza para la salud. Ante este problema global de falta de antimicrobianos, es perentorio intervenir de forma coordinada e idear fórmulas para incentivar la investigación a nivel internacional. Objetivo: Realizar una revisión actualizada sobre las causas y mecanismos de la resistencia a los antibióticos y la adaptación del sistema CRISPR/Cas para el desarrollo de innovadores antimicrobianos como parte esencial de una estrategia altamente específica en el tratamiento de infecciones producidas por bacterias resistentes. Métodos: Se realizó una revisión documental, se empleó la bibliografía nacional e internacional especializada publicada en los últimos 5 años. Se utilizó el motor de búsqueda Google Académico y se consultaron artículos de libre acceso en las bases de datos Pubmed, SciELO, LILACS, CUMED y HINARI, en el período comprendido entre marzo de 2020 hasta el mes de enero de 2021. Se revisaron un total de 41 artículos. Las consultas se hicieron en inglés y español. Para la búsqueda se tuvo en cuenta las palabras clave: eligobióticos; resistencia a antibióticos; CRISPR/Cas. Resultados: La evidencia recopilada sustenta que muchas enfermedades son inducidas por alteraciones del equilibrio de la microbiota humana y la técnica de edición genética CRISPR/Cas permitirá el desarrollo de novedosos antibióticos como los eligobióticos que eliminarán las bacterias patógenas multirresistentes y dejarán intacto el microbioma. Conclusiones: el esclarecimiento de los enigmas de la microbiota y su diseño con terapia génica permitirán el progreso de innovadores antibióticos con empleo del sistema CRISPR/Cas que ineludiblemente modificarán la práctica médica para siempre(AU)

Introduction: The increase in antimicrobial resistance is currently a dangerous threat to health. Faced with this global problem of lack of antimicrobials, it is imperative to intervene in a coordinated manner and devise formulas to encourage research at the international level. Objective: To review on the update causes and mechanisms of antibiotic resistance and the adaptation of CRISPR/Cas system for the development of innovative antimicrobials as an essential part of a highly specific strategy in the treatment of infections caused by resistant bacteria. Methods: A documentary review was carried out in the specialized national and international bibliography published in the last 5 years. Google Scholar search engine was used and free access articles were consulted in Pubmed, SciELO, LILACS, CUMED and HINARI databases, from March 2020 to January 2021. A total of 41 articles were retrieved. The consultations were made in English and Spanish. For the search, we took into account the keywords eligobiotics, antibiotic resistance, CRISPR/Cas. Results: The reviewed evidence supports that many diseases are induced by alterations in the balance of the human microbiota; and CRISPR/Cas gene editing technique will allow the development of novel antibiotics such as eligobiotics that will eliminate multi-resistant pathogenic bacteria and leave the microbiome intact. Conclusions: The clarification of the enigmas of the microbiota and its design with gene therapy will allow the progress of innovative antibiotics using CRISPR/Cas system that will inevitably change medical practice forever(AU)

Farmacoterapia da atrofia muscular espinhal / Pharmacotherapy of spinal muscular atrophy

Realizar uma revisão bibliográfica sobre os medicamentos específicos empregados no tratamento da Atrofia Muscular Espinhal (AME), uma doença genética neurodegenerativa caracterizada por fraqueza nos membros e atrofia muscular. Revisão narrativa de literatura, realizada por meio de um estudo descritivo, com abordagem qualitativa, baseada em estudos por meio de uma seleção classificatória de pesquisas sobre a farmacoterapia da AME. Os fármacos aprovados para o tratamento da AME fazem parte do arsenal da terapia gênica: nusinersena, onasemnogeno abeparvoveque e risdiplam. Com exceção do onasemnogeno abeparvoveque, utilizado em dose única, os demais devem ser utilizados pelo resto da vida. Todos eles, de maneiras distintas, elevam os níveis da proteína SMN (sobrevivência do neurônio motor), cuja deficiência leva à morte dos neurônios motores, causando aos sintomas progressivos da AME. Estes medicamentos apresentam custo elevado e são pouco acessíveis, sendo que apenas o nusinersena é disponibilizado pelo SUS. No momento as alternativas de tratamento farmacológico são escassas e de difícil acesso e a cura, apesar dos esforços da ciência, ainda está distante da realidade. No entanto, a terapia gênica se mostra como um diferencial para o tratamento e controle da AME, representando uma inovação e esperança para os pacientes com esta doença

Nanobubbles: A Novel Targeted Drug Delivery System

Abstract Nanobubbles are nanometer size bubbles having different constituents of varying physicochemical characteristic for the inner core and outer shell. Nanobubbles are mainly fabricated to improve the stability, bioavailability and improve the biodistribution of the delivered drug to the specific targeted site. Their small sizes bubbles allow the possibility of extravasation from blood vessels into the surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. Nanobubbles are developing as important contrast agents for imaging and carriers for drug delivery at targeted region. Sonication is the primary method for preparation of nanobubbles followed by thin-layer evaporation, high shear emulsification, mechanical agitation and coacervation or coalescence. With exposure to ultrasound/extracorporeal shock waves, the drug is liberated from the nanobubbles into the target cells. This review paper is an effort to reveal the different formulation development techniques briefly and varying shell and core content for developing nanobubbles.

O estado da arte das síndromes autoinflamatórias associadas à criopirina / The state of the art of cryopyrin-associated periodic syndromes

As síndromes autoinflamatórias associadas à criopirina (CAPS) compreendem um grupo espectral de doenças raras autoinflamatórias. Todas estas doenças estão relacionadas ao inflamassoma NLRP3, sendo que de 50-60% dos pacientes apresentam mutações ao longo do gene NLRP3. Clinicamente, febre recorrente associada à urticária neutrofílica e outros sintomas sistêmicos são o grande marco clínico, comum a todo o espectro. O bloqueio da interleucina-1 trouxe grande alívio ao tratamento destas desordens, mas variações na resposta clínica podem ser observadas, principalmente nos espectros mais graves. Neste trabalho os autores trazem uma revisão do estado da arte das doenças autoinflamatórias CAPS. Foi realizado levantamento de literatura e, ao final, 49 artigos restaram como base para construção do texto final. O trabalho traz de forma narrativa os principais pontos relacionados a imunofisiopatologia, manifestação clínica, diagnóstico, tratamento, complicações e novas armas diagnósticas, e terapia gênica.

Cryopyrin-associated periodic syndromes (CAPS) comprise a spectrum of rare autoinflammatory disorders. They are all related to the NLRP3 inflammasome, and 50-60% of the patients harbor mutations along the NLRP3 gene. Clinically, recurrent fever associated with neutrophilic urticaria and other systemic symptoms are a hallmark of all the disorders in the spectrum. Biologic drugs that can block interleukin-1 were a milestone for the treatment of such rare diseases, although variability in clinical response to this therapeutic intervention were observed, especially in those affected by severe phenotypes. In this paper, the authors provide a state-of-the-art review of CAPS. A literature search was performed and, finally, 49 articles remained for the construction of the final manuscript. The article presents a narrative review focused on the topics related to immune pathophysiology, clinical manifestations, diagnosis, treatment, complications and new therapeutic options, and gene therapy.

Limitations and advances in new treatments and future perspectives of corneal blindness / Limitações e avanços em novos tratamentos e perspectivas futuras na cegueira corneal

ABSTRACT This review is intended to describe the therapeutic approaches for corneal blindness, detailing the steps and elements involved in corneal wound healing. It also presents the limitations of the actual surgical and pharmacological strategies used to restore and maintain corneal transparency in terms of long-term survival and geographic coverage. In addition, we critically review the perspectives of anabolic agents, including vitamin A, hormones, growth factors, and novel promitotic and anti-inflammatory modulators, to assist corneal wound healing. We discuss the studies involving nanotechnology, gene therapy, and tissue reengineering as potential future strategies to work solely or in combination with corneal surgery to prevent or revert corneal blindness.(AU)

RESUMO O presente trabalho traz uma revisão das abordagens terapêuticas para a cegueira da córnea. O estudo detalha as etapas e os elementos envolvidos na cicatrização da córnea. Ele mostra as limitações das estratégias cirúrgicas e farmacológicas usadas para restaurar e manter a transparência da córnea em termos de sobrevida a longo prazo e alcance geográfico. As perspectivas dos agentes anabólicos, incluindo vitamina A, hormônios, fatores de crescimento e novos moduladores pró-mitóticos e anti-inflamatórios para auxiliar a cicatrização da ferida na córnea, são revisadas criticamente. Aqui, apresentamos estudos envolvendo nanotecnologia, terapia gênica e reengenharia de tecidos como possíveis estratégias futuras para atuar de maneira isolada ou combinada com a cirurgia da córnea para prevenir ou reverter a cegueira corneana.(AU)

Leucemia linfoide crónica: aspectos citogenéticos y moleculares / Chronic Lymphoid Leukemia: cytogenetic and molecular aspects

Introducción: La leucemia linfoide crónica es un trastorno linfoproliferativo caracterizado por la acumulación de linfocitos pequeños de aspecto maduro en sangre periférica, médula ósea y tejidos linfoides con un período de vida prolongado. Presenta una gran variabilidad clínica y genética. Objetivo: Describir los aspectos citogenéticos y moleculares de la leucemia linfoide crónica. Métodos: Se realizó revisión de la literatura en inglés y español, a través del sitio web PubMed y el motor de búsqueda Google académico, de artículos publicados en los últimos 5 años. Se hizo un análisis y resumen de la bibliografía revisada. Desarrollo: En la leucemia linfoide crónica están presentes alteraciones citogenéticas frecuentes como la deleción de los cromosomas 13q, 11q y 17p, así como la trisomía 12, que unido al conocimiento del estado mutacional del gen de la región variable de la cadena pesada de la inmunoglobulina, y otras mutaciones somáticas en diferentes genes, así como a variables clínicas y de laboratorio permiten la estratificación pronóstica de los pacientes. Conclusiones: El diagnóstico a través de los estudios citogenéticos convencionales estimulados con mitógenos, la hibridación in situ por fluorescencia y la secuenciación génica permite una mayor comprensión de la biología de la enfermedad, así como tomar decisiones terapéuticas más personalizadas(AU)

Introduction: Chronic B lymphoid leukemia is a lymphoproliferative disorder characterized by the accumulation of small, mature-looking lymphocytes in peripheral blood, bone marrow and lymphoid tissues with a long life span. It has great clinical and genetic variability. Objective: To describe the cytogenetic and molecular aspects of the disease. Methods: A review of the literature in English and in Spanish was carried out, in the PubMed website and using the search engine of Google Scholar, for articles published in the last five years. We performed analysis and summary of the reviewed bibliography. Development: In chronic lymphoid leukemia, frequent cytogenetic alterations are present such as deletion of chromosomes 13q, 11q and 17p, as well as trisomy 12, which together with the knowledge of the mutational status of the gene for the variable region of the immunoglobulin heavy chain and other somatic mutations in different genes, as well as clinical and laboratory variables allows prognostic stratification of patients. Conclusions: Diagnosis through conventional mitogen-stimulated cytogenetic studies, fluorescence in situ hybridization and gene sequencing allow a better understanding of the biology of the disease, as well as making more personalized therapeutic decisions(AU)

Global publication trends in ophthalmic genetics and gene therapy research: A Scientometric analysis / Tendências globais de publicação de pesquisas sobre genética oftálmica e terapia genética: uma análise cientométrica

ABSTRACT Objective: A scientometric analysis produced in ophthalmic genetics and gene therapy research is lacking. The purpose of this study is to present a holistic analysis of ophthalmic genetics literature. Methods: The data used in this study were obtained from the Web of Science (WoS) Core Collection. All published documents between 1975-2019 were included. The data exported from WoS enabled the extensive details of ophthalmic genetics related literature including countries, institutions, authors, citations and keywords. Scientometric network maps of keywords and also country and institution co-authorships were created with free software. Global contributions of the countries to the ophthalmic genetics literature were shown by a graphic. Results: The search query revealed a total of 2322 documents. Most of the documents were original articles (75.75%). USA was the leading country by producing 45.39% of all documents in ophthalmic genetics research followed by UK, Germany, China and France. Pennsylvania University was the most contributing institution in the literature (5.25%) followed by University College London and Moorfields Eye Hospital. The average citations per item was 29.4. The most used keywords over a 40-year period were 'family', 'cell', 'photoreceptor' and 'expression'. Conclusions: USA and UK dominated the ophthalmic genetics research. A substantial increase in the number of published documents in this field were observed after 2010.

RESUMO Objetivo: A literatura carece de análise cienciométrica produzida em genética oftálmica e de pesquisa em terapia genética. O objetivo deste estudo é apresentar uma análise holística da literatura genética oftálmica. Métodos: Os dados utilizados neste estudo foram obtidos na base de dados Web of Science (WoS) Core Collection. Todos os documentos publicados entre 1975 e 2019 foram incluídos na análise. Os dados exportados da WoS viabilizaram acesso a amplos detalhes da literatura relacionada à genética oftálmica, incluindo países, instituições, autores, citações e palavras-chave. Mapas de rede cienciométrica foram criados por meio de software gratuito, com base em palavras-chave e em coautorias de países e instituições. As contribuições globais dos países para a literatura sobre genética oftálmica foram apresentadas em gráfico. Resultados: a busca por pesquisas revelou um total de 2.322 documentos cuja maioria eram artigos originais (75,75%). Os EUA foram o país que mais produziu artigos sobre o tema, com 45,39% de todos os documentos em pesquisa genética oftálmica; ele foi seguido pelo Reino Unido, Alemanha, China e França. A Universidade da Pensilvânia foi a instituição que mais contribuiu para a literatura (5,25%), e foi seguida pela University College London e pelo Moorfields Eye Hospital. A média de citações por item foi de 29,4. As palavras-chave mais usadas em um período de 40 anos foram 'família', 'célula', 'fotorreceptor' e 'expressão'. Conclusões: Os EUA e o Reino Unido dominaram a pesquisa em genética oftálmica. Após 2010, observou-se um aumento substancial no número de documentos publicados nessa área.

Treatment of patients with immunodeficiency: Medication, gene therapy, and transplantation

Abstract Objectives: To provide an overview of drug treatment, transplantation, and gene therapy for patients with primary immunodeficiencies. Source of data: Non-systematic review of the literature in the English language carried out at PubMed. Synthesis of data: The treatment of patients with primary immunodeficiencies aims to control their disease, especially the treatment and prevention of infections through antibiotic prophylaxis and/or immunoglobulin replacement therapy. In several diseases, it is possible to use specific medications for the affected pathway with control of the condition, especially in autoimmune or autoinflammatory processes associated with inborn immunity errors. In some diseases, treatment can be curative through hematopoietic stem cell transplantation (HSCT); more recently, gene therapy has opened new horizons through new technologies. Conclusions: Immunoglobulin replacement therapy remains the main therapeutic tool. Precision medicine with specific drugs for altered immune pathways is already a reality for several immune defects. Advances in the management of HSCT and gene therapy have expanded the capacity for curative treatments in patients with primary immunodeficiencies.

CRISPR-Cas, el editor de genes / CRISPR, gene editor

El término CRISPR, por su acrónimo en inglés refiere a Clustered Regularly Interspaced Short Palindromic Repeats, es decir, repeticiones palindrómicas cortas, agrupadas y regularmente esparcidas, por sus características en el genoma, pertenece naturalmente al sistema de defensa de bacterias y arqueas. Este ha sido adaptado biotecnológicamente para la edición del ADN de células eucariotas, incluso de células humanas. El sistema CRISPR-Cas para editar genes consta, en forma generalizada, de dos componentes: una proteína nucleasa (Cas) y un ARN guía (sgRNA). La simplicidad del complejo lo hace una herramienta molecular reprogramable capaz de ser dirigida y de editar cualquier sitio en un genoma conocido. Su principal foco son las terapias para enfermedades hereditarias monogénicas y para el cáncer. Sin embargo, además de editor de genes, la tecnología CRISPR se utiliza para edición epigenética, regulación de la expresión génica y método de diagnóstico molecular. Este artículo tiene por objetivo presentar una revisión de las aplicaciones de la herramienta molecular CRISPR-Cas, particularmente en el campo biomédico, posibles tratamientos y diagnósticos, y los avances en investigación clínica, utilizando terapia génica con CRISPR/Cas más relevantes hasta la fecha. (AU)

CRISPR are Clustered Regularly Interspaced Short Palindromic Repeats, which naturally belong to the defense system of bacteria and archaea. It has been biotechnologically adapted for editing the DNA of eukaryotic cells, including human cells. The CRISPR-Cas system for editing genes generally consists of two components, a nuclease protein (Cas) and a guide RNA (sgRNA). The simplicity of the complex makes it a reprogrammable molecular tool capable of being targeted and editing any site in a known genome. Its main focus is therapies for monogenic inherited diseases and cancer. However, in addition to gene editor, CRISPR technology is used for epigenetic editing, regulation of gene expression, and molecular diagnostic methods. This article aims to present a review of the applications of the CRISPR-Cas molecular tool, particularly in the biomedical field, possible treatments and diagnoses, and the advances in clinical research, using the most relevant CRISPR-Cas gene therapy to date. (AU)

An epidermal growth factor motif of developmental endothelial locus 1 protein inhibits efficient angiogenesis in explanted squamous cell carcinoma in vivo

ABSTRACT Background: Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have long-term effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model. Objective: In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors. Materials and methods: Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis. Results: All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins. Conclusion: These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis. (REV INVEST CLIN. 2021;73(1):39-51)

Anti-micro rna-221 a promising genetic therapy of oral squamous cell carcinoma (scc-25)

Abstract Micro-RNA-221(miR-221) is one of oncogenic miRNAs that plays a vital role in the development and progression of oral cancers. The aim of this study is to introduce a new gene therapy for oral squamous cell carcinoma by blocking the expression of oncogenic miR-221 by its inhibitor. The present work was performed on squamous cell carcinoma cell line SCC-25 and anti-miR-221 was delivered to the cells using an ultrasound micro bubbles. Assessment of the effect of miR-221 inhibitor on SCC-25 cells was done using MTT assay, cell cycle analysis and apoptosis detection. In addition, reverse transcription-polymerase chain reaction was also used to detect the expression -miR-221 and its target genes. Using ANOVA, statistical analysis of the results showed significant inhibition of cell viability with and induction of cell apoptosis of SCC-25 cell line after transfection. Moreover, the expression of miR-221, Epidermal growth factor receptor (EGFR) and CDKNIB/p27 were downregulated without significant difference. Transfection of SCC-25 by inhibitor of miR-221 resulting in blockage of its expression leading to arresting of tumor growth. These results proved the effective role of micro-RNA inhibitors as novel therapeutic agent for oral cancers.

Resumo Micro-RNA-221 (miR-221) é um dos miRNAs oncogênicos que desempenham um papel vital no desenvolvimento e progressão de carcinomas orais. O objetivo deste estudo é apresentar uma nova terapia gênica para o carcinoma epidermóide oral por meio do bloqueio da expressão do miR-221 oncogênico por seu inibidor. O presente trabalho foi realizado na linhagem de células de carcinoma de células escamosas SCC-25 e o anti-miR-221 foi administrado às células usando micro-bolhas de ultrassom. A avaliação do efeito do inibidor miR-221 em células SCC-25 foi feita usando ensaio de MTT, análise do ciclo celular e detecção de apoptose. Além disso, a reação em cadeia da polimerase com transcrição reversa também foi usada para detectar a expressão -miR-221 e seus genes-alvo. Usando ANOVA, a análise estatística dos resultados mostrou inibição significativa da viabilidade celular e indução da apoptose celular da linhagem celular SCC-25 após a transfecção. Além disso, a expressão de miR-221, receptor do fator de crescimento epidérmico (EGFR) e CDKNIB/p27 foram regulados para baixo sem diferença significativa. A transfecção de SCC-25 por inibidor de miR-221 resultou no bloqueio de sua expressão, levando à interrupção do crescimento do tumor. Esses resultados comprovaram o papel eficaz dos inibidores de micro-RNA como novo agente terapêutico para carcinomas orais.

Síndrome de Hiper IgD: espectros clínicos, achados genéticos e condutas terapêuticas / Hyper-IgD syndrome: clinical spectrum, genetic findings, and therapeutic approaches

A deficiência de mevalonato quinase (MVK; MIM #142680; ORPHA #343) é uma doença genética, espectral, rara, associadas a mutações ao longo do gene MVK causando distúrbios na síntese do colesterol, que culminam em: inflamação sistêmica com febre, adenopatia, sintomas abdominais e outros achados clínicos. Enquanto no polo leve da doença os achados mais comuns são febres recorrentes com linfadenopatia, no polo mais grave adiciona-se o acometimento do sistema nervoso central (meningites assépticas, vasculites e atraso do desenvolvimento neuropsicomotor) e do sistema hematopoiético (síndrome de ativação macrofágica). Apesar de inúmeras terapêuticas, os bloqueadores da interleucina-1 ainda são os únicos medicamentos capazes de controlar a doença e de impedir a evolução para amiloidose. Os estudos atuais visam tentar novos tratamentos, como o transplante de células-tronco hematopoiéticas, ou mesmo a terapia gênica.

Mevalonate kinase deficiency (MVK; MIM #142680; ORPHA #343) is a rare spectral genetic disorder linked to mutations along the MVK gene leading to impaired cholesterol synthesis, clinically observed as systemic inflammation with fever, adenopathy, abdominal manifestations, and other clinical findings. While on mild forms recurrent fever with lymphadenopathy is commonly observed, severe forms add to that neurological (aseptic meningitis, vasculitis, and neuropsychomotor developmental delay) and hematopoietic involvement (macrophage activation syndrome). Despite of several therapeutic approaches, blocking interleukin-1 is the only effective method to control the disease and prevent the development of systemic amyloidosis. Ongoing studies aim to test new treatments, such as hematopoietic stem cell transplantation and gene therapy.

Células madre como alternativa al marcapaso transvenoso / Stem cells as an alternative to the transvenous pacemaker

Resumen Objetivo: describir el estado del arte del marcapasos biológico y las perspectivas para crear tejido cardíaco de marcapasos utilizando modernas tecnologías genéticas y de ingeniería de tejidos. Métodos: revisión sistemática de la literatura. Resultados: los marcapasos se han convertido en el tratamiento primordial para cierto tipo de arritmias o bloqueos avanzados sintomáticos. Somos testigos de mejoras continuas en la tecnología del dispositivo, con avances en el diseño del cable, el tamaño del generador, la longevidad de la batería y los algoritmos de software que se han traducido en dispositivos más pequeños con funcionalidad mejorada. En la actualidad existen muchos sistemas implantables de cardioestimulación capaces de reemplazar la función de los marcapasos fisiológicos (seno y nódulos aurículo-ventriculares) que incluyen los recientemente desarrollados marcapasos secuenciales y autoprogramables. En la última década la investigación ha confirmado que el marcapasos biológico se puede crear mediante la terapia génica y la terapia celular. Hoy existen dos enfoques para construir marcapasos biológicos: uno es para introducir genes de marcapasos en células madre mesenquimales, y el otro es para inducir células madre pluripotentes en las células del nódulo sinoauricular. Conclusiones: los marcapasos biológicos, actualmente en la etapa preclínica, podrían ser una alternativa a los dispositivos electrónicos para pacientes seleccionados en el futuro.

Abstract Objective: To describe the state of the art of biological pacemakers and the perspectives for creating cardiac pacing tissue using modern genetic and tissue engineering technologies. Methods: A systematic review of the literature. Results: Pacemakers have become the first line treatment for certain types of arrhythmias and advanced symptomatic blocks. We are witnessing continuous improvements in the technology of the device, with advances in the design of the cable, the size of the generator, the longevity of the battery, as well as the software algorithms that have led to smaller devices with improved functions. There are currently many cardiac stimulation implantable systems capable of replacing the function of physiological pacemakers systems (sinus and atrial-ventricular nodes) that include the recently developed sequential and self-programmable pacemakers. In the last ten years or so, studies have confirmed that biological pacemakers can be created using gene therapy and cell therapy. There are currently to main efforts to construct biological pacemakers. One is to introduce pacemaker genes in mesenchymal stem cells, and the other is to introduce pluripotent stem cells in cells of the sinoatrial node. Conclusions: Biological pacemakers, currently in the pre-clinical stage, could be an alternative to the electronic devices for selected patients in the future.

Terapia gênica de isquemia de membro é uma realidade? / Is gene therapy for limb ischemia a reality?

Resumo O conceito de terapia angiogênica surgiu no início da década de 90, o que pode ser feito com genes que codificam fatores de crescimento para promover a formação de novos vasos e o remodelamento de vasos colaterais. Como o procedimento dessa terapia geralmente consiste em apenas injeções locais de vetores, esse processo é pouco invasivo, rápido e de simples realização. Entretanto, desde as primeiras evidências clínicas do efeito de terapia gênica com o fator de crescimento de endotélio vascular (vascular endothelial growth factor, VEGF) vistos nos pacientes com doença arterial obstrutiva periférica até hoje, apenas dois fármacos de terapia angiogênica foram aprovados, um na Rússia e outro no Japão, o que parece um número muito pequeno diante do grande número de investimentos feitos por meio de estudos pré-clínicos e clínicos. Afinal, podemos considerar que a terapia angiogênica já é uma realidade?

Abstract The concept of angiogenic therapy emerged in the early 1990s. The method employs genes that encode growth factors to promote formation of new vessels and remodeling of collateral vessels. Since the procedure involved in this therapy usually only consists of local injections of vectors, the process is minimally invasive, quick, and simple to perform. However, since the first clinical evidence of the effects of gene therapy with vascular endothelial growth factor (VEGF) was observed in patients with peripheral artery disease, to date only two angiogenic therapy drugs have been approved, one in Russia and another in Japan, which seem a very small number, in view of the large volume of investment made in pre-clinical and clinical studies. After all, can we conclude that angiogenic therapy is a reality?

Intracoronary sarcoplasmic reticulum calcium-atpase gene therapy in advanced heart failure patients with reduced ejection fraction: a prospective cohort study

OBJECTIVE: Heart failure is a progressive and debilitating disease. Intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy may improve the function of cardiac muscle cells. This study aimed to test the hypothesis that intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy can improve outcomes and reduce the number of recurrent and terminal events in advanced heart failure patients with reduced ejection fraction. METHODS: A total of 768 heart failure patients with reduced ejection fraction and New York Heart Association classification II to IV were included in this prospective cohort study. Patients either underwent intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy (CA group, n=384) or received oral placebo (PA group; n=384). Data regarding recurrent and terminal event(s), treatment-emergent adverse effects, and outcome measures were collected and analyzed. RESULTS: After a follow-up period of 18 months, intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy reduced the number of hospital admissions (p=0.001), ambulatory treatments (p=0.0004), and deaths (p=0.024). Additionally, intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy improved the left ventricular ejection fraction (p<0.0001) and Kansas City Cardiomyopathy Questionnaire score (p<0.0001). The number of recurrent and terminal events/patients were higher in the PA group than in the CA group after the follow-up period of 18 months (p=0.015). The effect of the intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy was independent of the confounding variables. No new arrhythmias were reported in the CA group. CONCLUSIONS: Intracoronary sarcoplasmic reticulum calcium-ATPase gene therapy reduces the number of recurrent and terminal events and improves the clinical course of advanced heart failure patients with reduced ejection fraction.

p19Arf sensitizes B16 melanoma cells to interferon-β delivered via mesenchymal stem cells in vitro

The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-β (IFNβ) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNβ was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNβ production. MSCs used to produce IFNβ inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNβ produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNβ and applied as an anti-cancer strategy in combination with p19Arf gene therapy.

Respuesta inmune y terapia gó©nica con vectores virales adenoasociados / Immune response and gene therapy with adenoassociated viral vectors

En los ó;ºltimos aó;±os la terapia gó;©nica se ha posicionado como una opció;n real y segura en el desarrollo de alternativas terapó;©uticas para la cura y la prevenció;n de diferentes enfermedades. Consiste en la inserció;n de material genó;©tico en un tejido o có;©lula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unió;n y entrada a la có;©lula diana, la capacidad de transferencia del material genó;©tico al nó;ºcleo, la habilidad de expresió;n del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluació;n como opció;n terapó;©utica en un amplio nó;ºmero de enfermedades monogó;©nicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducció;n del vector e impiden la expresió;n del gen que transporta, limitando su aplicació;n clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia gó;©nica con vectores AAV. La presencia de NAb depende principalmente de la exposició;n al virus en la naturaleza y varía drásticamente segó;ºn edad, localizació;n geográfica y estado de salud de la persona evaluada.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient’s immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.