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1.
Rev. Hosp. Ital. B. Aires (2004) ; 41(1): 37-42, mar. 2021. ilus, tab
Article in Spanish | LILACS | ID: biblio-1178964

ABSTRACT

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)


Subject(s)
Humans , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , CRISPR-Cas Systems/genetics , Biotechnology , Genetic Therapy/methods , Gene Expression , Genome, Human/genetics , Gene Expression Regulation , Epigenomics/trends , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/therapeutic use , Genetic Diseases, Inborn/therapy , Neoplasms/therapy
2.
Int. j. morphol ; 37(4): 1564-1571, Dec. 2019. tab
Article in Spanish | LILACS | ID: biblio-1040170

ABSTRACT

Las glándulas salivales humanas pueden ser gravemente lesionadas por la radioterapia utilizada contra neoplasias de cabeza y cuello, produciendo hiposialia y xerostomía, las cuales afectan la salud oral y sistémica, mermando la calidad de vida de la persona. Los tratamientos convencionales actuales están diseñados para disminuir los síntomas, sin actuar sobre los cambios fisiopatológicos que se dan a nivel glandular. Esta revisión intenta analizar aquellas terapias preventivas y/o curativas que están desarrollándose en el campo biomolecular y que tienen un futuro prometedor por sus características innovadoras: terapia génica, terapia con células madre y terapia con factores de crecimiento. Se evidencia un aporte adicional de la nanotecnología, la cual está mejorando las vías de aplicación de los tratamientos.


Human salivary glands can be seriously injured by the radiotherapy used against head and neck neoplasms, producing hyposialia and xerostomy, which affect oral and systemic health, diminishing the person's quality of life. Current conventional treatments are designed to reduce symptoms, without acting on the pathophysiological changes that occur at the glandular level. This review attempts to analyze those preventive and /or curative therapies that are developing in the biomolecular field and that have a promising future due to their innovative features: Gene therapy, stem cell therapy and growth factor therapy. An additional contribution of nanotechnology is evident, which is improving the routes of treatment application.


Subject(s)
Humans , Radiotherapy/adverse effects , Salivary Gland Diseases/prevention & control , Stem Cells/physiology , Genetic Therapy/methods , Intercellular Signaling Peptides and Proteins/therapeutic use , Radiation Injuries/prevention & control , Radiation-Protective Agents/therapeutic use , Salivary Gland Diseases/therapy , Salivary Glands/radiation effects , Xerostomia/prevention & control , Nanotechnology
3.
Medicina (B.Aires) ; 79(6): 493-501, dic. 2019. ilus, tab
Article in Spanish | LILACS | ID: biblio-1056758

ABSTRACT

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.


Subject(s)
Humans , Male , Female , Genetic Therapy/methods , Dependovirus/genetics , Dependovirus/immunology , Parvoviridae Infections/genetics , Parvoviridae Infections/immunology , Parvoviridae Infections/virology , Antibodies, Neutralizing/analysis , Serogroup , Genetic Vectors , Antibodies, Viral/analysis
4.
Medicina (B.Aires) ; 79(supl.3): 77-81, set. 2019. ilus
Article in Spanish | LILACS | ID: biblio-1040555

ABSTRACT

La distrofia muscular de Duchenne es una enfermedad genéticamente determinada, ligada al cromosoma X y caracterizada clínicamente por producir debilidad muscular progresiva, con una incidencia de 1 por cada 3500-6000 varones nacidos. Es causada por la mutaciones en el gen DMD, el cual codifica la distrofina, una proteína sub-sarcolémica esencial para la estabilidad estructural del músculo. Los defectos genéticos en el gen DMD, se dividen en: deleciones (65%) duplicaciones (5-10%) y mutaciones puntuales (10-15%). Actualmente no se dispone de tratamiento curativo, el único fármaco que ha demostrado modificar la historia natural de la enfermedad (independientemente de la mutación genética) son los corticoides, los cuales están indicados en estadios tempranos de la enfermedad. En relación a los ensayos clínicos, en los últimos diez años se han experimentado grandes avances en el campo de las opciones terapéuticas, divididos en dos grandes dianas terapéuticas: 1) el área de las terapias génicas y 2) tratar de revertir o bloquear los procesos fisiopatológicos de la enfermedad, tales como inflamación, fibrosis, regeneración muscular, etc. Es probable que un tratamiento eficaz para la distrofia muscular de Duchenne requiera combinaciones que se apliquen tanto al defecto primario como las consecuencias fisiopatológicas secundarias.


Duchenne muscular dystrophy is a genetically determined disease, linked to the X chromosome, c haracterized clinically by producing progressive muscle weakness, with an incidence of 1 per 3500-6000 males born. It is caused by the mutation of the DMD gene, which encodes dystrophin, a sub-sarcolemmal protein essential for structural muscle stability. The genetic defects in the DMD gene are divided into: deletions (65%) duplications (5.10%) and point mutations (10-15%). At present there is no curative treatment, the only drug that has been shown to modify the natural history of the disease (independently of the genetic mutation) are corticosteroids, currently indicated in early stages of the disease. In relation to clinical trials, in the last ten years, has experienced great advances in the field of therapeutic options, divided into two major therapeutic targets: 1) the area of gene therapies and 2) trying to reverse or block the pathophysiological processes of the disease, such as inflammation, fibrosis, muscle regeneration, etc. It is likely that an effective treatment for Duchenne muscular dystrophy requires combinations of therapies that address both the primary defect and its secondary pathophysiological consequences.


Subject(s)
Humans , Animals , Rabbits , Genetic Therapy/methods , Muscular Dystrophy, Duchenne/therapy , Phenotype , Dystrophin/genetics , Mice, Inbred mdx , Muscular Dystrophy, Duchenne/genetics , CRISPR-Cas Systems , Genotype
5.
Neumol. pediátr. (En línea) ; 13(3): 118-121, sept. 2018. ilus
Article in Spanish | LILACS | ID: biblio-947631

ABSTRACT

Cystic fibrosis is an autosomal recessive multisystemic disease caused by a mutation in the gene encoding the CFTR protein (cystic fibrosis transmembrane conductance regulator). For decades treatments were focused on pulmonary and extrapulmonary symptoms, but in recent years new treatments based on genetics and CFTR mutations have been proposed. The first treatment to appear was genetic therapy, which did not show long-term benefits. These new treatments have allowed a more individualized scheme by using potentiators and modulators of CFTR. Phase III studies and systematic revisions have demonstrated pulmonary function improvement, lower rates of FEV1 decline, reduction in pulmonary exacerbations, BMI improvement and better chloride transport revealed by the sweat test. Recent literature has also shown that these effects persist in the long term.


La Fibrosis Quística es una enfermedad multisistémica autosómica recesiva causada por la mutación del gen que codifica al canal CFTR (proteína de regulación de transmembrana de Fibrosis Quística). Desde hace varias décadas se han utilizado tratamientos enfocados en síntomas pulmonares y extrapulmonares, pero los últimos años han surgido tratamientos basados en genética y mutaciones del CFTR. Inicialmente fue la terapia génica, la cual a largo plazo no demostró beneficios. Las nuevas terapias han permitido un tratamiento individualizado, mediante potenciadores y moduladores del CFTR, demostrándose en estudios fase III y revisiones sistemáticas mejoría en la función pulmonar, disminución de la velocidad de declinación del VEF1, reducción de exacerbaciones pulmonares, mejoría del IMC y del transporte de cloro medido en test del sudor, observándose efectos mantenidos a largo plazo.


Subject(s)
Humans , Child , Cystic Fibrosis/drug therapy , Genetic Therapy/methods , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Mutation
6.
Acta cir. bras ; 33(4): 386-395, Apr. 2018. tab, graf
Article in English | LILACS | ID: biblio-886279

ABSTRACT

Abstract Purpose: To investigate the safety and clinical, hemodynamic and tissue improvement ability in mini pigs undergoing cell and gene therapy for the treatment of acute myocardial infarction. Methods: Thirty-two mini pigs Br1 lineage, 12 months old, undergoing induction of acute myocardial infarction by occlusion of the diagonal branch of the paraconal coronary. They were divided into 4 groups: one control group and 3 treatment groups (cell therapy and gene cell therapy). Echocardiography reviews were performed on three occasions and histopathological analysis was performed after 4 weeks. Analysis of variance (ANOVA), Tukey and Wilcoxon tests, were performed. Results: Association of vascular endothelial growth factor (VEGF) with angiopoietin1 (Ang1) presented satisfactory results in the improvement of ventricular function following ischemic cardiomyopathy in mini pigs when compared to the results of the other treated groups. Conclusion: The therapy with VEGF and the combination of VEGF with Ang1, promoted recovered function of the myocardium, characterized by reduced akinetic area and induction of neovascularization.


Subject(s)
Animals , Genetic Therapy/methods , Ventricular Function/physiology , Angiopoietin-1/therapeutic use , Vascular Endothelial Growth Factor A/therapeutic use , Cell- and Tissue-Based Therapy/methods , Myocardial Infarction/therapy , Swine , Swine, Miniature , Wound Healing , Echocardiography , Reproducibility of Results , Treatment Outcome , Neovascularization, Physiologic , Disease Models, Animal , Hemodynamics/physiology , Myocardial Infarction/physiopathology , Myocardial Infarction/pathology , Necrosis
7.
Arq. neuropsiquiatr ; 76(4): 265-272, Apr. 2018. tab, graf
Article in English | LILACS | ID: biblio-888378

ABSTRACT

ABSTRACT Spinal muscular atrophy (SMA) is a severe and clinically-heterogeneous motor neuron disease caused, in most cases, by a homozygous mutation in the SMN1 gene. Regarding the age of onset and motor involvement, at least four distinct clinical phenotypes have been recognized. This clinical variability is, in part, related to the SMN2 copy number. By now, only supportive therapies have been available. However, promising specific therapies are currently being developed based on different mechanisms to increase the level of SMN protein; in particular, intrathecal antisense oligonucleotides that prevent the skipping of exon 7 during SMN2 transcription, and intravenous SMN1 insertion using viral vector. These therapeutic perspectives open a new era in the natural history of the disease. In this review, we intend to discuss the most recent and promising therapeutic strategies, with special consideration to the pathogenesis of the disease and the mechanisms of action of such therapies.


RESUMO A atrofia muscular espinhal (AME) é uma grave doença dos neurônios motores, de grande variabilidade clínica e causada na maioria dos casos por mutação em homozigose no gene SMN1. Pelo menos quatro fenótipos clínicos distintos são reconhecidos com base na idade de início e no grau de envolvimento motor. Tal variabilidade clínica é em parte relacionada com o número de cópias do gene SMN2. Até recentemente, apenas terapias de suporte estavam disponíveis. Atualmente, terapias especificas estão sendo desenvolvidas com base em diferentes mecanismos para aumentar o nível de proteína SMN; em particular oligonucleotídeos antissenso por via intratecal e inserção de cópia do gene SMN1, via endovenosa, usando vetor viral. Nesta revisão, objetivamos discutir as mais recentes e promissoras estratégias terapêuticas, com consideração especial aos aspectos patogênicos da doença e aos mecanismos de ação de tais terapias.


Subject(s)
Humans , Oligonucleotides/administration & dosage , Muscular Atrophy, Spinal/therapy , Genetic Therapy/methods , DNA, Antisense/administration & dosage , Survival of Motor Neuron 1 Protein/administration & dosage , Phenotype , Injections, Spinal , Mutation
8.
Clinics ; 73(supl.1): e476s, 2018. graf
Article in English | LILACS | ID: biblio-952839

ABSTRACT

Gene therapy has been evaluated for the treatment of prostate cancer and includes the application of adenoviral vectors encoding a suicide gene or oncolytic adenoviruses that may be armed with a functional transgene. In parallel, versions of adenoviral vector expressing the p53 gene (Ad-p53) have been tested as treatments for head and neck squamous cell carcinoma and non-small cell lung cancer. Although Ad-p53 gene therapy has yielded some interesting results when applied to prostate cancer, it has not been widely explored, perhaps due to current limitations of the approach. To achieve better functionality, improvements in the gene transfer system and the therapeutic regimen may be required. We have developed adenoviral vectors whose transgene expression is controlled by a p53-responsive promoter, which creates a positive feedback mechanism when used to drive the expression of p53. Together with improvements that permit efficient transduction, this new approach was more effective than the use of traditional versions of Ad-p53 in killing prostate cancer cell lines and inhibiting tumor progression. Even so, gene therapy is not expected to replace traditional chemotherapy but should complement the standard of care. In fact, chemotherapy has been shown to assist in viral transduction and transgene expression. The cooperation between gene therapy and chemotherapy is expected to effectively kill tumor cells while permitting the use of reduced chemotherapy drug concentrations and, thus, lowering side effects. Therefore, the combination of gene therapy and chemotherapy may prove essential for the success of both approaches.


Subject(s)
Humans , Male , Prostatic Neoplasms/therapy , Genetic Therapy/methods , Adenoviridae/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Genetic Vectors/therapeutic use , Lung Neoplasms/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/immunology , Tumor Suppressor Protein p53/biosynthesis , Prostate-Specific Antigen/genetics , Genes, Transgenic, Suicide , Neoplasm Proteins/genetics
9.
Clinics ; 73(supl.1): e479s, 2018. graf
Article in English | LILACS | ID: biblio-952830

ABSTRACT

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-β cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.


Subject(s)
Humans , Genetic Therapy/methods , Reading Frames/genetics , Interferon-beta/therapeutic use , Gene Transfer Techniques , Immunotherapy/methods , Neoplasms/therapy , Cell Death/genetics , Cyclin-Dependent Kinase Inhibitor p16/genetics , Tumor Suppressor Protein p14ARF/genetics , Neoplasms/immunology
10.
Int. braz. j. urol ; 43(6): 1167-1175, Nov.-Dec. 2017. tab, graf
Article in English | LILACS | ID: biblio-892932

ABSTRACT

ABSTRACT Objectives: To compare the effects and histopathological changes of botulinum neurotoxin type A and lysozyme gene injections into prostate tissue within a testosterone induced benign prostate hyperplasia rat model. Materials and Methods: 40 male Wistar rats were randomized into four Groups. Group-1: Control, Group-2: Testosterone replacement, Group-3: Testosterone+botulinum neurotoxin type A, Group-4: Testosterone+plazmid DNA/liposome complex. Results: Estimated prostate volume of the testosterone injected Groups were higher than the control (p <0.05). Actual prostate weight of the testosterone injected Groups was higher than the control Group (p <0.05). Testosterone undecanoate increased the prostate weight by 39%. Botulinum neurotoxin type A treatment led to an estimated prostate volume and actual prostate weights decreased up to 32.5% in rats leading to prostate apoptosis. Lysozyme gene treatment led to an estimated prostate volume and actual prostate weights decrease up to 38.7%. Conclusion: Lysozyme gene and botulinum neurotoxin type A treatments for prostate volume decreasing effect have been verified in the present study that could be anew modality of treatment in prostatic benign hyperplasia that needs to be verified in large randomized human experimental studies.


Subject(s)
Animals , Male , Rats , Prostatic Hyperplasia/drug therapy , Genetic Therapy/methods , Muramidase/genetics , Botulinum Toxins, Type A/therapeutic use , Prostatic Hyperplasia/chemically induced , Testosterone , Rats, Wistar , Disease Models, Animal
11.
Einstein (Säo Paulo) ; 15(3): 369-375, July-Sept. 2017. tab, graf
Article in English | LILACS | ID: biblio-891391

ABSTRACT

ABSTRACT The ability to make site-specific modifications to the human genome has been an objective in medicine since the recognition of the gene as the basic unit of heredity. Thus, gene therapy is understood as the ability of genetic improvement through the correction of altered (mutated) genes or site-specific modifications that target therapeutic treatment. This therapy became possible through the advances of genetics and bioengineering that enabled manipulating vectors for delivery of extrachromosomal material to target cells. One of the main focuses of this technique is the optimization of delivery vehicles (vectors) that are mostly plasmids, nanostructured or viruses. The viruses are more often investigated due to their excellence of invading cells and inserting their genetic material. However, there is great concern regarding exacerbated immune responses and genome manipulation, especially in germ line cells. In vivo studies in in somatic cell showed satisfactory results with approved protocols in clinical trials. These trials have been conducted in the United States, Europe, Australia and China. Recent biotechnological advances, such as induced pluripotent stem cells in patients with liver diseases, chimeric antigen receptor T-cell immunotherapy, and genomic editing by CRISPR/Cas9, are addressed in this review.


RESUMO A habilidade de fazer modificações pontuais no genoma humano tem sido o objetivo da medicina desde o conhecimento do DNA como unidade básica da hereditariedade. Entende-se terapia gênica como a capacidade do melhoramento genético por meio da correção de genes alterados (mutados) ou modificações sítio-específicas, que tenham como alvo o tratamento terapêutico. Este tipo de procedimento tornou-se possível por conta dos avanços da genética e da bioengenharia, que permitiram a manipulação de vetores para a entrega do material extracromossomal em células-alvo. Um dos principais focos desta técnica é a otimização dos veículos de entrega (vetores) que, em sua maioria, são plasmídeos, nanoestruturados ou vírus − sendo estes últimos os mais estudados, devido à sua excelência em invadir as células e inserir seu material genético. No entanto, existe grande preocupação referente às respostas imunes exacerbadas e à manipulação do genoma, principalmente em linhagens germinativas. Estudos em células somáticas in vivo apresentaram resultados satisfatórios, e já existem protocolos aprovados para uso clínico. Os principais trials têm sido conduzidos nos Estados Unidos, Europa, Austrália e China. Recentes avanços biotecnológicos empregados para o aprimoramento da terapia gênica, como células-tronco pluripotentes induzidas em pacientes portadores de doenças hepáticas, imunoterapia com células T do receptor do antígeno quimera e edição genômica pelos sistema CRISPR/Cas9, são abordados nesta revisão.


Subject(s)
Humans , Animals , Genetic Therapy/methods , CRISPR-Cas Systems/genetics , Gene Editing/methods , Receptors, Antigen, T-Cell/genetics , Genetic Therapy/trends , Genetic Vectors/genetics , Genetic Vectors/therapeutic use
12.
Clin. biomed. res ; 37(4): 330-333, 2017. ilus, graf
Article in English | LILACS | ID: biblio-876699

ABSTRACT

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder caused by deficiency of alpha-L-iduronidase (IDUA). Limitations such as the need for weekly injections, high morbidity and mortality, and high cost of current treatments show that new approaches to treat this disease are required. In this study, we aimed to correct fibroblasts from a patient with MPS I using non-viral gene therapy. Using a plasmid encoding the human IDUA cDNA, we achieved stable high IDUA levels in transfected fibroblasts up to 6 months of treatment. These results serve as proof of concept that a non-viral approach can correct the enzyme deficiency in cells of patients with lysosomal storage disorders, which can be used as a research tool for a series of disease aspects. Future studies should focus on showing if this approach can be useful in small animals and clinical trials (AU)


Subject(s)
Humans , Fibroblasts/enzymology , Gene Transfer Techniques , Genetic Vectors , Iduronidase/metabolism , Mucopolysaccharidosis I/therapy , DNA, Complementary , Genetic Therapy/methods , Iduronidase/genetics , Mucopolysaccharidosis I/genetics , Plasmids/genetics , Transfection/methods
13.
Rev. chil. pediatr ; 86(4): 251-256, ago. 2015. tab
Article in Spanish | LILACS | ID: lil-764081

ABSTRACT

Introducción: Autoconcepto es el conjunto de ideas y actitudes que se tiene acerca de sí mismo. Nuestro objetivo fue evaluar si existen diferencias en el nivel de autoconcepto de niños de 8-12 años con y sin secuelas de quemaduras, e identificar variables predictoras del autoconcepto en los niños con secuelas. Pacientes y método: Estudio comparativo, transversal de 109 niños con secuelas de quemaduras de 8 a 12 años de edad, con 109 niños sin secuelas de quemaduras, del mismo grupo de edad y nivel socioeconómico. Se utilizó la escala de autoconcepto de Piers-Harris, que entrega medida de autoconcepto general y dimensiones: conductual, estatus intelectual y escolar, apariencia y atributos físicos, ansiedad, popularidad, felicidad y satisfacción. Resultados: No hubo diferencias significativas en el nivel de autoconcepto general ni en sus dimensiones al comparar ambos grupos (p > 0,05). Dentro del grupo con secuelas de quemaduras la variable número de secuelas resultó ser un factor protector para las dimensiones ansiedad, popularidad, felicidad-satisfacción y autoconcepto general. La variable localización surgió como factor de riesgo para la dimensión conductual. Discusión: La ausencia de diferencias en autoconcepto entre niños con secuelas de quemaduras y sin ellas es similar a lo reportado por la literatura. El hallazgo en factores de riesgo y protectores motiva a continuar investigando, incorporando antecedentes premórbidos y familiares.


Introduction: Self-concept is the set of ideas and attitudes that a person has about him/herself. Objective: To evaluate whether there are differences in the level of self-concept in children 8-12 years old with and without burns sequelae. To identify predictive variables of self-concept in children with sequelae. Patients and method: A comparative cross-sectional study of self-concept in 109 children with burns sequelae, from 8 to 12 years old, with 109 children without burns sequelae, and of the same age and socioeconomic status. The Piers-Harris self-concept scale is used, which provides a general measurement of self-concept and behavioural, intellectual and school status, appearance, and physical attributes, anxiety, popularity, happiness and satisfaction dimensions. Results: There were no significant differences in the level of general self-concept or their dimensions (P > .05). In the group with burns sequelae, the protective factor was the variable number of sequels was associated with the dimensions of anxiety, popularity, happiness-satisfaction and general self-concept. The location variable emerged as a risk factor for the behavioural dimension. Discussion: The absence of differences in self-concept between children with burns sequelae and children without them is similar to that reported in the literature. The finding in the risk and protective factors encourages to further research, and perhaps incorporating pre-morbidity and family background.


Subject(s)
Humans , DNA , Quaternary Ammonium Compounds/chemistry , Serine/chemistry , Surface-Active Agents/chemistry , Amides/chemistry , Amines/chemistry , Cell Line, Tumor , Chemistry, Pharmaceutical/methods , Esters/chemistry , Gene Transfer Techniques , Genetic Therapy/methods , HeLa Cells , Lipids/chemistry , Transfection/methods
14.
Einstein (Säo Paulo) ; 13(1): 89-95, Jan-Mar/2015. tab, graf
Article in English | LILACS | ID: lil-745883

ABSTRACT

Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct. .


Objetivo Avaliar os efeitos da transferência gênica do VEGF165 no processo de remodelamento da matriz extracelular após infarto agudo do miocárdio. Métodos Ratos Wistar foram submetidos ao infarto do miocárdio por ligação da artéria coronária descendente esquerda, e a fração de ejeção de ventrículo esquerdo foi utilizada para classificar os infartos em grandes e pequenos. Os animais foram divididos em grupos de dez animais, de acordo com o tamanho do infarto (grande ou pequeno), e receberam ou não tratamento com o VEGF165. A avaliação dos diferentes marcadores foi realizada por imuno-histoquímica e quantificação digital. Os anticorpos primários utilizados foram antifibronectina, antivimentina, anti- CD44, anti-E-caderina, anti-CD24, anti-alfa-1-actina e anti-PCNA. Os resultados foram representados como média e erro padrão, e analisados por ANOVA, sendo considerado estatisticamente significativo se p≤0,05. Resultados Houve aumento significativo da expressão de marcadores de células indiferenciadas, como fibronectina (proteína presente na matriz extracelular) e CD44 (glicoproteína presente nas células endoteliais). Entretanto, houve diminuição da expressão de vimentina e PCNA, indicando possível diminuição do processo de proliferação celular após o tratamento com VEGF165. Os marcadores de células diferenciadas, E-caderina (proteína de adesão entre as células do miocárdio), CD24 (proteína presente nos vasos sanguíneos) e alfa-1-actina (marcador especifico de miócitos) também apresentaram maior expressão nos grupos submetidos à terapia gênica, comparativamente com o grupo não tratado. O valor obtido pela relação entre alfa-1-actina e vimentina foi aproximadamente três vezes maior nos grupos tratados com VEGF165, indicando maior diferenciação tecidual. Conclusão O papel dos miócitos se mostrou importante no processo de remodelamento tecidual, confirmando que o VEGF165 parece conferir um efeito protetor no tratamento do infarto agudo do miocárdio. .


Subject(s)
Animals , Female , Extracellular Matrix/physiology , Gene Transfer Techniques , Myocardial Infarction/therapy , Vascular Endothelial Growth Factor A/therapeutic use , Actins/analysis , /analysis , /analysis , Cadherins/analysis , Cell Proliferation/physiology , Disease Models, Animal , Fibronectins/analysis , Genetic Therapy/methods , Immunohistochemistry , Myocytes, Cardiac/physiology , Rats, Wistar , Reproducibility of Results , Treatment Outcome , Vascular Endothelial Growth Factor A/genetics , Vimentin/analysis
16.
Korean Journal of Urology ; : 197-204, 2015.
Article in English | WPRIM | ID: wpr-60934

ABSTRACT

PURPOSE: Electroporation is known to enhance the efficiency of gene transfer through a transient increase in cell membrane permeability. The aim of this study was to determine the optimal conditions for in vivo electroporation-mediated gene delivery into mouse corpus cavernosum. MATERIALS AND METHODS: Diabetes was induced in C57BL/6 mice by intraperitoneal injections of streptozotocin. After intracavernous injection of pCMV-Luc (100 microg/40 microL), different electroporation settings (5-50 V, 8-16 pulses with a duration of 40-100 ms) were applied to the penis to establish the optimal conditions for electroporation. Gene expression was evaluated by luciferase assay. We also assessed the undesired consequences of electroporation by visual inspection and hematoxylin-eosin staining of penile tissue. RESULTS: Electroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage-dependent manner. We observed electrical burn scars in the penis of normal mice who received electroporation with eight 40-ms pulses at a voltage of 50 V and sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 V. No detectable burn scars were noted in normal mice stimulated with eight 40-ms pulses at a voltage of 30 V. Electroporation also significantly induced gene expression in diabetic mice stimulated with 40-ms pulse at a voltage of 30 V without injury to the penis. CONCLUSIONS: We have established the optimal electroporation conditions for maximizing gene transfer into the corpus cavernosum of mice while avoiding damage to the erectile tissue. The electroporation-mediated gene delivery technique will be a valuable tool for gene therapy in the field of erectile dysfunction.


Subject(s)
Animals , Diabetes Mellitus, Experimental/complications , Electroporation/methods , Erectile Dysfunction/therapy , Gene Expression , Gene Transfer Techniques , Genes, Reporter , Genetic Therapy/methods , Luciferases/metabolism , Male , Mice , Mice, Inbred C57BL , Penile Erection/physiology , Penis/physiopathology , Transfection
17.
Article in English | WPRIM | ID: wpr-149090

ABSTRACT

Pulmonary arterial hypertension (PAH) is a rare but progressive and currently incurable disease, which is characterized by vascular remodeling in association with muscularization of the arterioles, medial thickening and plexiform lesion formation. Despite our advanced understanding of the pathogenesis of PAH and the recent therapeutic advances, PAH still remains a fatal disease. In addition, the susceptibility to PAH has not yet been adequately explained. Much evidence points to the involvement of epigenetic changes in the pathogenesis of a number of human diseases including cancer, peripheral hypertension and asthma. The knowledge gained from the epigenetic study of various human diseases can also be applied to PAH. Thus, the pursuit of novel therapeutic targets via understanding the epigenetic alterations involved in the pathogenesis of PAH, such as DNA methylation, histone modification and microRNA, might be an attractive therapeutic avenue for the development of a novel and more effective treatment. This review provides a general overview of the current advances in epigenetics associated with PAH, and discusses the potential for improved treatment through understanding the role of epigenetics in the development of PAH.


Subject(s)
Animals , DNA Methylation/drug effects , Drug Discovery/methods , Epigenesis, Genetic/drug effects , Genetic Therapy/methods , Humans , Hypertension, Pulmonary/genetics , MicroRNAs/genetics
18.
Dental press j. orthod. (Impr.) ; 19(6): 123-133, Nov-Dec/2014. graf
Article in English | LILACS | ID: lil-732439

ABSTRACT

Modulation of orthodontic tooth movement (OTM) is desirable not only to patients because it shortens treatment time, but also to orthodontists, since treatment duration is associated with increased risk of gingival inflammation, decalcification, dental caries, and root resorption. The increased focus on the biological basis of tooth movement has rendered Orthodontics a more comprehensive specialty that incorporates facets of all fields of medicine. Current knowledge raises the possibility of using new therapeutic modalities for modulation of OTM, such as corticotomy, laser therapy, vibration (low-intensity pulsed ultrasound), local injections of biomodulators and gene therapy; with the latter being applicable in the near future. They are intended to enhance or inhibit recruitment, differentiation and/or activation of bone cells, accelerate or reduce OTM, increase stability of orthodontic results, as well as assist with the prevention of root resorption. This article summarizes recent studies on each one of these therapeutic modalities, provides readers with information about how they affect OTM and points out future clinical perspectives.


A modulação do movimento dentário ortodôntico (MDO) é desejável para os pacientes, pois reduz o tempo de tratamento, e também para ortodontistas, uma vez que a duração do tratamento tem sido associada a um aumento do risco de inflamação gengival, descalcificação, cárie dentária e reabsorção radicular. O crescente foco sobre os mecanismos biológicos da movimentação dentária levou a Ortodontia a ser uma especialidade mais abrangente, que hoje incorpora aspectos de todas as áreas da Medicina. Com o conhecimento atual, o uso de novas modalidades terapêuticas que visam a modulação da MDO, como a corticotomia, terapia a laser de baixa intensidade e vibração (ultrassom pulsátil de baixa intensidade) já são uma realidade clínica. Outras, como injeções locais de biomoduladores e a terapia genética, serão utilizadas em breve. Elas destinam-se a aumentar ou inibir o recrutamento, à diferenciação e/ou ativação das células ósseas, a acelerar ou reduzir a MDO, a aumentar a estabilidade dos resultados ortodônticos, bem como auxiliar na prevenção da reabsorção radicular. Esse artigo resume os estudos mais recentes sobre cada uma dessas novas modalidades terapêuticas, fornecendo informações aos leitores a respeito de como afetam a MDO e aponta futuras perspectivas clínicas.


Subject(s)
Humans , Tooth Movement Techniques/methods , Genetic Therapy/methods , Immunologic Factors/therapeutic use , Laser Therapy/methods , Osteotomy/methods , Ultrasonics , Vibration/therapeutic use
19.
Article in English | IMSEAR | ID: sea-159354

ABSTRACT

Ultrasound (US), traditionally a diagnostic modality, and is emerging as a non-invasive therapy using local drug delivery and gene therapy. US exposure gene rates bio eff ects that result in shear stress, tissue heating, and cavitation eff ects, which are used in therapeutic applications. Sonoporation employs these eff ects to enhance delivery of large molecules such as DNA into the cells which is applied to muscle, head and neck tumor, in a cell disruption process called transformation and increases the permeability to bioactive materials, which is usually used in molecular biology and gene therapy. Nevertheless, it has recently become popular as a technique to enhance drug release from drug delivery systems. Th is review presents the main fi ndings in the fi eld of sonoporation, namely drug delivery, gene delivery and DNA transfer and its applications in dentistry.


Subject(s)
Dentistry/instrumentation , Dentistry/methods , Genetic Therapy/methods , Humans , Mouth Diseases/genetics , Mouth Diseases/therapy , Mouth Diseases/diagnostic imaging , Review Literature as Topic , Sonication/methods , Ultrasonography/methods
20.
Rev. Soc. Cardiol. Estado de Säo Paulo ; 24(1): 42-48, jan.-mar. 2014. ilus
Article in Portuguese | LILACS | ID: lil-729292

ABSTRACT

A despeito dos incontestáveis avanços no tratamento médico e em procedimentos de revascularização miocárdica (percutâneous e cirúrgicos), sintomas debilitantes relacionados à doença arterial coronária podem ocorrer devido à progressão da doença com envolvimento difuso arterial e oclusão de enxertos prévios ou reestenose pós-angioplastia, impossibilitando novos procedimentos de revascularização miocárdica. Característica desta condição (angina refratária) é o grande prejuízo dos afetados em termos de qualidade de vida, impedidos de realizar as atividades mais triviais do dia-a-dia (caminhar poucos metros no plano ou mesmo banha-se) sem que a dor anginosa ocorra; alguns pacientes são despertados frequentemente durante a noite por angina. Assim, para estes pacientes, o objetivo principal do tratamento é a melhoria na qualidade de vida, com maior tolerância ao esforço, e menor necessidade de hospitalizações e procedimentos diagnósticos ou terapêuticos. Neste contexto, elencaremos sucintamente as principais estratégias terapêuticas não farmacológica em desenvolvimento para o tratamento de pacientes com angina refratária, incluindo terapia gênica, terapia celular, revascularização transmiocárdica a laser, contrapulsação externa, estimulação de medula espinhal e revascularização miocárdica extracorpórea por ondas de choque.


Despite great advances in both medical management and myocardial revascularization procedures (percutaneous and surgical), disabling symptoms due to coronary artery disease may occur due to progression of the beds, grafts failures after successful bypass surgery, and/or stent restenosis, preventing further revacularization attempts. Patients with refractory angina have a mared impairment in quality of life, unable to perform any daily avtivity such as slowl walking or even taking a bath without chest pain; many patients are awaken during their sleep due to chest disconfort. For theses patients, the main objective f treatment is to improve their quality of life, with better exercise tolerance, and decreased number of hospitalizations and/or diagnosis/therapeutic procedures. In this paper, we briefly discuss new non-pharmacological therapeutic strategies being developed for patients with adavanced CAD including gene therapy, cell therapy, transmyocardial laser revascularization, enhanced external conter-pulsation, spinal cord stimulation and extracorporeal shockwave myocardial revascularization.


Subject(s)
Humans , Male , Female , Angina Pectoris/therapy , Coronary Artery Disease/therapy , Cardiovascular Diseases/mortality , Myocardial Revascularization/trends , Risk Factors , Implantable Neurostimulators , Cell- and Tissue-Based Therapy/trends , Genetic Therapy/methods
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