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1.
Buenos Aires; CONETEC; nov. 2019.
No convencional en Español | BRISA/RedTESA | ID: biblio-1048252

RESUMEN

INTRODUCCIÓN: La hipofosfatasia (HPP) es una enfermedad hereditaria "ultra rara" sumamente heterogénea, del metabolismo fosfo-cálcico generada por un déficit enzimático. Se presenta con múltiples formas clínicas, que van desde alteraciones dentales hasta falta de mineralización ósea generalizada, que lleva a una alta tasa de mortalidad. El pronóstico empeora cuanto más precoz es el inicio de los síntomas (formas perinatales e infantil). No se encuentran disponibles datos de prevalencia locales; se estima una incidencia de aproximadamente 2 casos nuevos de las formas severas cada año en Argentina. DESCRIPCIÓN DE LA TECNOLOGÍA: Asfotase alfa es una enzima de reemplazo diseñada para suplementar la actividad de la TN-SALP, la misma es obtenida por técnicas de ADN recombinante utilizando células de ovario de hámster chino. Se administra de forma subcutánea, la dosis recomendada por el fabricante es de 2 mg/kg tres veces por semana o 1 mg/kg seis veces por semana, aunque se ha utilizado en dosis superiores en los estudios encontrados. METODOLOGÍA: Se realizó una búsqueda en Medline, Lilacs, Cochrane, Tripdatabase, ClínicalTrials.gov, Orphanet y buscadores genéricos de internet. Se utilizaron las palabras clave "asfotase", "hypophosphatasia AND enzyme replacement", "asfotase AND hypophosphatasia", "strensiq". Se consideraron criterios de inclusión a ensayos clínicos controlados aleatorizados (ECCAs), ensayos clínicos no controlados, cohortes prospectivas, casos y controles y estudios de fase 2 o más avanzado, realizados en humanos; en idioma inglés, español, francés o portugués. RESULTADOS: Para la forma clínica perinatal severa e infantil, evidencia de muy baja calidad basada en estudios de fase 2 mostró un beneficio considerable para los desenlaces sobrevida, soporte ventilatorio y cambios radiológicos. Los costos directos anuales de la tecnología para el tratamiento de esta forma clínica se estimaron entre US$ 178.308 y US$ 217.620. En el caso de la forma clínica infanto-juvenil se incluyó un estudio de fase 2 donde se valoró un beneficio considerable para el desenlace cambios radiológicos. Sin embargo, el efecto del tratamiento fue incierto para los desenlaces sobrevida y calidad de vida. La calidad de la evidencia hallada en este caso también fue muy baja. Finalmente, para la forma clínica adulta, en el estudio de fase 2 incluido, no observó beneficio para los desenlaces mineralización ósea y dolor; y un efecto incierto del tratamiento para los desenlaces sobrevida y calidad de vida. La calidad de la evidencia hallada fue baja. CONCLUSIÓN: La información de este reporte contiene las opiniones y perspectivas de una cuidadora de un paciente pediátrico sobre la evolución de la enfermedad hasta llegar al tratamiento, y sobre la vida cotidiana. En el caso de este paciente de 6 años, con diagnostico a los 8 meses de vida, que recibe asfotase alfa hace 3 años, el tratamiento presentó buenos resultados en cuanto al dolor y al desarrollo de la marcha.


Asunto(s)
Humanos , ADN Recombinante/uso terapéutico , Terapia de Reemplazo Enzimático , Hipofosfatasia/tratamiento farmacológico , Evaluación de la Tecnología Biomédica , Análisis Costo-Beneficio/economía
3.
J Biol Chem ; 292(44): 18062-18074, 2017 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-28928221

RESUMEN

Parkinson's disease (PD) is one of the most epidemic neurodegenerative diseases and is characterized by movement disorders arising from loss of midbrain dopaminergic (DA) neurons. Recently, the relationship between PD and autophagy has received considerable attention, but information about the mechanisms involved is lacking. Here, we report that autophagy-related gene 5 (ATG5) is potentially important in protecting dopaminergic neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model in zebrafish. Using analyses of zebrafish swimming behavior, in situ hybridization, immunofluorescence, and expressions of genes and proteins related to PD and autophagy, we found that the ATG5 expression level was decreased and autophagy flux was blocked in this model. The ATG5 down-regulation led to the upgrade of PD-associated proteins, such as ß-synuclein, Parkin, and PINK1, aggravation of MPTP-induced PD-mimicking pathological locomotor behavior, DA neuron loss labeled by tyrosine hydroxylase (TH) or dopamine transporter (DAT), and blocked autophagy flux in the zebrafish model. ATG5 overexpression alleviated or reversed these PD pathological features, rescued DA neuron cells as indicated by elevated TH/DAT levels, and restored autophagy flux. The role of ATG5 in protecting DA neurons was confirmed by expression of the human atg5 gene in the zebrafish model. Our findings reveal that ATG5 has a role in neuroprotection, and up-regulation of ATG5 may serve as a goal in the development of drugs for PD prevention and management.


Asunto(s)
Proteína 5 Relacionada con la Autofagia/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Regulación de la Expresión Génica , Terapia Genética , Trastornos Parkinsonianos/prevención & control , Proteínas de Pez Cebra/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Autofagia/efectos de los fármacos , Proteína 5 Relacionada con la Autofagia/antagonistas & inhibidores , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/uso terapéutico , Conducta Animal/efectos de los fármacos , Biomarcadores/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , ADN Recombinante/uso terapéutico , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/patología , Embrión no Mamífero , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Larva , Microinyecciones , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/uso terapéutico , Neuroprotección/efectos de los fármacos , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética
4.
Ann N Y Acad Sci ; 1407(1): 5-16, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28905423

RESUMEN

The most advanced regulatory processes for complex biological products have been put in place in many countries to provide appropriate regulatory oversight of biotherapeutic products in general, and similar biotherapeutics in particular. This process is still ongoing and requires regular updates to national regulatory requirements in line with scientific developments and up-to-date standards. For this purpose, strong knowledge of and expertise in evaluating biotherapeutics in general and similar biotherapeutic products, also called biosimilars, in particular is essential. Here, we discuss the World Health Organization's international standard-setting role in the regulatory evaluation of recombinant DNA-derived biotherapeutic products, including biosimilars, and provide examples that may serve as models for moving forward with nonbiological complex medicinal products. A number of scientific challenges and regulatory considerations imposed by the advent of biosimilars are described, together with the lessons learned, to stimulate future discussions on this topic. In addition, the experiences of facilitating the implementation of guiding principles for evaluation of similar biotherapeutic products into regulatory and manufacturers' practices in various countries over the past 10 years are briefly explained, with the aim of promoting further developments and regulatory convergence of complex biological and nonbiological products.


Asunto(s)
Productos Biológicos/uso terapéutico , Biosimilares Farmacéuticos/uso terapéutico , ADN Recombinante/uso terapéutico , Industria Farmacéutica/normas , Evaluación de Medicamentos/normas , Industria Farmacéutica/tendencias , Quimioterapia/métodos , Quimioterapia/normas , Quimioterapia/tendencias , Guías como Asunto/normas , Humanos , Organización Mundial de la Salud
5.
Curr Top Med Chem ; 17(13): 1529-1541, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28017152

RESUMEN

Protein bioconjugates can be synthesized by using chemical reactions, enzymatic reactions or genetic engineering technologies. Naturally occurred protein fusion event is used on purpose in the development of better biopharmaceuticals by applying genetic engineering methodologies. This review will mainly focus on the types of fusion proteins produced with the use of recombinant DNA technology, by combining genes or parts of genes from the same or different organisms, in order to be used in pharmaceutical applications for several purposes. Main concerns for the development of better biopharmaceuticals include quality, efficacy, safety, immunogenicity and toxicity issues. Extending half-life of the drug to increase patient compliance, targeting the drugs to reduce toxicity, improving the manufacturing environment to reduce the costs and revealing protein interaction technologies to find novel and superior drugs are the main aims of fusion protein production. Here, related tags and examples of fusion methods for different purposes will be explained precisely.


Asunto(s)
ADN Recombinante/metabolismo , Preparaciones Farmacéuticas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , ADN Recombinante/química , ADN Recombinante/uso terapéutico , Humanos , Preparaciones Farmacéuticas/química , Proteínas Recombinantes de Fusión/química
6.
Cancer Control ; 23(1): 12-20, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27009452

RESUMEN

BACKGROUND: Locoregional advanced melanoma poses a complex clinical challenge that requires a multidisciplinary, patient-centered approach. Numerous agents have been studied for their suitability as intralesional therapy in the past decades, but few have successfully completed phase 3 clinical trial testing. METHODS: The relevant medical literature was searched for articles regarding use of intralesional therapies in metastatic melanoma. Therapies with data from phase 2 or higher studies were selected for review. This review also summarizes the mechanisms of action, adverse-event profiles, and clinical data for these agents. RESULTS: Intralesional therapies demonstrate promising effects in select patients with advanced melanoma. The optimal approach should be individually tailored and consist of a combination of intralesional therapies, regional perfusions, systemic immunotherapies, targeted therapies, and surgery, if necessary. CONCLUSIONS: Due to its relatively good local response rates and tolerable adverse-event profile, intralesional therapy may be a treatment option for select patients with unresectable, locally advanced or metastatic melanoma.


Asunto(s)
Terapia Genética , Inmunoterapia , Inyecciones Intralesiones/métodos , Melanoma/terapia , Virus Oncolíticos , Neoplasias Cutáneas/terapia , Administración Cutánea , Vacuna BCG/administración & dosificación , Vacuna BCG/efectos adversos , Vacuna BCG/uso terapéutico , ADN Recombinante/administración & dosificación , ADN Recombinante/uso terapéutico , Electroquimioterapia/métodos , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Antígeno HLA-B7/genética , Herpesvirus Humano 1 , Humanos , Interleucina-2/administración & dosificación , Interleucina-2/uso terapéutico , Lípidos/administración & dosificación , Lípidos/uso terapéutico , Melanoma/genética , Rosa Bengala/administración & dosificación , Rosa Bengala/uso terapéutico , Neoplasias Cutáneas/genética
7.
Expert Opin Biol Ther ; 16(5): 619-26, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26898656

RESUMEN

INTRODUCTION: Intralesional immunotherapy supplements systemic treatments and often achieves higher remission rates as compared to systemic therapy. Its indication is metastatic melanoma with limited tumor burden, particularly in loco-regional metastasis and distant soft tissue metastasis. AREAS COVERED: This review describes intralesional immunotherapy with talimogene laherparepvec (T-VEC), with velimogene aliplasmid (Allovectin-7) and with intralesional interleukin-2. These therapies function exclusively by activating the immune system. Furthermore, Rose Bengal and electrochemotherapy have been included, as bystander effects have been observed with these treatments. EXPERT OPINION: Objective remissions are achieved in a higher percentage with intralesional immunotherapies, such as intralesional interleukin-2 with up to 69% of complete remissions, as compared to systemic treatment. Therefore, intralesional immunotherapy may act as supplement in the armament against metastatic melanoma. In particular, for patients with multiple cutaneous and subcutaneous metastases (20­≥ 100) and in patients with subcutaneous bulky disease intralesional immunotherapy can improve the disease outcome. The exact role of intralesional immunotherapy in the age of immune checkpoint blockade has still to be determined. A number of clinical trials are on the way in order to better understand synergistic actions of intralesional and systemic immunotherapy.


Asunto(s)
Antineoplásicos/uso terapéutico , Inmunoterapia , Melanoma/tratamiento farmacológico , Melanoma/patología , Neoplasias Cutáneas/tratamiento farmacológico , ADN Recombinante/uso terapéutico , Humanos , Inyecciones Intralesiones , Lípidos/uso terapéutico , Viroterapia Oncolítica , Inducción de Remisión , Neoplasias Cutáneas/patología
8.
Methods Mol Biol ; 1349: 151-61, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26458835

RESUMEN

The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is used to generate capripoxvirus knockout viruses (KO), and is based on the targeting a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses.


Asunto(s)
Capripoxvirus/genética , ADN Recombinante/genética , Técnicas de Inactivación de Genes/métodos , Vacunas Sintéticas/genética , Capripoxvirus/inmunología , Clonación Molecular , ADN Recombinante/inmunología , ADN Recombinante/uso terapéutico , Escherichia coli/genética , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Regiones Promotoras Genéticas , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéutico
9.
Adv Exp Med Biol ; 871: 31-47, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26374211

RESUMEN

The National Institutes of Health (NIH) oversight of human gene transfer research, which is defined as the deliberate transfer of recombinant and/or synthetic nucleic acid molecules to humans, originates with the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines). The NIH Guidelines, which were first published in the Federal Register almost 40 years ago, have been amended numerous times to remain responsive to scientific progress and to clearly define the responsibilities of NIH, the Recombinant DNA Advisory Committee (RAC), investigators, and institutions. Human gene transfer trials conducted at clinical sites in the United States (USA) are subject to the NIH Guidelines if they are conducted at, or sponsored by, an institution that receives any support for recombinant or synthetic nucleic acid research from the NIH. Human gene transfer trials conducted either in the USA or abroad are also subject to the NIH Guidelines if the investigational agent was developed with NIH funds and the institution that developed the investigational materials sponsors or participates in these projects. Trials are registered with the NIH Office Biotechnology Activities (OBA) and there are ongoing reporting requirements. Each new trial is reviewed by the RAC, and those that are novel or raise unique ethical or social issues are selected for review at quarterly public RAC meetings. The RAC also advises the NIH on policy and other matters relating to clinical gene transfer research and biosafety.


Asunto(s)
ADN Recombinante/uso terapéutico , Control de Medicamentos y Narcóticos/legislación & jurisprudencia , Terapia Genética/legislación & jurisprudencia , Vectores Genéticos/uso terapéutico , Investigación en Medicina Traslacional/legislación & jurisprudencia , Animales , Ensayos Clínicos como Asunto , Terapia Genética/ética , Humanos , Consentimiento Informado/ética , Consentimiento Informado/legislación & jurisprudencia , National Institutes of Health (U.S.) , Seguridad del Paciente/legislación & jurisprudencia , Guías de Práctica Clínica como Asunto , Proyectos de Investigación , Investigación en Medicina Traslacional/ética , Estados Unidos
10.
Adv Exp Med Biol ; 871: 49-71, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26374212

RESUMEN

Health Canada regulates gene therapy products and many cell therapy products as biological drugs under the Canadian Food and Drugs Act and its attendant regulations. Cellular products that meet certain criteria, including minimal manipulation and homologous use, may be subjected to a standards-based approach under the Safety of Human Cells, Tissues and Organs for Transplantation Regulations. The manufacture and clinical testing of cell and gene therapy products (CGTPs) presents many challenges beyond those for protein biologics. Cells cannot be subjected to pathogen removal or inactivation procedures and must frequently be administered shortly after final formulation. Viral vector design and manufacturing control are critically important to overall product quality and linked to safety and efficacy in patients through concerns such as replication competence, vector integration, and vector shedding. In addition, for many CGTPs, the value of nonclinical studies is largely limited to providing proof of concept, and the first meaningful data relating to appropriate dosing, safety parameters, and validity of surrogate or true determinants of efficacy must come from carefully designed clinical trials in patients. Addressing these numerous challenges requires application of various risk mitigation strategies and meeting regulatory expectations specifically adapted to the product types. Regulatory cooperation and harmonisation at an international level are essential for progress in the development and commercialisation of these products. However, particularly in the area of cell therapy, new regulatory paradigms may be needed to harness the benefits of clinical progress in situations where the resources and motivation to pursue a typical drug product approval pathway may be lacking.


Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/ética , Control de Medicamentos y Narcóticos/legislación & jurisprudencia , Terapia Genética/legislación & jurisprudencia , Investigación en Medicina Traslacional/legislación & jurisprudencia , Animales , Productos Biológicos/uso terapéutico , Canadá , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , ADN Recombinante/uso terapéutico , Aprobación de Drogas , Terapia Genética/ética , Vectores Genéticos/uso terapéutico , Humanos , Seguridad del Paciente/legislación & jurisprudencia , Guías de Práctica Clínica como Asunto , Proyectos de Investigación , Investigación en Medicina Traslacional/ética , Estudios de Validación como Asunto
11.
Adv Exp Med Biol ; 871: 73-85, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26374213

RESUMEN

Advanced therapy medicinal products, a new class of products with promising therapeutic effects, have been classified as medicinal products and as such should be developed according to a well-structured development plan, to establish their quality, safety and efficacy profile and conclude, at the time of the marketing authorisation evaluation, on a positive risk/benefit balance for patients. An important part of this development plan is achieved through clinical trials, which have also to be approved according to a well-established regulatory process, prior any initiation. This chapter is dedicated to describe the regulatory pathway to be followed in France, before initiating any clinical trial with those investigational advanced therapy medicinal products. In France, to get the final authorisation to initiate a clinical trial, the legislation imposes to run in parallel two independent but complementary authorisation procedures. The first procedure is aimed at assessing the ethical aspect of the biomedical research, while the second has to review the safety and regulatory aspects. A third procedure has to be envisaged where in case the investigational product consists or contains a genetically modified organism. The French system herein described is in line with the EU regulation on clinical trial and follows the respective deadlines for granting the final approval. The complexity of the procedure is in fact more due to the complexity of the products and protocols to be assessed than to the procedure itself which is now very close to the well-known procedure applied routinely for more conventional chemical or biological candidate medicinal products.


Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/ética , Aprobación de Drogas/legislación & jurisprudencia , Control de Medicamentos y Narcóticos/legislación & jurisprudencia , Terapia Genética/legislación & jurisprudencia , Investigación en Medicina Traslacional/legislación & jurisprudencia , Animales , Productos Biológicos/uso terapéutico , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Ensayos Clínicos como Asunto , ADN Recombinante/uso terapéutico , Francia , Terapia Genética/ética , Vectores Genéticos/uso terapéutico , Humanos , Aplicación de Nuevas Drogas en Investigación/legislación & jurisprudencia , Seguridad del Paciente/legislación & jurisprudencia , Guías de Práctica Clínica como Asunto , Proyectos de Investigación , Investigación en Medicina Traslacional/ética
12.
Ann N Y Acad Sci ; 1350: 17-28, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26273800

RESUMEN

Among the five enzyme complexes in the oxidative phosphorylation system, NADH-coenzyme Q oxidoreductase (also called complex I) is the largest, most intricate, and least understood. This enzyme complex spans the inner mitochondrial membrane and catalyzes the first step of electron transfer by the oxidation of NADH, and thereby provides two electrons for the reduction of quinone to quinol. Complex I deficiency is associated with many severe mitochondrial diseases, including Leber hereditary optic neuropathy and Leigh syndrome. However, to date, conventional treatments for the majority of genetic mitochondrial diseases are only palliative. Developing a reliable and convenient therapeutic approach is therefore considered to be an urgent need. Targeted proteins fused with the protein transduction domain of human immunodeficiency virus 1 transactivator of transcription (TAT) have been shown to enter cells by crossing plasma membranes while retaining their biological activities. Recent developments show that, in fusion with mitochondrial targeting sequences (MTSs), TAT-MTS-bound cargo can be correctly transported into mitochondria and restore the missing function of the cargo protein in patients' cells. The available evidence suggests that the TAT-mediated protein transduction system holds great promise as a potential therapeutic approach to treat complex I deficiency, as well as other mitochondrial diseases.


Asunto(s)
ADN Mitocondrial/metabolismo , Complejo I de Transporte de Electrón/deficiencia , Complejo I de Transporte de Electrón/metabolismo , Enfermedades Mitocondriales/metabolismo , Modelos Biológicos , Señales de Clasificación de Proteína , Transducción de Señal , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , ADN Mitocondrial/uso terapéutico , ADN Recombinante/metabolismo , ADN Recombinante/uso terapéutico , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/uso terapéutico , Terapia de Reemplazo Enzimático/métodos , VIH-1/metabolismo , Humanos , Enfermedades Mitocondriales/genética , Fosforilación Oxidativa , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Reparación del Gen Blanco/métodos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética
13.
PLoS Pathog ; 11(5): e1004907, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25996983

RESUMEN

Herpes simplex virus (HSV) enters cells by means of four essential glycoproteins - gD, gH/gL, gB, activated in a cascade fashion by gD binding to one of its receptors, nectin1 and HVEM. We report that the engineering in gH of a heterologous ligand - a single-chain antibody (scFv) to the cancer-specific HER2 receptor - expands the HSV tropism to cells which express HER2 as the sole receptor. The significance of this finding is twofold. It impacts on our understanding of HSV entry mechanism and the design of retargeted oncolytic-HSVs. Specifically, entry of the recombinant viruses carrying the scFv-HER2-gH chimera into HER2+ cells occurred in the absence of gD receptors, or upon deletion of key residues in gD that constitute the nectin1/HVEM binding sites. In essence, the scFv in gH substituted for gD-mediated activation and rendered a functional gD non-essential for entry via HER2. The activation of the gH moiety in the chimera was carried out by the scFv in cis, not in trans as it occurs with wt-gD. With respect to the design of oncolytic-HSVs, previous retargeting strategies were based exclusively on insertion in gD of ligands to cancer-specific receptors. The current findings show that (i) gH accepts a heterologous ligand. The viruses retargeted via gH (ii) do not require the gD-dependent activation, and (iii) replicate and kill cells at high efficiency. Thus, gH represents an additional tool for the design of fully-virulent oncolytic-HSVs retargeted to cancer receptors and detargeted from gD receptors.


Asunto(s)
ADN Recombinante/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Simplexvirus/fisiología , Anticuerpos de Cadena Única/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Tropismo Viral , Internalización del Virus , Animales , Sitios de Unión , Línea Celular , Supervivencia Celular , ADN Recombinante/química , ADN Recombinante/uso terapéutico , Terapia Genética , Humanos , Ligandos , Mutación , Neoplasias/metabolismo , Neoplasias/terapia , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/uso terapéutico , Ingeniería de Proteínas , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/uso terapéutico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Transducción de Señal , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/uso terapéutico , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/uso terapéutico
14.
Adv Exp Med Biol ; 848: 131-48, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25757619

RESUMEN

There is no licensed vaccine or cure for human cytomegalovirus (CMV), a ubiquitous ß-herpes virus that infects 60-95 % of adults worldwide. Infection is a major cause of congenital abnormalities in newborns, contributes to development of childhood cerebral palsy and medulloblastoma, can result in severe disease in immunocompromised patients, and is a major impediment during successful organ transplantation. While CMV has been increasingly associated with numerous inflammatory diseases and cancers, only recently has it been correlated with increased risk of heart disease in adults, the number-one killer in the USA. These data, among others, suggest that subclinical CMV infection, or microinfection, in healthy individuals may play more of a causative role than an epiphenomenon in development of CMV-associated pathologies. Due to the myriad of diseases and complications associated with CMV, an efficacious vaccine would be highly valuable in reducing human morbidity and mortality as well as saving billions of dollars in annual health-care costs and disability adjusted life years (DALY) in the developing world. Therefore, the development of a safe efficacious CMV vaccine or immune therapy is paramount to the public health. This review aims to provide a brief overview on aspects of CMV infection and disease and focuses on current vaccine strategies. The use of new synthetic DNA vaccines might offer one such approach to this difficult problem.


Asunto(s)
Clonación Molecular/métodos , Infecciones por Citomegalovirus/terapia , Vacunas contra Citomegalovirus/uso terapéutico , Inmunoterapia Activa/métodos , Vacunas de ADN/uso terapéutico , Adulto , Animales , Citomegalovirus/genética , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/inmunología , Vacunas contra Citomegalovirus/genética , ADN Recombinante/genética , ADN Recombinante/uso terapéutico , Humanos , Vacunas de ADN/genética
15.
Immunol Cell Biol ; 91(10): 642-51, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24145856

RESUMEN

Recombinant DNA vaccines represent a novel method for generating in situ expression of vaccine antigens. Intramuscular injections of naked DNA are able to elicit potent humoral and cellular immune responses but still numerous factors limit the immunogenicity of DNA vaccines. Co-expression of cytokines with antigen encoding genes in DNA vectors can improve the immune responses and modify Th1/Th2 balance. In this study, the immunomodulatory effect of Interleukin 22 (IL-22) as an adjuvant was studied by DNA vaccination with S. Typhi Heat shock protein 60 (HSP60/GroEL) in mice. Further, DNA construct of IL-22 gene fused with GroEL was developed and immunization studies were carried out in mice. DNA vaccination with GroEL alone stimulated humoral and cell-mediated immune responses. Co-immunization (IL-22+GroEL) further resulted in increase in T-cell proliferative responses, antibody titres (IgG, IgG1, IgG2a) and secretion of IFNγ (Th1), IL-1ß and Th2 (IL-4, IL-6) cytokines. Co-expression (IL-22-GroEL DNA) also promoted antibody titres and cytokine levels were significantly higher as compared to co-immunized group. A reduction in bacterial load in spleen, liver and intestine was seen in all the immunized groups as compared to control, with least organ burden in fusion DNA construct group (co-expression). Improved protective efficacy (90%) against lethal challenge by Salmonella was observed with IL-22-GroEL co-expressing DNA vector as compared with plasmid encoding GroEL only (50-60%) or co-immunization group (75-80%). This study thus shows that co-expression of IL-22 and GroEL genes enhances the immune responses and protective efficacy, circumventing the need of any adjuvant.


Asunto(s)
Chaperonina 60/genética , Inmunidad/genética , Interleucinas/genética , Salmonelosis Animal/genética , Salmonelosis Animal/inmunología , Animales , Formación de Anticuerpos/inmunología , Carga Bacteriana/inmunología , Proliferación Celular , Citocinas/metabolismo , ADN Recombinante/genética , ADN Recombinante/uso terapéutico , Femenino , Expresión Génica , Vectores Genéticos/metabolismo , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/biosíntesis , Biosíntesis de Proteínas , Salmonelosis Animal/tratamiento farmacológico , Salmonelosis Animal/prevención & control , Linfocitos T/citología , Linfocitos T/inmunología , Transcripción Genética , Resultado del Tratamiento , Vacunas de ADN/inmunología , Vacunas de ADN/uso terapéutico
17.
Hum Reprod ; 28(9): 2398-406, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23820419

RESUMEN

STUDY QUESTION: Is targeted adenovirus vector, Ad-SSTR-RGD-TK (Adenovirus -human somatostatin receptor subtype 2- arginine, glycine and aspartate-thymidine kinase), given in combination with ganciclovir (GCV) against immortalized human leiomyoma cells (HuLM) a potential therapy for uterine fibroids? SUMMARY ANSWER: Ad-SSTR-RGD-TK/GCV, a targeted adenovirus, effectively reduces cell growth in HuLM cells and to a significantly greater extent than in human uterine smooth muscle cells (UtSM). WHAT IS KNOWN ALREADY: Uterine fibroids (leiomyomas), a major cause of morbidity and the most common indication for hysterectomy in premenopausal women, are well-defined tumors, making gene therapy a suitable and potentially effective non-surgical approach for treatment. Transduction of uterine fibroid cells with adenoviral vectors such as Ad-TK/GCV (herpes simplex virus thymidine kinase gene) decreases cell proliferation. STUDY DESIGN, SIZE, DURATION: An in vitro cell culture method was set up to compare and test the efficacy of a modified adenovirus vector with different multiplicities of infection in two human immortalized cell lines for 5 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immortalized human leiomyoma cells and human uterine smooth muscle cells were infected with different multiplicities of infection (MOI) (5-100 plaque-forming units (pfu)/cell) of a modified Ad-SSTR-RGD-TK vector and subsequently treated with GCV. For comparison, HuLM and UtSM cells were transfected with Ad-TK/GCV and Ad-LacZ/GCV. Cell proliferation was measured using the CyQuant assay in both cell types. Additionally, western blotting was used to assess the expression of proteins responsible for regulating proliferation and apoptosis in the cells. MAIN RESULTS AND THE ROLE OF CHANCE: Transduction of HuLM cells with Ad-SSTR-RGD-TK/GCV at 5, 10, 50 and 100 pfu/cell decreased cell proliferation by 28, 33, 45, and 84%, respectively (P < 0.05) compared with untransfected cells, whereas cell proliferation in UtSM cells transfected with the same four MOIs of Ad-SSTR-RGD-TK/GCV compared with that of untransfected cells was decreased only by 8, 23, 25, and 28%, respectively (P < 0.01). Western blot analysis showed that, in comparison with the untargeted vector Ad-TK, Ad-SSTR-RGD-TK/GCV more effectively reduced expression of proteins that regulate the cell cycle (Cyclin D1) and proliferation (PCNA, Proliferating Cell Nuclear Antigen), and it induced expression of the apoptotic protein BAX, in HuLM cells. LIMITATIONS, REASONS FOR CAUTION: Results from this study need to be replicated in an appropriate animal model before testing this adenoviral vector in a human trial. WIDER IMPLICATIONS OF THE FINDINGS: Effective targeting of gene therapy to leiomyoma cells enhances its potential as a non-invasive treatment of uterine fibroids.


Asunto(s)
ADN Recombinante/metabolismo , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos/metabolismo , Leiomioma/metabolismo , Miometrio/metabolismo , Transducción Genética , Neoplasias Uterinas/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Adenoviridae/patogenicidad , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Proliferación Celular , ADN Recombinante/efectos adversos , ADN Recombinante/uso terapéutico , Femenino , Terapia Genética/efectos adversos , Terapia Genética/métodos , Vectores Genéticos/efectos adversos , Vectores Genéticos/uso terapéutico , Humanos , Leiomioma/terapia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Uterinas/terapia
18.
Pharmacol Rep ; 65(5): 1075-85, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24399704

RESUMEN

Pharmaceutical biotechnology has a long tradition and is rooted in the last century, first exemplified by penicillin and streptomycin as low molecular weight biosynthetic compounds. Today, pharmaceutical biotechnology still has its fundamentals in fermentation and bioprocessing, but the paradigmatic change affected by biotechnology and pharmaceutical sciences has led to an updated definition. The biotechnology revolution redrew the research, development, production and even marketing processes of drugs. Powerful new instruments and biotechnology related scientific disciplines (genomics, proteomics) make it possible to examine and exploit the behavior of proteins and molecules. Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. This technology, now approximately 25 years old, is becoming one of the most important technologies developed in the 20(th) century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances regarding rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we describe these processes.


Asunto(s)
Biotecnología/métodos , ADN Recombinante/biosíntesis , Ingeniería Genética , Proteínas Recombinantes/biosíntesis , Animales , ADN Recombinante/genética , ADN Recombinante/uso terapéutico , Darbepoetina alfa , Eritropoyetina/análogos & derivados , Eritropoyetina/biosíntesis , Eritropoyetina/uso terapéutico , Fibrinolíticos/metabolismo , Fibrinolíticos/uso terapéutico , Genómica , Hematínicos/metabolismo , Hematínicos/uso terapéutico , Humanos , Hipoglucemiantes/metabolismo , Hipoglucemiantes/uso terapéutico , Insulina Regular Humana/biosíntesis , Insulina Regular Humana/uso terapéutico , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Proteómica , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Somatostatina/biosíntesis , Somatostatina/uso terapéutico , Activador de Tejido Plasminógeno/biosíntesis , Activador de Tejido Plasminógeno/uso terapéutico
19.
Cancer Gene Ther ; 19(12): 811-7, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23037806

RESUMEN

Allovectin (velimogene aliplasmid) is a cancer immunotherapeutic currently completing a pivotal phase 3 study for metastatic melanoma. Consisting of a bicistronic plasmid encoding both major histocompatibility complex (MHC) class I heavy and light chains (HLA-B7 and ß2-microglobulin, respectively) formulated with a cationic lipid-based system, it is designed for direct intratumoral administration. Following injection into a single lesion, the product is intended to induce anti-tumor immune responses against both treated and distal lesions. Both the plasmid and lipid components of Allovectin contribute to the biological activity of the drug product, and its therapeutic activity is hypothesized to derive from multiple mechanisms of actions (MOAs). These include the induction of both cytotoxic T-cell and innate immune responses directed against allogeneic as well as tumor-derived targets, consequences of both an increased MHC class I expression on tumor cells and the induction of a localized immune/inflammatory response. In this paper, we review Allovectin's proposed MOAs, placing their contributions in the context of anti-tumor immunity and highlighting both preclinical and clinical supporting data.


Asunto(s)
ADN Recombinante/uso terapéutico , Lípidos/uso terapéutico , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/inmunología , Animales , ADN Recombinante/farmacología , Antígeno HLA-B7/inmunología , Humanos , Inmunoterapia/métodos , Lípidos/farmacología , Melanoma/patología , Neoplasias Cutáneas/patología , Resultado del Tratamiento
20.
Ther Deliv ; 3(7): 835-56, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22900466

RESUMEN

Gene therapy is gaining momentum as a method of treating human disease. Initially conceived as a strategy to complement defective genes in monogenic disorders, the scope of gene therapy has expanded to encompass a variety of applications. Likewise, the molecular tools for gene delivery have evolved and diversified to meet these various therapeutic needs. Recombinant adeno-associated virus (rAAV) has made significant strides toward clinical application with an excellent safety profile and successes in several clinical trials. This review covers the basic biology of rAAV as a gene therapy vector as well as its advantages compared with other methods of gene delivery. The status of clinical trials utilizing rAAV is also discussed in detail. In conclusion, methods of engineering the vector to overcome challenges identified from these trials are covered, with emphasis on modification of the viral capsid to increase the tissue/cell-specific targeting and transduction efficiency.


Asunto(s)
ADN Recombinante/uso terapéutico , Dependovirus/genética , Terapia Genética/métodos , Transducción Genética , Animales , Dependovirus/crecimiento & desarrollo , Terapia Genética/efectos adversos , Humanos , Seguridad del Paciente , Medición de Riesgo , Factores de Riesgo , Replicación Viral
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