Paul Berg and the origins of recombinant DNA.

In fall 1972, Paul Berg's laboratory published articles in PNAS describing two methods for constructing recombinant DNAs in vitro. He received half of the 1980 Nobel Prize in Chemistry for this landmark accomplishment. Here, we describe how this discovery came about, revolutionizing both biological research and the pharmaceutical industry.

Building biosecurity for synthetic biology.

The fast-paced field of synthetic biology is fundamentally changing the global biosecurity framework. Current biosecurity regulations and strategies are based on previous governance paradigms for pathogen-oriented security, recombinant DNA research, and broader concerns related to genetically modified organisms (GMOs). Many scholarly discussions and biosecurity practitioners are therefore concerned that synthetic biology outpaces established biosafety and biosecurity measures to prevent deliberate and malicious or inadvertent and accidental misuse of synthetic biology's processes or products. This commentary proposes three strategies to improve biosecurity: Security must be treated as an investment in the future applicability of the technology; social scientists and policy makers should be engaged early in technology development and forecasting; and coordination among global stakeholders is necessary to ensure acceptable levels of risk.

Asfotase alfa en el tratamiento de la hipofosfatasia / Asphotase alfa in the treatment of hypophosphatasia

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.

LeRoy Walters's Legacy of Bioethics in Genetics and Biotechnology Policy.

LeRoy Walters was at the center of public debate about emerging biological technologies, even as "biotechnology" began to take root. He chaired advisory panels on human gene therapy, the human genome project, and patenting DNA for the congressional Office of Technology Assessment. He chaired the subcommittee on Human Gene Therapy for NIH's Recombinant DNA Advisory Committee. He was also a regular advisor to Congress, the executive branch, and academics concerned about policy governing emerging biotechnologies. In large part due to Prof. Walters, the Kennedy Institute of Ethics was one of the primary sources of talent in bioethics, including staff who populated policy and science agencies dealing with reproductive and genetic technologies, such as NIH and OTA. His legacy lies not only in his writings, but in those people, documents, and discussions that guided biotechnology policy in the United States for three decades.

Alienation from the Objectives of the Patent System: How to Remedy the Situation of Biotechnology Patent.

Some fundamental biotechnologies hold unprecedented potential to eradicate many incurable diseases. However, in absence of regulations, the power of patent makes the future use of some important biotechnology in few institution's hands. The excessive patents restrict researcher access to the fundamental technologies. It generates concerns and complaints of deteriorating the public health and social welfare. Furthermore, intellectual curiosities, funding, respect among colleagues etc., rather than patents, are the real motivations driving a major ground-breaking discoveries in biotechnology. These phenomena reveal that some biotechnology patents are alienated from the purpose of patent system. Therefore, it is necessary to take some approaches to stop over-patenting these fundamental biotechnology inventions. This article proposes a model regulatory framework for controlling biotechnology patent alienating from the purpose of patent system.

Laronidasa para el tratamiento de pacientes con mucopolisacaridosis tipo 1 / Laronidase for the treatment of patients with mucopolysaccharidosis type 1

INTRODUCCIÓN: a) Cuadro clínico: La mucopolisacaridosis tipo I (MPS I) es una enfermedad autosómica recesiva dentro del grupo de errores innatos del metabolismo con depósito lisosomal de diversos tipos de glucosaminoglucanos (GAG). Este cúmulo de GAG (dermatán sulfato y heparan sulfato) puede darse en cualquier órgano y es provocado por la deficiencia de la enzima -L-iduronidasa, conllevando a síntomas progresivos multisistémicos y potencialmente mortales. b) Tecnología sanitaria: Laronidasa (Aldurazyme®, BioMarin Pharmaceutical Inc) es una variante polimórfica de la enzima humana -L-iduronidasa que se produce mediante tecnología de ADN recombinante. Su objetivo es sustituir la -L-iduronidasa ausente en MPS I proporcionando una enzima exógena para la absorción en los lisosomas. De esta forma aumenta el catabolismo de GAG y disminuye su acumulación. OBJETIVO: Evaluar la eficacia y seguridad, así como documentos relacionados a la decisión de cobertura de laronidasa para pacientes con mucopolisacaridosis tipo I. METODOLOGÍA: Se realizó una búsqueda en las principales bases de datos bibliográficas: MEDLINE (PubMed), LILACS, COCHRANE, así como en buscadores genéricos de Internet incluyendo Google Scholar y TRIPDATABASE. En primer lugar se seleccionaron ensayos clínicos aleatorizados (ECAs) y revisiones sistemáticas (RS) que evalúen la eficacia y seguridad de la tecnología. Debido a la escasez de estudios, se incluyen también estudios observacionales. Adicionalmente, se hizo una búsqueda dentro de la información generada por las principales instituciones internacionales de enfermedades raras; y agencias que realizan RS, evaluación de tecnologías sanitarias (ETS) y guías de práctica clínica (GPC). RESULTADOS: Se seleccionaron dos RS, dos GPC, un consenso y cinco ETS. Una RS publicada en el año 2017, incluyó dos ECAs y siete estudios no aleatorizados que incluyeran más de cinco pacientes. El primer ECA seleccionado compara laronidasa en dosis de 100 U/kg (0,58 mg/kg) endovenosa y placebo en niños de con una edad promedio de 15,5 años (rango: 6 a 43 años) teniendo como desenlaces primarios a la capacidad vital forzada (CVF) y la prueba de caminata de 6 minutos (PC6m). Los autores reportan que después de 26 semanas, los pacientes que recibieron laronidasa mostraron mejoras estadísticamente significativas en la CVF, mas no en la distancia de PC6m. También redujo significativamente la hepatomegalia y los niveles de GAG urinarios y, en pacientes más gravemente afectados, mejoró la apnea/hipopnea del sueño y la flexión del hombro. En el segundo ECA se compararon distintas dosis de laronidasa, no encontrando diferencias significativas en la reducción de la excreción urinaria de GAG o el volumen hepático. En ambos estudios laronidasa tuvo un perfil de seguridad aceptable. Debido a la heterogeneidad de estos dos ECAs, no se pudo realizar un meta-análisis. CONCLUSIONES: La evidencia con respecto a la eficacia y seguridad de laronidasa en MPS-I es escasa y de baja calidad metodológica; se basa en dos ECAs comparando laronidasa frente a placebo o distintas dosis de laronidasa entre sí. Si bien se ha intentado analizar los desenlaces clínicos combinando los resultados de los ECAs y estudios observacionales, la calidad de estos resultados es cuestionable y tiene que ser tomada con precaución. Los beneficios demostrados con la evidencia disponible son moderados, sin incluir un beneficio en desenlaces primordiales como mortalidad o calidad de vida. La mayoría de GPC y consensos consideran el uso de laronidasa después del trasplante de células hematopoyéticas. No existe consenso en las recomendaciones de las ETS seleccionadas, dos de ellas consideran justificable el reembolso de laronidasa después de la evaluación de la evidencia y análisis presupuestarios. Las cinco ETS coinciden en que la evidencia disponible es de baja calidad metodológica.

WHO standards for biotherapeutics, including biosimilars: an example of the evaluation of complex biological products.

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.

Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost.

Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides.

FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium-Copy Number Plasmids in Escherichia coli.

Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Unfortunately, overexpression of fabI cannot be used to select medium-copy number plasmids, typically used for the expression of heterologous proteins in E. coli. Here we report that Vibrio cholera FabV, a functional homologue of E. coli FabI, can be used as a suitable marker for the selection and maintenance of both high and medium-copy number plasmid vectors in E. coli.

Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.

New molecular biologist perspective and insight: DNA topoisomerases production by recombinant DNA technology for medical laboratory application and pharmaceutical industry

DNA topoisomerases are essential enzymes that control the topological state of DNA replication during mitosis. These enzymes are classified based on their mechanisms and physical properties. During mitosis, superhelical DNA must be unwound or relaxed by DNA topoisomerases prior to a decoding step by DNA processing enzymes, such as DNA polymerase and RNA polymerase. By blocking the reaction of resealing the breaks in the DNA ultimately can result in cellular death. Compounds that inhibit the catalytic function of these enzymes can serve as potential anticancer agents. DNA topoisomerases are found in nature and used as high quality and well-validated targets for the screening of potential anticancer agents. Our current work focuses on determining potential anticancer agents from natural resources using DNA topoisomerases as the screening targets. Large scale production of these enzymes using recombinant DNA technology in our academic laboratory is utilised to avoid dependence on expensive commercially available enzymes. The in-house produced enzymes can also be used to enhance our research in the field of molecular medicine by providing an enzyme source that can be used to screen potential anticancer agents, and for other newly developed diagnostic and medical research projects in the near future as well as a step in moving our efforts into the industrial sector.

Stanley N. Cohen - an interview by Judy Peng.

Biotechnology Journal talks to Prof. Stanley Cohen after his plenary lecture at the International Biotechnology Symposium, Sept 2012, in Daegu, Korea.

Enhanced recovery and purification of P(3HB-co-3HHx) from recombinant Cupriavidus necator using alkaline digestion method.

A simple, efficient and economical method for the recovery of P(3HB-co-3HHx) was developed using various chemicals and parameters. The initial content of P(3HB-co-3HHx) in bacterial cells was 50-60 wt%, whereas the monomer composition of 3HHx used in this experiments was 3-5 mol%. It was found that sodium hydroxide (NaOH) was the most effective chemical for the recovery of biodegradable polymer. High polyhydroxyalkanoate purity and recovery yield both in the range of 80-90 wt% were obtained when 10-30 mg/ml of cells were incubated in NaOH at the concentration of 0.1 M for 60-180 min at 30 °C and polished using 20 % (v/v) of ethanol.

EMMA, a cost- and time-effective diagnostic method for simultaneous detection of point mutations and large-scale genomic rearrangements: application to BRCA1 and BRCA2 in 1,525 patients.

The detection of unknown mutations remains a serious challenge and, despite the expected benefits for the patient's health, a large number of genes are not screened on a routine basis. We present the diagnostic application of EMMA (Enhanced Mismatch Mutation Analysis(®) , Fluigent, Paris, France), a novel method based on heteroduplex analysis by capillary electrophoresis using innovative matrices. BRCA1 and BRCA2 were screened for point mutations and large rearrangements in 1,525 unrelated patients (372 for the validation step and 1,153 in routine diagnosis) using a single analytical condition. Seven working days were needed for complete BRCA1/2 screening in 30 patients by one technician (excluding DNA extraction and sequencing). A total of 137 mutations were found, including a BRCA2 duplication of exons 19 and 20, previously missed by Comprehensive BRACAnalysis(®) . The mutation detection rate was 11.9%, which is consistent with patient inclusions. This study therefore suggests that EMMA represents a valuable short-term and midterm option for many diagnostic laboratories looking for an easy, reliable, and affordable strategy, enabling fast and sensitive analysis for a large number of genes.

Developments in high-yield system expressed vaccines and immunotherapy.

Conventional vaccine production techniques are outdated, leaving the world defenseless to viruses and pathogens. Successful protection necessitates the innovation of strategies that can generate an induced defensive humoral and cellular response with: ease of mass production, nominal side-effects, and controlled design specificity, all while being cost effective. Fortunately, technology exists to facilitate such advances in this billion dollar industry and this review is focused on recent publications and patents which hold promise to revolutionize the fight against pathogenic illnesses.