RESUMEN
The European Pharmacopoeia (Ph. Eur.) general chapter 5.14. Gene transfer medicinal products for human use suggests the use of absorbance measurements at 260 nm to determine the DNA concentration of plasmid vectors used for the preparation of gene therapy products for human use. An international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to confirm the suitability of UV spectrophotometry for the quantification of plasmid vectors used in gene therapy (GT). Three Official Medicine Control Laboratories (OMCLs of the European OMCL Network) and members of the OMCL Working Group for GT products took part in the study, in which various types of spectrophotometers were assessed using common test samples. Results of the study demonstrated that UV spectrophotometry can be considered suitable for the quantification of plasmid DNA in GT products regardless of the instrument used.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/análisis , Plásmidos/análisis , Espectrofotometría Ultravioleta , Calibración , Europa (Continente) , Terapia Genética/normas , Vectores Genéticos/genética , Vectores Genéticos/normas , Humanos , Modelos Lineales , Variaciones Dependientes del Observador , Plásmidos/genética , Plásmidos/normas , Control de Calidad , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrofotometría Ultravioleta/normasRESUMEN
Many gene therapy medicinal products and also some vaccines consist of, or contain, genetically modified organisms (GMOs), which require specific consideration in the environmental risk assessment (ERA) before marketing authorisation or clinical trial applications. The ERA is performed in order to identify the potential risks for public health and the environment, which may arise due to the clinical use of these medicinal products. If such environmental risks are identified and considered as not acceptable, the ERA should go on to propose appropriate risk management strategies capable to reduce these risks. This article will provide an overview of the legal basis and requirements for the ERA of GMO-containing medicinal products in the context of marketing authorisation in the EU and clinical trials in Germany. Furthermore, the scientific principles and methodology that generally need to be followed when preparing an ERA for GMOs are discussed.
Asunto(s)
Terapia Biológica/efectos adversos , Trasplante de Células/legislación & jurisprudencia , Conservación de los Recursos Naturales/legislación & jurisprudencia , Ingeniería Genética/legislación & jurisprudencia , Terapia Genética/legislación & jurisprudencia , Organismos Modificados Genéticamente , Ensayos Clínicos como Asunto/legislación & jurisprudencia , Terapia Genética/efectos adversos , Comercialización de los Servicios de Salud/legislación & jurisprudencia , Medición de RiesgoRESUMEN
Tenascin-R, an extracellular matrix constituent expressed by oligodendrocytes and some neuronal cell types, may contribute to the inhibition of axonal regeneration in the adult central nervous system. Here we show that outgrowth of embryonic and adult retinal ganglion cell axons from mouse retinal explants is significantly reduced on homogeneous substrates of tenascin-R or a bacterially expressed tenascin-R fragment comprising the epidermal growth factor-like repeats (EGF-L). When both molecules are presented as a sharp substrate border, regrowing adult axons do not cross into the tenascin-R or EGF-L containing territory. All in vitro experiments were done in the presence of laminin, which strongly promotes growth of embryonic and adult retinal axons, suggesting that tenascin-R and EGF-L actively inhibit axonal growth. Contrary to the disappearance of tenascin-R from the regenerating optic nerve of salamanders (Becker et al., J Neurosci 19:813-827, 1999), the molecule remains present in the lesioned optic nerve of adult mice at levels similar to those in unlesioned control nerves for at least 63 days post-lesion (the latest time point investigated), as shown by immunoblot analysis and immunohistochemistry. In situ hybridization analysis revealed an increase in the number of cells expressing tenascin-R mRNA in the lesioned nerve. We conclude that, regardless of the developmental stage, growth of retinal ganglion cell axons is inhibited by tenascin-R and we suggest that the continued expression of the protein after an optic nerve crush may contribute to the failure of adult retinal ganglion cells to regenerate their axons in vivo.
Asunto(s)
Regeneración Nerviosa/efectos de los fármacos , Proteínas del Tejido Nervioso/fisiología , Traumatismos del Nervio Óptico/patología , Nervio Óptico/efectos de los fármacos , Tenascina/fisiología , Animales , Axones/ultraestructura , Western Blotting , Técnica del Anticuerpo Fluorescente Indirecta , Hibridación in Situ , Ratones , Ratones Endogámicos , Compresión Nerviosa , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Oligodendroglía/metabolismo , Nervio Óptico/fisiología , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/farmacología , Células Ganglionares de la Retina/patología , Tenascina/biosíntesis , Tenascina/química , Tenascina/genéticaRESUMEN
The adhesion molecule L1 is a member of the immunoglobulin superfamily. L1 is involved in various recognition processes in the CNS and PNS, and binding to L1 can activate signal transduction pathways. Mutations in the human L1 gene are associated with a variable phenotype, including mental retardation and anomalous development of the nervous system, referred to as 'CRASH' (corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus). We generated an animal model of these conditions by gene targetting. Mutant mice were smaller than wild-type and were less sensitive to touch and pain, and their hind-legs appeared weak and uncoordinated. The size of the corticospinal tract was reduced and, depending on genetic background, the lateral ventricles were often enlarged. Non-myelinating Schwann cells formed processes not associated with axons and showed reduced association with axons. In vitro, neurite outgrowth on an L1 substrate and fasciculation were impaired. The mutant mouse described here will help to elucidate the functions of L1 in the nervous system and how these depend on genetic influences.