RESUMO
In spite of continuous development of gene therapy vectors with thousands of drug candidates in clinical drug trials there are only a small number approved on the market today stressing the need to have characterization methods to assist in the validation of the drug development process. The level of packaging of the vector capsids appears to play a critical role in immunogenicity, hence an objective quantitative method assessing the content of particles containing a genome is an essential quality measurement. As transmission electron microscopy (TEM) allows direct visualization of the particles present in a specimen, it naturally seems as the most intuitive method of choice for characterizing recombinant adeno-associated virus (rAAV) particle packaging. Negative stain TEM (nsTEM) is an established characterization method for analysing the packaging of viral vectors. It has however shown limitations in terms of reliability. To overcome this drawback, we propose an analytical method based on CryoTEM that unambiguously and robustly determines the percentage of filled particles in an rAAV sample. In addition, we show that at a fixed number of vector particles the portion of filled particles correlates well with the potency of the drug. The method has been validated according to the ICH Q2 (R1) guidelines and the components investigated during the validation are presented in this study. The reliability of nsTEM as a method for the assessment of filled particles is also investigated along with a discussion about the origin of the observed variability of this method.
Assuntos
Dependovirus , Terapia Genética , Capsídeo , Dependovirus/genética , Vetores Genéticos/genética , Reprodutibilidade dos TestesRESUMO
Nano-drugs based on nanoparticles (NP) or on nano-assemblies as carriers of the active pharmaceutical ingredient (API) are often expected to perform better compared to conventional dosage forms. Maximum realization of this potential though requires optimization of multiple physico-chemical, including structural and morphological, parameters. Meaningful distributions of these parameters derived from sufficient populations of individual NPs rather than ensemble distributions are desirable for this task, provided that relevant high-resolution data is available. In this study we demonstrate powerful capabilities of the up-to-date cryogenic transmission electron-microscopy (cryo-TEM) as well as correlations with other techniques abundant in the nano-research milieu. We explored Doxil®-like (an anticancer drug and the first FDA-approved nano-drug) (75-100 nm) PEGylated liposomes encapsulating single doxorubicin-sulfate nano-rod-crystals (PLD). These crystals induce liposome sphere-to-ellipsoid deformation. Doxil® was characterized by a multitude of physicochemical methods. We demonstrate, that accompanied by advanced image-analysis means, cryo-TEM can successfully enable the determination of multiple structural parameters of such complex liposomal nano-drugs with an added value of statistically-sound distributions. The latter could not be achieved by most other physicochemical approaches. It seems that cryo-TEM is capable of quantitative description of individual liposome morphological features, including meaningful distributions of all structural elements, with averages that correlate with other physical methods. Here it is demonstrated that such quantitative cryo-TEM analysis is a powerful tool in determining what is the optimal drug to lipid ratio in PLD, which is found to be the drug to lipid ratio existing in Doxil®.
RESUMO
The Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene encodes an integral membrane protein, called seipin, of unknown function localized to the endoplasmic reticulum of eukaryotic cells. Seipin is associated with the heterogeneous genetic disease BSCL2, and mutations in an N-glycosylation motif links the protein to two other disorders, autosomal-dominant distal hereditary motor neuropathy type V and Silver syndrome. Here, we report a topological study of seipin using an in vitro topology mapping assay. Our results suggest that the predominant form of seipin is 462 residues long and has an N(cyt)-C(cyt) orientation with a long luminal loop between the two transmembrane helices.
Assuntos
Retículo Endoplasmático/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Membranas Intracelulares/metabolismo , Motivos de Aminoácidos/genética , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/metabolismo , Diabetes Mellitus Lipoatrófica/genética , Diabetes Mellitus Lipoatrófica/metabolismo , Retículo Endoplasmático/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Humanos , Modificação Traducional de Proteínas/genética , Estrutura Terciária de Proteína/genéticaRESUMO
The human cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR), translocates its long amino-terminal (N-terminal) domain across the endoplasmic reticulum (ER) membrane in a C-to-N terminal direction. Using the Semliki Forest virus (SFV) expression system, CB1 was expressed in baby hamster kidney (BHK) cells. It was found that a large fraction of the CB1 molecules were N-terminally truncated prior to ER translocation. Truncation was fast and independent of the proteasome. It is concluded that the truncation process might be a way to create a novel type of CB1.