RESUMEN
Recombinant adeno-associated virus (rAAV) has been utilized successfully for in vivo gene delivery for treatment of a variety of human diseases. To sustain the growth of recombinant AAV gene therapy products, there is a critical need for the development of accurate and robust analytical methods. Fifty percent tissue culture infectious dose (TCID50) assay is an in vitro cell-based method widely used to determine AAV infectivity, and this assay is historically viewed as a challenge due to its high variability. Currently, quantitative PCR (qPCR) serves as the endpoint method to detect the amount of replicated viral genome after infection. In this study, we optimize the TCID50 assay by adapting endpoint detection with droplet digital PCR (ddPCR). We performed TCID50 assays using ATCC AAV-2 reference standard stock material across 18 independent runs. The cell lysate from TCID50 assay was then analyzed using both qPCR and ddPCR endpoint to allow for direct comparison between the two methods. The long-term 1-year side-by-side comparison between qPCR and ddPCR as endpoint measurement demonstrated improved interassay precision when the ddPCR method was utilized. In particular, after the addition of a novel secondary set threshold for infectivity scoring of individual wells, the average infectious titer of 18 runs is 6.45E+08 with % coefficient of variation (CV) of 42.5 and 5.63E+08 with % CV of 34.9 by qPCR and ddPCR, respectively. In this study, we offer improvements of infectious titer assay with (1) higher interassay precision by adapting ddPCR as an endpoint method without the need of standard curve preparation; (2) identification of a second "set threshold" value in infectivity scoring that improves assay precision; and (3) application of statistical analysis to identify the acceptance range of infectious titer values. Taken together, we provide an optimized TCID50 method with improved interassay precision that is important for rAAV infectious titer testing during process development and manufacturing.
Asunto(s)
Dependovirus , Genoma Viral , Humanos , Dependovirus/genética , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodosRESUMEN
Repetitive sequence-based PCR (rep-PCR) has been recognized as an effective method for bacterial strain typing. Recently, rep-PCR has been commercially adapted to an automated format known as the DiversiLab system to provide a reliable PCR-based typing system for clinical laboratories. We describe the adaptations made to automate rep-PCR and explore the performance and reproducibility of the system as a molecular genotyping tool for bacterial strain typing. The modifications for automation included changes in rep-PCR chemistry and thermal cycling parameters, incorporation of microfluidics-based DNA amplicon fractionation and detection, and Internet-based computer-assisted analysis, reporting, and data storage. The performance and reproducibility of the automated rep-PCR were examined by performing DNA typing and replicate testing with multiple laboratories, personnel, instruments, DNA template concentrations, and culture conditions prior to DNA isolation. Finally, we demonstrated the use of automated rep-PCR for clinical laboratory applications by using isolates from an outbreak of Neisseria meningitidis infections. N. meningitidis outbreak-related strains were distinguished from other isolates. The DiversiLab system is a highly integrated, convenient, and rapid testing platform that may allow clinical laboratories to realize the potential of microbial DNA typing.
Asunto(s)
Técnicas de Tipificación Bacteriana , Reacción en Cadena de la Polimerasa/instrumentación , Reacción en Cadena de la Polimerasa/métodos , Secuencias Repetitivas de Ácidos Nucleicos/genética , Automatización , Medios de Cultivo , Humanos , Infecciones Meningocócicas/epidemiología , Infecciones Meningocócicas/microbiología , Epidemiología Molecular , Neisseria meningitidis/clasificación , Neisseria meningitidis/genética , Neisseria meningitidis/aislamiento & purificación , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
BACKGROUND: Melanoma is an aggressive tumor with a propensity to rapidly metastasize. The PTEN gene encodes a phosphatase with an unusual dual specificity for proteins and lipids. Mutations of PTEN have been found in various human cancers, including glioblastoma, prostate, breast, lung, and melanoma. Here we investigate in vitro the effects of blocking PI3K signaling using adenoviral-delivered PTEN (Ad-PTEN) in cell lines derived from both early- and late-stage melanoma. MATERIALS AND METHODS: Ad-PTEN transduced melanoma cell lines or normal cells were assayed for cell death, apoptosis, gene expression, invasion and migration, and regulation of angiogenesis. RESULTS: The PTEN locus from RGP and metastatic melanoma cell lines was sequenced; no coding region mutations were found. Adenoviral transfer of PTEN into melanoma cells containing wild-type PTEN alleles led to tumor-specific apoptosis and growth inhibition, with coordinate inhibition of AKT phosphorylation. Ad-PTEN suppressed cell migration by metastatic melanoma cells with concomitant increase in the level of cell surface E-cadherin. Immunohistochemical and confocal analyses localized PTEN to the cytoplasm and demonstrated enrichment at the cell membrane. Ad-PTEN inhibited angiogenesis as demonstrated by the tube formation assay using human vascular endothelial cells. CONCLUSIONS: These studies indicate that Ad-PTEN can inhibit tumor cells via multiple mechanisms and has pro-apoptotic, anti-metastatic, and anti-angiogenic properties. Thus, PI3K blockade via Ad-PTEN may be a promising approach for the treatment of early- and late-stage melanoma, even in tumors that do not harbor PTEN mutations.