Ten simple rules for implementing open and reproducible research practices after attending a training course.

Open, reproducible, and replicable research practices are a fundamental part of science. Training is often organized on a grassroots level, offered by early career researchers, for early career researchers. Buffet style courses that cover many topics can inspire participants to try new things; however, they can also be overwhelming. Participants who want to implement new practices may not know where to start once they return to their research team. We describe ten simple rules to guide participants of relevant training courses in implementing robust research practices in their own projects, once they return to their research group. This includes (1) prioritizing and planning which practices to implement, which involves obtaining support and convincing others involved in the research project of the added value of implementing new practices; (2) managing problems that arise during implementation; and (3) making reproducible research and open science practices an integral part of a future research career. We also outline strategies that course organizers can use to prepare participants for implementation and support them during this process.

Evaluation of the SH-SY5Y cell line as an in vitro model for potency testing of a neuropeptide-expressing AAV vector.

Viral vectors have become important tools for basic research and clinical gene therapy over the past years. However, in vitro testing of vector-derived transgene function can be challenging when specific post-translational modifications are needed for biological activity. Similarly, neuropeptide precursors need to be processed to yield mature neuropeptides. SH-SY5Y is a human neuroblastoma cell line commonly used due to its ability to differentiate into specific neuronal subtypes. In this study, we evaluate the suitability of SH-SY5Y cells in a potency assay for neuropeptide-expressing adeno-associated virus (AAV) vectors. We looked at the impact of neuronal differentiation and compared single-stranded (ss) AAV and self-complementary (sc) AAV transduction at increasing MOIs, RNA transcription kinetics, as well as protein expression and mature neuropeptide production. SH-SY5Y cells proved highly transducible with AAV1 already at low MOIs in the undifferentiated state and even better after neuronal differentiation. Readouts were GFP or neuropeptide mRNA expression. Production of mature neuropeptides was poor in undifferentiated cells. By contrast, differentiated cells produced and sequestered mature neuropeptides into the medium in a MOI-dependent manner.

Insight and Development of Advanced Recombinant Adeno-Associated Virus Analysis Tools Exploiting Single-Particle Quantification by Multidimensional Droplet Digital PCR.

Recombinant adeno-associated virus (rAAV) has become the most widely used vector in the gene therapy field with hundreds of clinical trials ongoing and already several products on the market. AAV's physicochemical stability, and the various natural and engineered serotypes allow for targeting a broad range of cell types and tissue by diverse routes of administration. Progressing from early clinical studies to eventual market approval, many critical quality attributes have to be defined and reproducibly quantified, such as AAV stability, purity, aggregates, empty/full particles ratio, and rAAV genome titration. Droplet digital PCR (ddPCR) is becoming the tool of choice to perform absolute quantification of rAAV genomes. In the present study, we have identified critical parameters that could impact AAV titration and characterization accuracy, such as Poisson distribution confidence interval, primers/probe position, and potential aggregates. Our work presents how ddPCR can help to better characterize AAV vectors on the single particle level and highlights challenges that we are facing today in terms of AAV titration.