A comparison of AAV-vector production methods for gene therapy and preclinical assessment.

Adeno Associated Virus (AAV)-mediated gene expression in the brain is widely applied in the preclinical setting to investigate the therapeutic potential of specific molecular targets, characterize various cellular functions, and model central nervous system (CNS) diseases. In therapeutic applications in the clinical setting, gene therapy offers several advantages over traditional pharmacological based therapies, including the ability to directly manipulate disease mechanisms, selectively target disease-afflicted regions, and achieve long-term therapeutic protein expression in the absence of repeated administration of pharmacological agents. Next to the gold-standard iodixanol-based AAV vector production, we recently published a protocol for AAV production based on chloroform-precipitation, which allows for fast in-house production of small quantities of AAV vector without the need for specialized equipment. To validate our recent protocol, we present here a direct side-by-side comparison between vectors produced with either method in a series of in vitro and in vivo assays with a focus on transgene expression, cell loss, and neuroinflammatory responses in the brain. We do not find differences in transduction efficiency nor in any other parameter in our in vivo and in vitro panel of assessment. These results suggest that our novel protocol enables most standardly equipped laboratories to produce small batches of high quality and high titer AAV vectors for their experimental needs.

Optimization of production and transgene expression of a retrogradely transported pseudotyped lentiviral vector.

BACKGROUND: To target specific neuronal populations by gene transfer is challenging. A complicating fact is that populations of neurons may have opposing roles despite being found adjacent to each other. One example is the medium spiny neurons of the striatum. These cells have different projection patterns, a trait used in this study to specifically target one population. NEW METHOD: Here we present a way of labeling and further studying neurons based on their projections. This was achieved by pseudotyping lentiviral vectors with a chimeric glycoprotein allowing for retrograde transport in combination with optimizing the promoter element used. RESULTS: We transduced on average 4000 neurons of the direct pathway in the striatum, with the viral vector allowing for microscopy and miRNA immunoprecipitation. In addition, we were able to optimize vector production, reducing the time and material used. COMPARISON WITH EXISTING METHOD: The optimized protocol is more reproducible compared to previously published protocols. Alternative methods to study specific populations of neurons are transgenic animals or, if available, specific promoter elements. However, very specific promoter elements are rarely available and often large, limiting the usefulness in viral vectors. Our optimized retrograde vectors allow for selection based on neuronal projections and are therefore independent of such elements. CONCLUSION: We have developed a method that allows for specific analysis of neuronal subpopulations in the brain either by microscopy or by biochemical methods e.g. immunoprecipitation. This method is simple to use and can be combined with transgenic animals for studying disease models.

GDNF-mediated rescue of the nigrostriatal system depends on the degree of degeneration.

Glial cell-line derived neurotrophic factor (GDNF) is a promising therapeutic molecule to treat Parkinson's disease. Despite an excellent profile in experimental settings, clinical trials testing GDNF have failed. One of the theories to explain these negative outcomes is that the clinical trials were done in late-stage patients that have advanced nigrostriatal degeneration and may therefore not respond to a neurotrophic factor therapy. Based on this idea, we tested if the stage of nigrostriatal degeneration is important for GDNF-based therapies. Lentiviral vectors expressing regulated GDNF were delivered to the striatum of rats to allow GDNF expression to be turned on either while the nigrostriatal system was degenerating or after the nigrostriatal system had been fully lesioned by 6-OHDA. In the group of animals where GDNF expression was on during degeneration, neurons were rescued and there was a reversal of motor deficits. Turning GDNF expression on after the nigrostriatal system was lesioned did not rescue neurons or reverse motor deficits. In fact, these animals were indistinguishable from the control groups. Our results suggest that GDNF can reverse motor deficits and nigrostriatal pathology despite an ongoing nigrostriatal degeneration, if there is still a sufficient number of remaining neurons to respond to therapy.