Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers.Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days.Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP + neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.
Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers.Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days.Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP + neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.
Neutralizing antibody (NAb) activity against the viral capsid of adeno-associated viral (AAV) vectors decreases transduction efficiency, thus limiting transgene expression. Several reports have mentioned a variation in NAb prevalence according to age, AAV serotype, and, most importantly, geographic location. There are currently no reports specifically describing the anti-AAV NAb prevalence in Latin America. Here, we describe the prevalence of NAb against different serotypes of AAV vectors (AAV1, AAV2, and AAV9) in Colombian patients with heart failure (HF) (referred to as cases) and healthy individuals (referred to as controls). The levels of NAb were evaluated in serum samples of 60 subjects from each group using an in vitro inhibitory assay. The neutralizing titer was reported as the first dilution inhibiting ≥50% of the transgene signal, and the samples with neutralizing titers at ≥1:50 dilution were considered positive. The prevalence of NAb in the case and control groups were similar (AAV2: 43% and 45%, respectively; AAV1 33.3% in each group; AAV9: 20% and 23.2%, respectively). The presence of NAb for two or more of the serotypes analyzed was observed in 25% of the studied samples, with the largest amount in the positive samples for AAV1 (55–75%) and AAV9 (93%), suggesting serial exposures, cross-reactivity, or coinfection. Moreover, patients in the HF group exhibited more common combined seropositivity for NAb against AAV1 d AAV9 than those in the control group (91.6% vs. 35.7%, respectively; p = 0.003). Finally, exposure to toxins was significantly associated with the presence of NAb in all regression models. These results constitute the first report of the prevalence of NAb against AAV in Latin America, being the first step to implementing therapeutic strategies based on AAV vectors in this population in our region.
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