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Adeno-associated virus (AAV) vectors have emerged as key clinical candidates for gene therapy. Yet, the efficiency and safety of these 20-25 nm biological nanoparticles remain difficult to harmonize across pre-clinical studies due to the limitations of current analytical tools. The presence of residual DNA, protein contaminants, empty particles and VP subunits resulting from incomplete capsid assembly are variables that can strongly modulate the reliability of in vivo data and that, therefore, need to be closely monitored in AAV research laboratories. In this work, 70 AAV preparations, obtained with various production (baculo/Sf9 and triple transfection system) and purification (iodixanol gradient and double cesium-chloride gradient) techniques were analyzed using a thermal shift assay based on the fluorescent dye Sypro® Orange. The fluorescence fingerprint obtained did not only allow to discriminate various AAV serotypes based on their capsid melting temperatures, but also enabled to probe the homogeneity and purity of AAV vector preparations, investigated in parallel using dynamic light scattering (DLS) and polyacrylamide gel electrophoresis. In particular, a double fluorescence transition indicated the presence of capsid-associated protein contaminants whereas a high initial fluorescence background correlated with the presence of free protein contaminants and capsid subunits, possibly resulting from capsid degradation during vector purification or storage. The variability, sensitivity and precision of this assay were further investigated in two different AAV research laboratories. This simple, fast (analysis of 94 preps in ~6 hrs) and low-cost assay emerges as a relevant tool for characterization of AAV vector preparations and will help to increase the reliability of in vivo gene transfer studies.

A 2010 report that Adeno-associated virus 2 (AAV2) encodes an additional protein in the second open reading frame of the cap(sid) gene significantly changed our perception of the biology of this virus. This so-called assembly-activating protein or AAP appears to interact with the major viral capsid protein 3 (VP3) and to chaperone its assembly into virus-like particles, via mechanisms that remain unknown. Moreover, it was shown that deliberate mutation of the AAP gene diminishes AAV particle production.As the AAP open reading frame fully overlaps with that of the AAV capsid proteins VP1/VP2 (and partially VP3), it is likely that the AAP gene is inadvertently disrupted or mutated during molecular AAV evolution protocols that involve capsid gene shuffling (homology-directed recombination of cap gene fragments). To study this possibility comprehensively and to provide solutions, if needed, we first investigated the role of AAP for 9 further AAV serotypes other than AAV2. Therefore, we knocked out AAP expression in serotypes 1, 3-9 and rh10 while leaving the rep and cap genes intact. Indeed, all mutants were markedly impaired in particle production, confirming that functional AAP is critically required for at least 10 different AAV serotypes.Notably, all vectors were fully restored upon co-delivery of the corresponding AAP via an extra plasmid during particle production. Furthermore, when testing the cross-reactivity of all AAPs with the AAV2-AAP mutant (AAV2mut), we observed a broad interchangeability except for AAP4 and 5 which predominantly rescued their cognate serotype.These results were confirmed independently in a baculovirus AAV2 vector production system where we also observed a dramatic reduction in particle yields upon AAP mutation. Interestingly, in both, mammalian and insect cells, we consistently noted that absence of AAP reduced the steady-state levels of VP proteins, suggesting a similar biological function of AAP in the two heterologous cellular systems. As we could rule out an effect on the transcriptional level, we postulate that AAP-mediated chaperoning of AAV virion assembly indirectly stabilizes VP proteins in cells of different species.To explicitly dissect the role of AAP in the context of molecular AAV evolution, we produced five different AAV capsid libraries with increasing complexities, subcloned 46 randomly selected shuffled AAPs from these and tested them with the AAV-AAP mutants. Out of these 46 shuffled AAPs, 37 could rescue AAV2mut, and two more efficiently complemented an AAV5-AAP mutant. In total, 84.8 % of all tested AAPs were functional.Of note, the highest proportion of defect AAPs was found in libraries that contained the diverse capsids of AAV4 and 5. We thus shuffled serotypes 2, 4, 5, 8 and 9 and produced viral libraries with or without a cocktail of excess AAP from all five serotypes. Strikingly, albeit up to 40% of all AAP genes were predicted to be non-functional as a result of inadvertent shuffling, AAP trans-complementation had no significant effect on particle yields.Collectively, our data suggest that albeit AAP is disrupted to some extent during AAV capsid gene shuffling, this is likely not a major restriction for the efficiency and success of this technology.

Type 1 diabetes results from autoimmune destruction of the insulin-producing -cells of pancreatic islets. Although islet transplantation leads to successful insulin delivery, shortage of donors limits a broad application of this therapy, and thereby new sources of -cells are required. Bone marrow is an important source of easily procurable adult stem cells, and cells derived from bone marrow compartment have shown to possess pluripotent capabilities. Indeed, some studies have found an important contribution of bone marrow-derived cells to the pancreatic -cells, while other studies did not reach the same conclusions. It has been discussed that these controversial results could be due to different experimental conditions, such as bone marrow transplantation protocols, methods used for the measurements, and the model of pancreatic damage. Overexpression of IGF-I specifically in -cells in transgenic mice (RIP/IGF-I) lead to recovery from type 1 diabetes by increasing -cell neogenesis and replication, and by counteracting apoptosis in -cells after STZ treatment. Here we studied the contribution of bone marrow-derived cells to the -cell mass of STZ-treated and non-treated RIP/IGF-I transgenic mice. Bone marrow from GFP transgenic mouse was transplanted into control and RIP/IGF-I transgenic mice. One month later, diabetes was induced by STZ injection in 50% control and 50% transgenic mice. Blood glucose levels of control mice reach values over 400mg/dl, while transgenic mice remained mild hyperglycemic (about 200mg/dl). All animals were killed 4 months after the transplant and both mice under steady-state conditions and STZ-treated were analyzed for chimerism in pancreatic tissue. Chimerism in peripheral blood cells was >80% one month after the transplant and was maintained at the endpoint of the study. Immunohistochemical analysis of the pancreas revealed that most of the GFP+ cells were found in the interstitium of both exocrine and endocrine tissue, and some groups were detected near the ducts. However, no significant in vivo differentiation of bone marrow into -cells was observed, neither in healthy nor in STZ-treated RIP/IGF-I mouse. Thus, neither pancreatic damage induced by STZ nor growth factor overexpression was sufficient to recruit and differentiate bone marrow cells into endocrine cells. In conclusion, our results suggest that bone marrow stem cells have a limited capacity to differentiate into -cells in vivo, and thereby they do not contribute to endocrine pancreas regeneration of RIP/IGF-I transgenic mice.

Type 1 diabetes results from autoimmune destruction of pancreatic β cells. This process might be reverted by genetically engineering endocrine pancreas in vivo to express factors that induce β-cell replication and neogenesis and counteract the immune response. However, the pancreas is difficult to manipulate and pancreatitis is a serious concern, which has made effective gene transfer to this organ elusive. Thus, new approaches for gene delivery to pancreas in vivo are required. Here we show that mouse pancreatic β-cells were efficiently transduced, and they expressed β-galactosidase after systemic injection of adenovirus with clamped hepatic circulation. Seven days after vector administration about 70% of pancreatic islets showed β-gal expression. In these islets, about 20% of the β-cells were transduced by the adenoviruses. Furthermore, scattered acinar cells expressing β-gal were also observed. In addition to mouse, we also examined the ability of adenovirus to transduce dog pancreas. We have observed that acinar, ductal and endocrine pancreatic cells were transduced after injection of adenovirus in dogs underwent local clamping of pancreatic circulation. Thus, these approaches may be used to transfer genes of interest to pancreas for both the study of islet biology and the gene therapy of diabetes and other pancreatic disorders.