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These reports describe recombinant AAVs that express reporter genes under the control of cell-specific transcriptional enhancers (enhancer rAAV), and their use to label target cells in the CNS with a high level of specificity and sensitivity. The approach is validated in cortical, striatal, and spinal cord neurons, as well as in non-neuronal cells of the glia and the vascular endothelium.
Somatic AAV gene transfer of reporter or therapeutic transgenes has numerous advantages over the current gold-standard methods using transgenic animals with Cre-mediated induction of cell specific reporters. Above all, enhancer rAAVs can be used to probe and manipulate brain circuits in different species, including primates, whereas the transgenic studies are mostly confined to rodents.
This new tool kit is exciting because it will enable studies of brain connectivity and development in ways that have not been possible up to now. It will also help design more precise and safer therapeutic modalities in the CNS. The new AAV-based tools are validated by measuring, in the target cells of the same animal, the signal generated by chromosomal Cre-mediated expression, and the one obtained from the rAAV genome. A high level of specificity and sensitivity is generally reported. However, these numbers vary, depending on the target cell type and the enhancer rAAV used. Specificities as low as 20 percent are reported, pointing to limitations of the approach.
In discussing these shortcomings, the authors note that the outcome of the studies can vary with the dose of rAAV, the age of the animal, and the route of administration. Also, the introduction of multiple copies of an enhancer sequence in a cell can induce squelching of transcription factors. Although no evidence of this is reported, it is indeed a source of concern, and it may be enhancer dependent.
Users of the technology should keep in mind that other factors are likely to limit or bias the precision of the rAAV tools. Toxicities associated with the quality of the rAAV preparation can have an impact, calling for careful analysis of vector quality. Certain cell types may not be transduced efficiently (or at all) when using certain capsids. Furthermore, it is now well-demonstrated that capsid proteins themselves can radically influence the formation of chromatin around the rAAV genome when it is delivered to the nucleus of the target cell. Cases of complete transcriptional shut-off have been described and that may counteract the activity of the enhancers. This bias can be cell- and species-specific.
With such a powerful tool kit, future investigators will need to go through a detailed validation for each novel combination of rAAV, enhancer, species, and route of administration. The technology is groundbreaking but not (yet) turnkey.
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