Selecting Novel Retino-tropic AAV Capsids by Directed Evolution.

The selection of novel AAV variants for retinal transduction has been successful for several model systems in the past. Despite this, the publication of a full directed evolution protocol that explains all steps from basic library generation techniques to the selection of suitable candidates for further testing is lacking. The following methods chapter provides a detailed protocol for the generation of an AAV library and the recovery of the most functional candidates using highly predictive transgene expression-driven selection platform. It is important to note that multiple selection platforms have been developed to date, each with unique set of strengths and weaknesses. However, the choice of a selection platform most suitable for given AAV bioengineering application is beyond the scope of this chapter. For the purpose of this chapter, we will describe steps involved in generation of an exemplar peptide insertion library into the variable region VIII of the AAV2 capsid, but the steps outlined can be extended to all other serotypes and other surface loops. Furthermore, the technologies described work independently of the model system and do not require any major specialist equipment.