E Pluribus Unum: 50 Years of Research, Millions of Viruses, and One Goal—Tailored Acceleration of AAV Evolution  Dirk Grimm, Sergei Zolotukhin  Molecular Therapy  Volume 23, Issue 12, Pages 1819-1831 (December 2015) DOI: 10.1038/mt.2015.173 Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Schematic diagram depicting one round of directed molecular evolution which involves three basic steps: diversification, selection, and amplification. Molecular Therapy 2015 23, 1819-1831DOI: (10.1038/mt.2015.173) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Methods for AAV capsid diversification. Depicted are the three strategies that are predominantly found in the literature—DNA family shuffling (DFS), peptide display (PD), and error-prone PCR (epPCR). Also indicated are typical results or recent optimizations, respectively, of each technology. DFS: (1) “good” capsid combining properties of the parental viruses; (2) “bad” capsid in which too many diverse fragments have disrupted functionality; (3) “superior” capsid which has gained novel useful features (pink) not found in any parent. PD: (1) depletion of library from heparin binders (outside the circle), to improve retargeting; (2) use of a chimeric capsid as scaffold for peptide display; (3) same as (2), but using a different AAV wild type. epPCR: (1) rare example of a “winner” capsid which has been improved by a few point mutations; (2) typical wealth of nonfunctional capsids (“losers”) resulting from disruptive mutations; (3) focused PCR randomization in a surface-exposed capsid region. Molecular Therapy 2015 23, 1819-1831DOI: (10.1038/mt.2015.173) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Typical workflow for directed AAV capsid evolution. Depicted from top to bottom are the main steps, from cap gene diversification via different methods (see also Figure 2) and packaging of the resulting mutant pool as a viral library, to selection in cells or animals (positive selection pressure) and, if desired, in the presence of neutralizing anti-AAV antibodies (negative selection pressure). Also indicated is that the ensuing enriched library can be subjected to further rounds of selection after amplification (dashed line) and, again if desired, after additional diversification (dotted line). Eventually, up to five of these iterative cycles will result in one (ideal outcome) or a few mutant capsids that fulfill all requirements and are best tailored for a given application. This figure contains clipart from Servier Medical Art. Molecular Therapy 2015 23, 1819-1831DOI: (10.1038/mt.2015.173) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Schematic depiction of four possible 3D fitness landscapes resulting from plotting of AAV capsid sequence diversity (horizontal axes) against particle fitness (vertical axis). Indicated on the left bottom in each panel is a starting AAV library that is subjected to positive and negative selection pressures, in order to enrich a lead candidate (always circled in red) that managed to climb the highest fitness peak. (a–d) From a to d, the complexity of the landscapes increases which in turn augments chances that other particles may also start to become enriched (such as those in yellow or green), whereas others may be rapidly lost (the one in blue in panel d). An example for panel a is a simple library selection in a single cell type in culture without any additional pressure, whereas the setting in panel d is representative for in vivo biopanning in a live animal. Obviously, such a complex landscape with its many peaks and pits, and hence numerous potential outcomes, puts significant demands on the experimenter to foster the eventual selection of desired capsids. See main text for possible strategies. Molecular Therapy 2015 23, 1819-1831DOI: (10.1038/mt.2015.173) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions