Figure 2.

Design of a Cre-Dependent Selection Strategy for AAV-Directed Evolution

(A) Modifications to the AAV viral genome enable Cre-dependent selection. Mutant loxP sites lox66 and lox71 flank the cap gene. Upon Cre inversion of cap, primer ISF changes from a reverse primer to a forward primer, and primer NSF changes from a forward primer to a reverse primer. Primer R remains a reverse primer. Amplification of inverted cap is achieved using the primer pair ISF and R, whereas primers NSF and R selectively amplify non-inverted cap. The translation initiation codons for the Rep open reading frames are knocked out. (B) Cre-dependent AAV selection strategy to target adult neural stem cells. AAV libraries were generated through DNA shuffling or other methods, packaged into AAV virions, and administered to GFAP-Cre 73.12 mice through a unilateral i.c.v. injection. Three weeks later, genomic DNA was extracted from brain tissue of the contralateral hemisphere, and inverted cap variants were selectively amplified using the primers ISF and R for the next round of selection. (C) Viral genomic titers are not significantly different when rep is supplied in trans to package wild-type AAV2. Data are presented as mean ± SEM; n = 3. (D) Selective amplification of inverted cap from GFAP-Cre mice. NS, not significant.