Directed Evolution of Shuffled AAV Capsids

A shuffled AAV capsid was created as described^{17 (link)} using the following parent capsids as starting material: AAV1, AAV2, AAV2i8,^{21 (link)} AAV2.5,^{22 (link)} AAV6, AAV8, AAV9, AAV9.47,^{23 (link)} AAVrh10 and capsids recovered from unrelated ongoing shuffling projects.³² The shuffled library was injected intravenously into rats that previously received a striatal 6-OHDA treatment. Three days later, the rats were killed and striatal cells were mechanically dissociated. DNA was recovered from neuron-enriched samples using the Qiagen DNeasy blood and tissue kit (Venlo, Netherlands) and subsequently concentrated by ethanol precipitation. Intact capsid library sequences were recovered by PCR using a high-fidelity polymerase, Pfu Ultra II (Agilent, Santa Clara, CA, USA), with primers SwaICapUP 5′-GCGAATGATTTAAATCAGGTATGGCTGC-3′ and P4ext 5′-AAGCTCTAGACGGACACCAAAGTTCAACT-3′. A subsequent error-prone PCR step was employed to further diversify the library between rounds. Cloning and library amplification between selection rounds was carried out as previously described.^{17 (link)} A total of two rounds of selection were performed. Recovered clones after each round were subcloned into rAAV pXR2 backbones and SSV9 replication-competent backbones and sequenced.