AAV Production and Characterization

A CD19 chimeric antigen receptor sequence containing the FMC63 scFv, hinge and transmembrane domain of CD8α, and intracellular domains of 4-1BB and CD3ζ³² was synthesized (Integrated DNA Technologies). This sequence was cloned into a plasmid that contained a pair of inverted terminal repeat (ITR) sequences for production of AAV. The AAV plasmid was initially prepared by cloning two homology arms from the TRAC genomic sequence sequentially in between the ITRs. The first homology arm included half of the nuclease target site and 985 bp of upstream genomic sequence. The second homology arm included the other half of the nuclease target site and 763 bp of downstream genomic sequence. A JeT promoter was synthesized from overlapping oligonucleotides in a PCR reaction and cloned between the homology arms. An SV40 polyadenylation sequence was amplified by PCR and cloned downstream of the JeT promoter. Finally, the synthesized CAR was inserted between the JeT promoter and the SV40 polyadenylation sequence. All AAV6 vectors used in Figures 1, ​,2,2, ​,3,3, ​,4,4, and ​and5A5A were produced using a triple transfection protocol and were purified using a CsCl gradient before dialysis in 1× PBS.42 The AAVs used in Figures 5B, 5C, and ​and66 were produced by Virovek. Vectors were aliquoted and stored at −80°C until use.