GoKawiilEthics statement
Leukopaks from deidentified healthy donors with Institutional Review Board-approved consent forms and protocols were purchased from StemCell Technologies (200-0092). Residuals from leukoreduction chambers after Trima Apheresis from deidentified healthy donors with Institutional Review Board-approved consent forms and protocols were purchased from Vitalant.
All of the mice in this study were treated following a protocol (AN182757) approved by the UCSF Institutional Animal Care and Use Committee (IACUC).
Plasmids
For transient GFP expression, a scAAV plasmid with a CMV enhancer chicken B-actin intron (CAG) promoter was used (scAAV-CAG-GFP, Addgene, 83279). A similar ssAAV (ssAAV-CAG-GFP) has been used for biodistribution studies (Addgene, 28014).
A plasmid encoding the HIV protein gag fused to Cas9 expressing four Nuclear Localization Signal (4×NLS) was used to package Cas9 into EDVs59. For integrating genes at the TRAC locus, we used the same homology arms and sgRNA sequence as previously described1. An EGFRt sequence was cloned in following a 1928z-1XX sequence33. To potentially improve nuclease activity when combined with a Cas9-containing EDV, a U6 promoter expressing the sgRNA for targeting TRAC was cloned into the plasmids upstream of the left homology arm (pAAV-U6/TRAC-TRAC-1928-1XX-P2A-EGFRt). The extracellular domain of a BCMA-targeting CAR was used to replace the CD19-binding sequence to generate a pAAV-U6/TRAC-TRAC-BCMA-1XX-P2A-EGFRt.
Plasmids for the B7H3 experiment were designed by cloning the scFv targeting B7H3 into a 28z-1XX CAR. The construct was flanked by the same homology arms used previously targeting TRAC.
To generate a GFP fusion at the Clta N terminus, the GFP gene was cloned into an AAV plasmid containing homology arms targeting the CLTA exon 1 start codon (pAAV-CLTA-GFP). The design homology arm design and sgRNA sequences were taken from a previous study41.
To produce lentiviruses delivering an anti-CD19 CAR, a second-generation lentivirus comprising a transfer plasmid, a packaging plasmid and an envelope plasmid were used. The exact CAR sequence from pAAV-U6/TRAC-TRAC-1928-1XX-P2A-EGFRt cloned into a lentiviral transfer plasmid, under an EF1-α promoter, was used. The AAV-hT7 capsid variant generated in this study has been made available at Addgene (252215).
Cell lines
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