Extended Data Fig. 10: The HCMV UL36/UL37×1/A151 cocktial promotes T cell expansion after non-viral genome engineering.
a and b, Delivery efficiency (a) and expression intensity (b) 48 h after electroporation with 1 μg of a plasmid expressing PEmax-Npart, 1 μg of a plasmid expressing PEmax-Cpart-T2A-mCherry, along with various combinations of A151 and mRNAs encoding HCMV components UL31, UL36, and UL37x1 in primary T cells. Plasmid-only treatment was performed as a control. c and d, Percentage of T cells expressing EGFP (c) and fold change in EGFP + T cells over input (d) 7 days post-electroporation with the PRIME-In editing components for targeted integration of the EF1α-EGFP transgene, along with varying doses of UL mRNAs and A151. e and f, T cell viability (e) and fold change in total recovered T cells (f) 7 days after non-viral PRIME-In editing for targeted integration of a 5.8-kb transgene expressing CD19 CAR and EGFP. PRIME-In 2.0-engineered T cells treated with DMSO, apoptosis inhibitors Z-DEVD-FMK, Z-VAD-FMK and innate immune inhibitors BX795, Ruxolitinib during the first two days post-electroporation were examined and compared with the UL36/UL37x1-treated and UL36/UL37x1/A151-treated groups. g and h, Subproportions (g) and differentiation states (h) of CAR T cells after PRIME-In-mediated engineering with or without UL36/UL37x1/A151 cocktail treatment. All experiments were independently replicated using primary T cells from n = 3 individual biological donors. Statistical analyses were done using one-way ANOVA with Dunnett’s multiple comparisons test in a-d and g-h, and two-way ANOVA with Bonferroni’s multiple comparisons test in e and f. The results are presented as the mean ± SEM. ns, no significance. Tn, naive T cells; Tcm, central memory T cells; Tem, effector memory T cells; Teff, effector T cells.
