Sleeping Beauty System to Redirect T-cell Specificity for Human Applications

Abstract

The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electro-transfer of CD19-specific SB DNA plasmids in PBMC and propagation on CD19+ artificial antigen presenting cells (aAPC) was used to numerically expand CD3+ T cells expressing CAR. By Day 28 of co-culture >90% of expanded CD3+ T cells expressed CAR. CAR+ T cells specifically killed CD19+ target cells and consisted of subsets expressing biomarkers consistent with central memory, ieffector memory, and effector phenotypes. CAR+ T cells contracted numerically in the absence of CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCRVα and TCRVβ repertoire. Quantitative fluorescence in situ hybridization (Q-FISH) revealed that CAR+ T cells preserved telomere length. Quantitative PCR (Q-PCR) and FISH showed CAR transposon integrated on average once per T-cell genome. CAR+ T cells in peripheral blood can be detected by Q-PCR at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the non-viral SB system for the investigational treatment of CD19+ B-cell malignancies (currently under three INDs #: 14193, 14577, and 14739).

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

“…These included transcription factors known to be expressed in naive and memory cells (Lef1, FoxP1), integrin ITGB1, and type 1 cytokine receptor IL7R; costimulatory molecule ICOS; multifunctional protein BIRC2 that regulates not only caspases and apoptosis, but also modulates inflammatory sig- However, no specific gene set was enriched (FDR < 5% or < 1%) either in day-0 or day-28 cells (data not shown). To avoid transposition after infusion, the presence of SB11 was excluded by PCR at the time of cryopreservation in all infused products (Supplemental Table 2) (35,38). In addition, aliquots of each CAR T cell product were tested for autonomous growth according to our published methods (38), and no aberrant growth was seen (Supplemental Figure 5).…”

“…We note that in these studies a transposase of intermediate activity (SB11; ref. 35) was used, rather than the more active SB100X transposase that may direct multiple integrations of transposons into individual cells (68,69). Another advantage of our approach is that we avoided the expense and inconvenience associated with steady-state apheresis, as the electroporation and propagation of T cells was accomplished from PB obtained by venipuncture.…”

“…To produce CAR T cells for infusion, peripheral blood (PB) mononuclear cells (PBMCs) simply obtained by venipuncture (Supplemental Table 1), were electroporated with SB transposon and transposase plasmids encoding CD19RCD28 and SB11. Electroporated cells were expanded on γ-irradiated clone 4 AaPCs for a median of 28 days (autologous: average 29 days, SD 1.4 days; allogeneic: average 28.5 days, SD 3.9 days) with IL-2 and IL-21, enabling selective growth of T cells stably encoding the CD19-specific CAR (Figure 2A), following methods reported previously (34)(35)(36)(37)(38). This process resulted in the selective outgrowth of T cells with integrated CAR (average cell expansion: ~2,200-fold autologous and ~2,500-fold allogeneic).…”

Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection

“…We genetically modified primary circulating T cells using our SB transposon/transposase system that we adapted for human gene transfer. Indeed, we have achieved institutional and federal regulatory approvals for four clinical trials (NCT00968760, NCT01497184, NCT01362452, NCT01653717) infusing autologous and allogeneic T cells genetically modified with an SB encoding a CD19-specific CAR and propagation on aAPC (clone #4) (21,22,37). To generate clinically relevant numbers of D-CAR + T cells for adoptive transfer, T cells were expanded numerically on "designer" aAPC.…”

“…Thus, using a gene transfer and propagation approach adapted for human application (21,22), we have demonstrated that an innate immune response to fungi can be harnessed by bioengineering cytotoxic T cells. This demonstration provides the foundation for testing whether D-CAR + T cells can be infused to improve the survival of immunocompromised patients who develop opportunistic IA infection.…”