Investigational therapy can be successfully undertaken using viral- and nonviral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently, the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR+ T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease.
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Transposition was applied to practice in 2012 using the SB system to enforce expression of a second generation 170 CD19-specific CAR in T cells. The CAR redirects T-cell specificity for CD19 independent of major histocompatibility complex as antigen-recognition is mediated through the single-chain variable fragment domain of a CD19-specific monoclonal antibody and T-cell activation is initiated though chimeric endodomain. Adoptive transfer of CD19-specific T-cells in early clinical trials has shown therapeutic efficacy the based on expected loss of normal CD19 + B cells and significant destruction of large numbers of malignant B cells. 171-175 Patients with large tumor burdens that are rendered lymphopenic through prior conditioning chemotherapy can experience the numeric expansion of infused CAR + T cells. If synchronous activation of the infused T cells occurs, then there can be supraphysiologic elevation of inflammatory cytokines and systemic toxicities. These adverse events may be temporarily delayed from the time of infusion as the numbers of in vivo CAR + T cells swells. However, the signs and symptoms can be managed through the administration of systemic corticosteroids as well as biologic agents that interrupt the inflammatory cascade. Infusion of CD19-specific CAR + T cells is currently warranted in patients at high risk from dying due to progressive B-cell malignancies. At present, most trials targeting CD19 infuse T cells that express CAR as a result of viral transduction. Indeed, hundreds of transductions and infusions of genetically modified T cells have been undertaken without apparent evidence of insertional mutagenesis. 176 Furthermore, targeting CD19 in patients with aggressive leukemias and lymphomas is justified as the “on-target” side effects because of lysis of normal B cells, and associated hypogammaglobulinemia is tolerated given the potential benefit of controlling or curing the underlying B-cell malignancy. Thus, the use of T cells as a cellular substrate and the expression of a CD19-specific CAR provide an appealing platform to assess the safety and feasibility of using the SB system for gene therapy. The first-in-human trials using the SB system are currently underway at MD Anderson Cancer Center (INDs 14193, 14577, 14739, 15180). Three of these pilot studies infuse patient- and donor-derived CD19-specific CAR + T cells after standard-of-care autologous and allogeneic hematopoietic stem-cell transplantation, including umbilical cord blood transplantation. A fourth trial infuses autologous CD19-specific CAR + T cells after lymphodepleting chemotherapy. These trials have passed institutional (Clinical Research Center, Institutional Review Board and Institutional Biosafety Committee) and federal (National Institutes of Health Office of Biotechnology Activities and the U.S. Food and Drug Administration) regulatory committees. The standard operating procedures and associated worksheets are in place to manufacture and release the genetically modified T cells in compliance with current good manufacturing practice for phase I/II trials. In preparation for these trials, data was accumulated regarding the ability of the SB system to enforce expression of a CAR. The CAR itself was modified to not only redirect T-cell specificity to CD19 but to employ an endodomain with 2 chimeric signaling motifs to achieve a fully-competent T-cell activation event defined as CAR-mediated proliferation, cytokine production, and specific lysis. The introduction of the CAR was achieved by electro-transfer (using a commercially-available Nucleofector device from Lonza) of supercoiled DNA coding for SB transposon, and transposition was achieved by co-electro-transfer of a supercoiled DNA plasmid coding for the hyperactive SB transposase, SB11. 177 T cells expressing stable integrants of CAR could be readily achieved through co-culture on designer artificial antigen-presenting cells (aAPC) in the presence of the soluble recombinant cytokines interleukin IL-2 and IL-21. The aAPC, derived from K562, were genetically modified to co-express CD19 as well as CD64 and the T-cell co-stimulatory molecules CD86, CD137L, and a membrane-bound mutein of IL-15. The recursive addition of these γ-irradiated aAPC to the electroporated T cells results in the reproducible, rapid, and massive numeric expansion of CAR + T cells. The electroporation uses defined ratios of T cells, SB transposon, and SB transposase resulting in between 1 and 2 copy numbers of CAR per T-cell genome. 178 The propagation also uses defined ratios of T cells and aAPC to ensure efficient outgrowth of T cells with at least 80% expressing CAR emerging within a few weeks of electroporation. Long-term co-culture (28 days) with aAPC does not affect T-cell activity or function because the T cells can effectively lyse target tumor cells and produce cytokine in an antigen-dependent manner while maintaining a naïve/memory phenotype. The CAR + T-cells can further effectively control B-cell tumor in an immunocompromised NOD-scid-gamma-mouse model, thus, providing evidence for their persistence. Moreover, the T-cell telomere lengths are not shortened after co-culture. 179