The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk for genome instability, and by the inability to use transposase protein directly. Here, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Merkel cell carcinoma (MCC) is a rare and highly aggressive skin cancer associated with the Merkel cell polyomavirus (MCV). As MCC cell lines show oncogene addiction to the MCV T antigens, pharmacologic interference of the large T antigen (LTA) may represent an effective therapeutic approach for this deadly cancer. In this study, we investigated the effects of IFNs on MCC cell lines, especially on MCV-positive (MCV þ ) lines. Type I IFNs (i.e., Multiferon, a mix of different IFN-a subtypes, and IFN-b) strongly inhibited the cellular viability. Cellcycle analysis showed increased sub-G fractions for these cells upon IFN treatment indicating apoptotic cell death; these effects were less pronounced for IFN-g. Notably, this inhibitory effect of type I IFNs on MCV þ MCC cell lines was associated with a reduced expression of the MCV LTA as well as an increased expression of promyelocytic leukemia (PML) protein, which is known to interfere with the function of the LTA. In addition, the intratumoral application of Multiferon resulted in a regression of MCV þ but not MCV À MCCs in vivo. Together, our findings show that type I IFNs have a strong antitumor effect, which is at least in part explained by modulation of the virally encoded LTA. Cancer Res; 72(8); 2120-8. Ó2012 AACR.

It has been demonstrated, that DNA from the Merkel cell polyoma virus (MCV) is monoclonally integrated in the genome of Merkel cell carcinoma (MCC) cells in the majority of tumors.1 In this respect, Bathia et al. recently reported an observation in THE JOURNAL which suggests that two subgroups of MCC can be distinguished on the basis of the abundance of MCV; moreover, these subgroups differed in their expression of cancer related proteins, i.e. the Retinoblastoma protein (RB).2 The authors report that MCV DNA load was less then one copy per 300 cells in 14 of 23 MCC tumors (60%), and that these tumors were characterized also by loss of RB expression. In the remaining samples, which were characterized by high levels of RB expression, the estimated viral load was always higher than 1 copy per 20 cells. Noteworthy, only in two cases viral load was higher than 1 copy per 2 cells. In consequence, the authors speculate on a possible mechanism in MCC with a minority of infected cells contributing to transformation of uninfected neighbouring cells by paracrine mechanisms.2 However, the results of Bathia and colleagues are in contrast to another recently published paper describing the viral load in MCV þ MCCs to be gener- (Fig. 1a). Moreover, we analyzed genomic DNA purified from 50 MCC tissue samples for the presence of MCV by Real time PCR. Relative quantification of the samples was calculated by the DDC t method normalized to the repetitive DNA elements LINE1; genomic DNA of a MCC cell line that harbours at least two concatemerized copies of the MCV genome in every cell served as calibrator which allowed to approximate the minimal copy number per cell in the tissues. To this end, MCV DNA was undetectable in only 7 of the 50 samples (14%). In those samples with detectable MCV DNA the median of the estimated minimal copy numbers is approximately 1 copy per cell (Fig. 1b). Indeed, 63% of the MCV positive cases (27 samples) demonstrate minimal copy numbers higher than 0.5 copies/cell. Even some of the values below 0.5 may still be in accordance with 1 MCV copy per

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Despite promising clinical results in a small subset of malignancies, therapies based on engineered chimeric antigen receptor and T-cell receptor T cells are associated with serious adverse events, including cytokine release syndrome and neurotoxicity. These toxicities are sometimes so severe that they significantly hinder the implementation of this therapeutic strategy. For a long time, existing preclinical models failed to predict severe toxicities seen in human clinical trials after engineered T-cell infusion. However, in recent years, there has been a concerted effort to develop models, including humanized mouse models, which can better recapitulate toxicities observed in patients. The Accelerating Development and Improving Access to CAR and TCR-engineered T cell therapy (T2EVOLVE) consortium is a public–private partnership directed at accelerating the preclinical development and increasing access to engineered T-cell therapy for patients with cancer. A key ambition in T2EVOLVE is to design new models and tools with higher predictive value for clinical safety and efficacy, in order to improve and accelerate the selection of lead T-cell products for clinical translation. Herein, we review existing preclinical models that are used to test the safety of engineered T cells. We will also highlight limitations of these models and propose potential measures to improve them.