The V(D)J Recombinase Efficiently Cleaves and Transposes Signal Joints

V(D)J recombination generates two types of products: coding joints, which constitute the rearranged variable regions of antigen receptor genes, and signal joints, which often form on immunologically irrelevant, excised circular molecules that are lost during cell division. It has been widely believed that signal joints simply convert reactive broken DNA ends into safe, inert products. Yet two curious in vivo observations made us question this assumption: signal ends are far more abundant than coding ends, and signal joints form only after RAG expression is downregulated. In fact, we find that signal joints are not at all inert; they are cleaved quite efficiently in vivo and in vitro by a nick-nick mechanism and form an excellent substrate for RAG-mediated transposition in vitro, possibly explaining how genomic stability in lymphocytes may be compromised

Anti-Tumor Effects after Adoptive Transfer of IL-12 Transposon-Modified Murine Splenocytes in the OT-I-Melanoma Mouse Model

<div><p>Adoptive transfer of gene modified T cells provides possible immunotherapy for patients with cancers refractory to other treatments. We have previously used the non-viral <i>piggyBac</i> transposon system to gene modify human T cells for potential immunotherapy. However, these previous studies utilized adoptive transfer of modified human T cells to target cancer xenografts in highly immunodeficient (NOD-SCID) mice that do not recapitulate an intact immune system. Currently, only viral vectors have shown efficacy in permanently gene-modifying mouse T cells for immunotherapy applications. Therefore, we sought to determine if <i>piggyBac</i> could effectively gene modify mouse T cells to target cancer cells in a mouse cancer model. We first demonstrated that we could gene modify cells to express murine interleukin-12 (p35/p40 mIL-12), a transgene with proven efficacy in melanoma immunotherapy. The OT-I melanoma mouse model provides a well-established T cell mediated immune response to ovalbumin (OVA) positive B16 melanoma cells. B16/OVA melanoma cells were implanted in wild type C57Bl6 mice. Mouse splenocytes were isolated from C57Bl6 OT-I mice and were gene modified using <i>piggyBac</i> to express luciferase. Adoptive transfer of luciferase-modified OT-I splenocytes demonstrated homing to B16/OVA melanoma tumors <i>in vivo</i>. We next gene-modified OT-I cells to express mIL-12. Adoptive transfer of mIL-12-modified mouse OT-I splenocytes delayed B16/OVA melanoma tumor growth <i>in vivo</i> compared to control OT-I splenocytes and improved mouse survival. Our results demonstrate that the <i>piggyBac</i> transposon system can be used to gene modify splenocytes and mouse T cells for evaluating adoptive immunotherapy strategies in immunocompetent mouse tumor models that may more directly mimic immunotherapy applications in humans.</p></div

Homing of <i>piggyBac</i>-modified mouse splenocytes to tumor sites <i>in vivo</i>.

<p>OT-I mouse splenocytes were transfected with pCMV-m7pB and pT-effLuc-Thy1.1. 5 X 10⁵ B16/OVA cells into the flank of C57Bl6 mice (day –8). <i>piggyBac</i>-modified splenocytes were adoptively transferred via tail vein injection on day 0 and day +8. Localization of infused splenocytes was visualized via <i>in vivo</i> imaging of luciferase expression on day +11. Show are 3 of 6 representative animals.</p

Cancer predisposition and hematopoietic failure in Rad50(S/S) mice

Mre11, Rad50, and Nbs1 function in a protein complex that is central to the metabolism of chromosome breaks. Null mutants of each are inviable. We demonstrate here that hypomorphic Rad50 mutant mice (Rad50(S/S) mice) exhibited growth defects and cancer predisposition. Rad50(S/S) mice died with complete bone marrow depletion as a result of progressive hematopoietic stem cell failure. Similar attrition occurred in spermatogenic cells. In both contexts, attrition was substantially mitigated by p53 deficiency, whereas the tumor latency of p53(−/−) and p53(+/−) animals was reduced by Rad50(S/S). Indices of genotoxic stress and chromosomal rearrangements were evident in Rad50(S/S) cultured cells, as well as in Rad50(S/S) and p53(−/−)Rad50(S/S) lymphomas, suggesting that the Rad50(S/S) phenotype was attributable to chromosomal instability. These outcomes were not associated with overt defects in the Mre11 complex's previously established double strand break repair and cell cycle checkpoint regulation functions. The data indicate that even subtle perturbation of Mre11 complex functions results in severe genotoxic stress, and that the complex is critically important for homeostasis of proliferative tissues

Vector schematics.

<p>The hyperactive (m7pB) <i>piggyBac</i> transposase was used in combination with various transposons for mIL-12 and/or reporter gene (venus or luciferase) expression <i>in vitro</i> or <i>in vivo</i>. CMV, cytomegalovirus immediate early enhancer/promoter; <i>piggyBac</i>, transposase; pA, SV40 polyadenylation signal; mIL-12, murine IL-12; 2A, 2A sequence; venus, reporter gene; effLuc, enhanced luciferase reporter gene; stop, stop codon; IRES, internal ribosomal entry site; Thy1.1, mouse Thy1.1 antigen; WPRE, woodchuck hepatitis post-transcriptional regulatory element; eGFP, enhanced green fluorescent protein.</p

IL-12 transfected OT-I cells produce IL-12 and produce IFNγ when co-cultured with B16/OVA cells.

<p><b>A</b>, OT-I splenocytes were transfected with pT-eGFP (control) or pT-mIL12 and co-cultured with B16 or B16/OVA cells. Flow cytometry confirmed the presence of eGFP expressing CD8 positive OT-I cells at the end of the co-culture. Shown is a representative of 3 independent experiments. <b>B</b>, cytometric bead analysis was used to measure mIL-12 (au, arbitrary units) in the media derived from the co-culture. *, p<0.05 comparing mIL-12 groups to eGFP. <b>C</b>, cytometric bead analysis was used to measure INFg production from transfected OT-I cells in the presence of B16 or B16/OVA cells. *, p<0.05 comparing B16/OVA groups to B16 (without OVA).</p

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

10.3109/14653249.2012.700767Cytotherapy1491131-1143CYTR