We have used the previously developed technologies of RNA optimization to optimize expression of IL-15 cytokine, and have shown that we can over-produce bioactive cytokine after DNA delivery in mice and macaques. We explored the biology of IL-15 and showed that efficient production of IL-15 is possible only by co-expression in the same cell with the so-called IL-15 Receptor-alpha. We also showed that a second form of IL-15 (SSP IL-15) previously identified in humans and rodents as intracellular or nuclear IL-15 is also efficiently secreted from the cells when co-expressed with the IL-15 Receptor alpha. These results shed new light in the biology and regulation of IL-15 and provide methods for the efficient production and clinical application of this cytokine. Cell lines overproducing soluble bioactive IL-15/IL-15 Receptor alpha heterodimers have been constructed and were used for the production of the authentic bioactive form of IL-15 found in the body. IL-15 purified from over-producing human cells was injected in mice and shown to be bioactive. IL-15 is of interest due to its ability to stimulate the growth, activation and survival of CD8 and NK cells. Thus, IL-15 has been considered for cancer immunotherapy and for support of the growth of cytotoxic cell clones after adoptive transfer. Other proposed uses of IL-15 are in toxic shock and as vaccine adjuvant. We have shown that IL-15 injection accelerates the recovery of lymphocytes in mice rendered lymphopenic after treatment with cytotoxic drugs. We have used optimized expression vectors to express IL-12 cytokine in animals. Efficient expression results in bioactive levels, which increase immune response after DNA vaccination, thus becoming important molecular adjuvant for our vaccines. This work established methods to optimize expression of the IL-12 family of cytokines (IL-12, IL-23, IL-27, IL-35). Efficient expression of IL-27 after DNA delivery demonstrated synergy with IL-2 in the elimination of neuroblastoma metastases in mice. We have previously identified an extensive family of RNA transport elements (RTE) in the mouse genome able to replace the HIV-1 Rev/RRE posttranscriptional regulatory system, using a mutated HIV-1 DNA proviral clone as a novel molecular trap. This is general methodology for the identification of cis-acting posttranscriptional control elements in the mammalian genome. We have identified the cellular factor responsible for binding to RTE and linking it to the NXF1 export pathway. This protein, the RNA binding motif protein 15 (RBM15), had no previous assigned function. Our analysis revealed direct interaction of RBM15 and a related protein, OTT3, with the essential nuclear export factor NXF1 via their C-terminal regions. Biochemical and subcellular localization studies showed that OTT3 and RBM15 also interact with each other in vivo, further supporting a shared function. Genetic knock-down of RBM15 in mouse is embryonic lethal, indicating that OTT3 cannot compensate for the RBM15 loss, which supports the notion that these proteins, in addition to sharing similar activities, have distinct biological roles. RBM15 is an important factor facilitating the function of other proteins on RNA. The DEAD family RNA helicase Dbp5 is essential for nuclear export of mRNA and is thought to dissociate Mex67 from mRNP upon translocation, thereby generating directional passage to the cytoplasm. The molecular mechanism by which Dbp5 recognizes Mex67-containing mRNP is not clear. We found that RBM15 binds specifically to human DBP5 and facilitates its direct contact with mRNA in vivo. These results contribute significantly to the further understanding of the basic mechanisms of nucleocytoplasmic traffic of macromolecules.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010750-06
Application #
8349138
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2011
Total Cost
$813,040
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Bergamaschi, Cristina; Watson, Dionysios C; Valentin, Antonio et al. (2018) Optimized administration of hetIL-15 expands lymphocytes and minimizes toxicity in rhesus macaques. Cytokine 108:213-224
Watson, Dionysios C; Yung, Bryant C; Bergamaschi, Cristina et al. (2018) Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes. J Extracell Vesicles 7:1442088
Watson, Dionysios C; Moysi, Eirini; Valentin, Antonio et al. (2018) Treatment with native heterodimeric IL-15 increases cytotoxic lymphocytes and reduces SHIV RNA in lymph nodes. PLoS Pathog 14:e1006902
Ng, Sinnie Sin Man; Nagy, Bethany A; Jensen, Shawn M et al. (2017) Heterodimeric IL15 Treatment Enhances Tumor Infiltration, Persistence, and Effector Functions of Adoptively Transferred Tumor-specific T Cells in the Absence of Lymphodepletion. Clin Cancer Res 23:2817-2830
Hong, Enping; Usiskin, Ilana M; Bergamaschi, Cristina et al. (2016) Configuration-dependent Presentation of Multivalent IL-15:IL-15R? Enhances the Antigen-specific T Cell Response and Anti-tumor Immunity. J Biol Chem 291:8931-50
Thaysen-Andersen, M; Chertova, E; Bergamaschi, C et al. (2016) Recombinant human heterodimeric IL-15 complex displays extensive and reproducible N- and O-linked glycosylation. Glycoconj J 33:417-33
Li, Jinyao; Valentin, Antonio; Ng, Sinnie et al. (2015) Differential effects of IL-15 on the generation, maintenance and cytotoxic potential of adaptive cellular responses induced by DNA vaccination. Vaccine 33:1188-96
Bergamaschi, C; Kulkarni, V; Rosati, M et al. (2015) Intramuscular delivery of heterodimeric IL-15 DNA in macaques produces systemic levels of bioactive cytokine inducing proliferation of NK and T cells. Gene Ther 22:76-86
Chertova, Elena; Bergamaschi, Cristina; Chertov, Oleg et al. (2013) Characterization and favorable in vivo properties of heterodimeric soluble IL-15·IL-15R? cytokine compared to IL-15 monomer. J Biol Chem 288:18093-103
Ng, Sinnie Sin Man; Li, Andrew; Pavlakis, George N et al. (2013) Viral infection increases glucocorticoid-induced interleukin-10 production through ERK-mediated phosphorylation of the glucocorticoid receptor in dendritic cells: potential clinical implications. PLoS One 8:e63587

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