1) We have developed two novel technologies for depletion of unwanted subsets of cells at will. The strategy is based on the nature of chemokines to deliver antigens to the cytosol of cells expressing respective chemokine receptors. One technology designated chemotoxin, a chimeric chemokine fused with toxic moieties, can specifically kill cells expressing respective chemokine receptors. TARC-chemotoxin efficiently eradicates established leukemia (CEM cells) generating almost 100% tumor- free mice . We propose that the strategy may be applied for treatment of human T cell malignant diseases when patients immune system is severely immunocompromised. The results of this study have been recently published (Baatar et al, 2007a). We have also utilized this technology to specifically deplete Tregs in mice to augment immune responses to cancer vaccines. Part one of this ongoing project demonstrates that TARC-chemotoxin-mediated depletion of Tregs enhance CD8+ T cell responses to melanoma antigen gp100. This is an encouraging result, although its clinical significance remains to be evaluated. This technology is being successfully used to understand chemokine/chemokine receptor axis in regulation of metastasis (see AG000443-02). It was also used in our recent study on the mechanism of Treg-mediated suppression of resting T cells (Baatar et al.,2009). 2) Despite significant potency and attractiveness of siRNA and shRNA-mediated gene silencing methods, the technology can not be efficiently used in vivo. The major problem is that there is almost no practical way that delivers/targets oligonucleotides to cells of interest. The inability to deliver siRNA into primary cells precludes its utilization in vivo. To solve this, we have hypothesized that this can be achieved by generating modified chemokines that contain RNA binding domains (RBD). Although this technology designated chemoarp is still under development, we have successfully demonstrated proof of principle by delivering siRNA and knocking off expression of genes in various cells, including tumor cells. To date, we have generated three different chemoarps which deliver siRNA by targeting chemokine receptors, such as CCR2, CCR4 and CCR5. However and importantly, this technology enables delivery of siRNA into primary immune cells inactivating their genes of interest. We have also established a simplified but efficient yeast production system and created chemoarp purification methods. Overall, this truly novel project, which was recently applied for patenting from NIA, is not only progressing well, but also has attracted significant interest from commercial enterprises such as ECI, Inc., Japan. and Ambion, Life Technologies, USA. At present, we are completing two studies on the preclinical use of chemoarp. Our data indicate that potent anticancer responses can be elicited by delivering siRNA into Tregs and tBregs with the help of chemoarps (Olkhanud et al., and Bodogai et al., manuscript in preparation).
