Aiming to specifically inactivate the cells of interest in vivo, we developed two novel technologies. Both strategies are based on the nature of chemokines to deliver antigens to the cytosol of cells expressing respective chemokine receptors. 1) The technology designated chemotoxin, a chimeric chemokine fused with toxic moieties, specifically kills cells expressing respective chemokine receptors. For example, using TARC-chemotoxin we could eradicates established leukemia in mice. This technology is for treatment of human T cell malignant diseases when patients immune system is severely immunocompromised. The results of this study was published (Baatar et al, 2007a). The technology can also be used to specifically deplete Tregs in mice and to augment immune responses to cancer vaccines. TARC-chemotoxin eliminated Tregs and enhanced CD8+ T cell responses to gp100-based vaccines in mice. This technology is also being successfully used to understand the role of regulatory immune cells in cancer metastasis in mice (see AG000443-02). The results generated using TARC-chemotoxin were published in several our papers (Baatar et al.,2009;Olkhanud et al., 2009). 2) We developed novel technology designated Chemoarp to specifically deliver siRNA/miRNA into immune cells in vivo. The technology overcomes the major stumbling block of the use of siRNA/miRNA, i.e. the lack of ways to deliver them into immune cells in vivo. We created chemokines modified to bind siRNA/miRNA by hypothesizing that this will enable us to specifically deliver siRNA/miRNA into immune cells. Indeed, we demonstrate that modified chemokine CCL17 (TARC-arp) efficiently silences expression of genes in immune cells by delivering inhibitory oligonucleotides via their chemokine receptors. In modeling studies using mice with established 4T1.2 breast cancer, we show that IL10 produced by FoxP3+CCR4+ Tregs plays an important role in lung metastasis. As such, TARC-arp-mediated silencing of IL-10 or FoxP3 in CCR4+ regulatory T cells (Tregs) is sufficient to block lung metastasis. Thus, we provide a simple solution that circumvents the problems of RNAi use in vivo, indicating that a disease outcome can be successfully controlled by delivering inhibitory oligonucleotides with chemokines to inactivate a selective subset of immune cells, such as CCR4+ Tregs. The manuscript has been recently submitted for publication (Biragyn et al., 2012). To date, we have generated three different chemoarps which deliver siRNA by targeting chemokine receptors, such as CCR2, CCR4 and CCR5. 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.
