Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States. In spite of recent therapeutic advances, long term survival in PDAC is often limited to patients who have had surgery in early stage of the disease. The biological aggressiveness of PDAC is due, in part, to the tumor's resistance to chemotherapy and to its propensity to metastasize even when the primary tumor is small. PDAC is also characterized by a high frequency of mutations in the Kras gene, as well as by the inactivation of several tumor suppressor genes such as p16Ink4a, p53 and Smad4 (Kern, S. E. 2000. Med. Clin. North Am. 84:691-695). PDAC also displays abnormal upregulation of multiple mitogenic and angiogenic growth factors and their cognate high affinity receptors (Korc, M. 1998. Surg. Oncol. Clin. N. Am. 7:25-41). Together, these alterations serve to enhance the biological aggressiveness of PDAC.
Cyclin D1, a cell cycle regulator, is known to be overexpressed in several human cancers including PDAC (Gansauge, S. et al. 1997. Cancer Res. 57:1634-1637). In response to mitogenic growth factors, normal cells exhibit cyclin D1 accumulation by early to mid phase of G1. Cyclin D1 then activates its cognate cyclin dependent kinases, CDK4 and CDK6. Activated CDK4/6 initiates the phosphorylation-dependent inactivation of the retinoblastoma protein (RB) which then promotes progression through the G1-S phase of the cell cycle (Fu, M. et al. 2004. Endocrinology 145:5439-5447). Cyclin D1 also has CDK-independent functions (Bernards, R. 1999. Biochim. Biophys. Acta 1424:M17-M22). Thus, cyclin D1 can associate with and enhance the activity of transcription factors by recruiting cofactors that govern chromatin remodeling (Chan, H. M. et al. 2001. Nat. Cell Biol. 3:667-674), and contribute to the regulation of cell metabolism, differentiation and migration (Fu, M. et al. 2004. Endocrinology 145:5439-5447).
PDACs express high levels of cyclin D1 (Poch et al. 2001. Pancreas 23:280-287), and this overexpression has been correlated with decreased patient survival (Kornmann, M. et al. 1998. Oncology 55:363-369). Moreover, suppression of cyclin D1 expression by an antisense-based strategy resulted in marked inhibition of pancreatic cancer cell growth in vitro and in vivo (Kornmann, M. et al. 1998. J. Clin. Invest. 101:344-352; Yasui, M. et al. 2006. Clin. Cancer Res. 12:4720-4729). However, such an antisense strategy is not readily amenable for use in therapeutic regimens. RNA interference (RNAi) has rapidly become a powerful tool for gene silencing, drug discovery and target validation (Nishikura, K. 2001. Cell 107:415-418), and vectors that synthesize small hairpin (sh) RNA permit sustained gene silencing (Grimm, D. et al. 2007. Am. Soc. Hematol. Educ. Program 473-481). These vectors allow for the synthesis of 50 base pair (bp)-long single stranded RNAs that fold in 21-23 bp dsRNA with a small hairpin in the middle, and that are subsequently processed to siRNAs by the cellular machinery. These shRNA expression vectors can be engineered to contain selectable markers to generate stable transfectants, to co-express reporter genes, and to be incorporated into viral vectors such as a lentiviral vector (Hannon, G. J. et al. 2004. Methods Mol. Biol. 257:255-266). This virus is modified to be non-replicative, and is efficiently transduced into human cells, including non-dividing cells (Hannon, G. J. et al. 2004. Methods Mol. Biol. 257:255-266; Amado, R. G. and I. S. Chen. 1999. Science 285:674-676). It then integrates into the host genome, resulting in long-term and stable expression of the shRNA (Sikorski, R. et al. 1998. Science 282:1438).
U.S. Patent Application No. 2005/0164224 discloses modulating cyclin D1 expression through the use of short-interfering nucleic acid molecules, including shRNA, to treat disease such as cancer. However, the patent application fails to teach use of a lentivirus based shRNA molecule and injection directly into pancreatic tumor cells.
U.S. Patent Application 2006/0269921 discloses methods of diagnosing pancreatic cancer in a patient that involves detecting the presence of a pancreatic cancer-associated transcript, such as cyclin D1. The patent application fails to teach or suggest modulating activity of cyclin D1 to inhibit growth of cancer cells.
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