Abstract A196: Characterization of stability and biological activity of the cancer gene therapy biologic SNS01 following storage at ambient and freezing temperatures

Abstract Introduction: The eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to be regulated by post-translational addition of a hypusine residue. Both hypusinated eIF5A and the enzyme that mediates eIF5A hypusination have been identified as markers of neoplastic growth and metastasis. However, recent studies have indicated that, in its unhypusinated form, eIF5A is pro-apoptotic and thus functionally distinct from hypusine-modified eIF5A. In vitro cell studies and in vivo xenograft studies have demonstrated that simultaneous suppression of eIF5A expression and overexpression of a non-hypusinable mutant of eIF5A potently induces apoptosis in multiple cancer cell types. SNS01 is a nanoparticle specifically designed for the treatment of multiple myeloma. SNS01 is comprised of three components: a DNA vector containing a B-cell-specific (B29) promoter driving expression of a pro-apoptotic mutant of eIF5A (eIF5AK50R) that cannot be hypusinated; an siRNA that targets the native hypusinated eIF5A that promotes growth of cancer cells; and a synthetic polymer called polyethylenimine (PEI). Methods: Ethidium-bromide agarose gel electrophoresis was used to detect heparin-mediated release of nucleic acids from nanoparticles. Dynamic light scattering (DLS) was used to assess the short-term stability of SNS01 by monitoring changes in size distribution, polydispersity, and zeta potential. In vitro biological activity of SNS01 was assessed using RT-qPCR to measure eIF5A and eIF5AK50R transgene expression as well as FACS detection of AnnexinV-FITC labeled apoptotic cells. Anti-tumoral activity of SNS01 was evaluated using a subcutaneous human myeloma model (KAS-6/1 cells) in SCID mice with twice-weekly intra-venous injections of SNS01. Results: SNS01 nanoparticles were found to be relatively stable for as long as 48 hours at room temperature with no significant loss in nucleic acid integrity. The nanoparticles had an average diameter of 146.2 nm, a polydispersity of 0.287 and an average zeta potential of 35.3+0.7 mV. These parameters were quite stable over 48 hours. SNS01 could also be frozen at − 80°C for at least one month with no observable loss in biological activity. Twice-weekly intra-venous injections into myeloma tumor-bearing mice of a frozen SNS01 preparation was found to have anti-tumoral activity comparable to freshly prepared SNS01 (frozen SNS01: 89 % tumor inhibition, p = 0.00158; SNS01: 84 % tumor inhibition, p = 0.00003). Conclusions: SNS01 is relatively stable at room temperature and was also found to be stable and biologically active following freezing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A196.

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