Cell-selective delivery and sensitivity to serum nucleases remain major hurdles to the clinical application of RNA-based oligonucleotide therapeutics, such as siRNA. Spider silk shows great potential as a biomaterial due to its biocompatibility and biodegradability. Self-assembling properties of silk proteins allow for processing into several different morphologies such as fibers, scaffolds, films, hydrogels, capsules and spheres. Moreover, bioengineering of spider silk protein sequences can functionalize silk by adding peptide moieties with specific features including binding or cell recognition domains. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics, such as CpG-siRNA. The MS2 bioengineered silk was functionalized with poly-lysine domain (KN) to generate hybrid silk MS2KN. CpG-siRNA efficiently bound to MS2KN in contrary to control MS2. Both MS2KN complexes and spheres protected CpG-siRNA from degradation by serum nucleases. CpG-siRNA molecules encapsulated into MS2KN spheres were efficiently internalized and processed by TLR9-positive macrophages. Importantly, CpG-STAT3siRNA loaded in silk spheres showed delayed and extended target gene silencing compared to naked oligonucleotides. The prolonged Stat3 silencing resulted in the more pronounced downregulation of interleukin 6 (IL-6), a proinflammatory cytokine and upstream activator of STAT3, which limits the efficacy of TLR9 immunostimulation. Our results demonstrate the feasibility of using spider silk spheres as a carrier of therapeutic nucleic acids. Moreover, the modified kinetic and activity of the CpG-STAT3siRNA embedded into silk spheres is likely to improve immunotherapeutic effects in vivo.

SPIN1 is necessary for normal meiotic progression in mammals. It is overexpressed in human ovarian cancers and some cancer cell lines. Here, we examined the functional significance and regulation of SPIN1 and SPIN3 in the TCam-2 human seminoma cell line. We found that while SPIN1 overexpression reduced apoptosis in these cells, SPIN3 overexpression induced it. Similarly, SPIN1 upregulated and SPIN3 downregulated CYCD1, which is a downstream target of the PI3K/AKT pathway and contributes to apoptosis resistance in cancer cell lines. It appears that SPIN1 is pro-oncogenic and SPIN3 acts as a tumor suppressor in TCam-2 cells. To our knowledge, this is the first report of SPIN3 tumor suppressor activity. However, both SPIN1 and SPIN3 stimulated cell cycle progression. In addition, using luciferase reporters carrying SPIN1 or SPIN3 mRNA 3′UTRs, we found that PUM1 and PUM2 targeted and repressed SPINs. We also found that PUM1 itself strongly stimulated apoptosis and moderately slowed cell cycle progression in TCam-2 cells, suggesting that PUM1, like SPIN3, is a tumor suppressor. Our findings suggest that acting, at least in part, through SPIN1 and SPIN3, PUM proteins contribute to a mechanism promoting normal human male germ cell apoptotic status and thus preventing cancer.

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