In vivoSafety and Antitumor Efficacy of Bifunctional Small Hairpin RNAs Specific for the Human Stathmin 1 Oncoprotein

Abstract

Bifunctional small hairpin RNAs (bi-shRNAs) are functional miRNA/siRNA composites that are optimized for posttranscriptional gene silencing through concurrent mRNA cleavage-dependent and -independent mechanisms (Rao et al., 2010 ). We have generated a novel bi-shRNA using the miR30 scaffold that is highly effective for knockdown of human stathmin (STMN1) mRNA. STMN1 overexpression well documented in human solid cancers correlates with their poor prognosis. Transfection with the bi-shSTMN1-encoding expression plasmid (pbi-shSTMN1) markedly reduced CCL-247 human colorectal cancer and SK-Mel-28 melanoma cell growth in vitro (Rao et al., 2010 ). We now examine in vivo the antitumor efficacy of this RNA interference-based approach with human tumor xenografted athymic mice. A single intratumoral (IT) injection of pbi-shSTMN1 (8 μg) reduced CCL-247 tumor xenograft growth by 44% at 7 days when delivered as a 1,2-dioleoyl-3-trimethyl-ammoniopropane:cholesterol liposomal complex. Extended growth reductions (57% at day 15; p < 0.05) were achieved with three daily treatments of the same construct. STMN1 protein reduction was confirmed by immunoblot analysis. IT treatments with pbi-shSTMN1 similarly inhibited the growth of tumorgrafts derived from low-passage primary melanoma (≥70% reduction for 2 weeks) and abrogated osteosarcoma tumorgraft growth, with the mature bi-shRNA effector molecule detectable for up to 16 days after last injection. Antitumor efficacy was evident for up to 25 days posttreatment in the melanoma tumorgraft model. The maximum tolerated dose by IT injection of >92 μg (Human equivalent dose [HED] of >0.3 mg/kg) in CCL-247 tumor xenograft-bearing athymic mice was ∼10-fold higher than the extrapolated IC(50) of 9 μg (HED of 0.03 mg/kg). Healthy, immunocompetent rats were used as biorelevant models for systemic safety assessments. The observed maximum tolerated dose of <100 μg for intravenously injected pbi-shSTMN1 (mouse equivalent of <26.5 μg; HED of <0.09 mg/kg) confirmed systemic safety of the therapeutic dose, hence supporting early-phase assessments of clinical safety and preliminary efficacy.

Stathmin 1, a Therapeutic Target in Esophageal Carcinoma?

“…Interestingly, Stathmin 1 silencing reduced the migration capacity of the esophageal carcinoma cell lines (Liu et al, 2013). Accordingly, studies performed by Wang and colleagues (Wang et al, 2011) and by Akhtar and colleagues (Akhtar et al, 2014b) demonstrated that Stathmin 1 silencing is able to reverse the malignant phenotype of esophageal carcinoma cells, including reduced cell proliferation, cell cycle progression, migration, invasion and tumorigenicity, and increased apoptosis.…”

“…For instance, the GDP366, a dual inhibitor for Stathmin 1 and Survivin, reduced dramatically the malignant phenotype of cancer cells in vitro and in vivo (Shi et al, 2010). In another study, Phadke and colleagues (Phadke et al, 2011), using small hairpin RNA approach in vivo, showed that inhibition of Stathmin 1 reduced xenograft tumor growth in a mice model. Accordingly, Akhtar and colleagues (Akhtar et al, 2014c) tested the local injection of lentivirus-delivered shRNA targeting Stathmin 1 as approach, and found a significant regression in the cell growth of pre-established xenograft tumors in mice.…”

A New Approach to Cancer Therapy: The Tumor Microenvironment as Target

“…Thus, delivery strategies for RNAi become the main hurdle that must be resolved prior to the full-scale clinical development of siRNA therapeutics [454][455][456][457][458]. As some examples, oncolytic adenoviral delivery of siRNA offers the potential benefits of restricted and renewable siRNA expression within the tumor microenvironment with an additive antitumor effect through viral oncolysis and siRNA-mediated oncogene silencing [459,460]. Significant advances have been achieved with sterically stabilized lipid-based nanocarriers such as the stabilized nucleic acid lipid particles (SNALP).…”

Bifunctional Short Hairpin RNA (bi-shRNA): Design and Pathway to Clinical Application

“…There are numerous delivery systems designed speci fi cally for systemic applications in vivo ( 22,(33)(34)(35) . We have utilized the fusogenic, cationic DOTAP:cholesterol bilamellar invaginated vesicle lipoplex (BIV) for our in vivo studies ( 22,36 ) and have successfully translated it to the clinic ( 37 ) . We are currently developing modi fi cation strategies for more focused biodistribution, targeted delivery, and enhanced intracellular uptake.…”