In vivo safety and antitumor efficacy of bifunctional small hairpin RNAs specific for the human Stathmin 1 oncoprotein.

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.

Safety and in vivo expression of a GNE-transgene: a novel treatment approach for hereditary inclusion body myopathy-2.

Hereditary inclusion body myopathy-2 (HIBM2) is an adult-onset, muscular disease caused by mutations in the GNE gene. HIBM2-associated GNE mutations causing hyposialyation have been proposed to contribute to reduced muscle function in patients with HIBM2, though the exact cause of this disease is unknown. In the current studies we examined pre-clinical in vivo toxicity, and expression of the plasmid-based, CMV driven wild-type GNE plasmid vector. The plasmid vector was injected intramuscularly (IM) or systemically (IV) into BALB/c mice, following encapsulation in a cationic liposome (DOTAP:Cholesterol). Single IM injections of the GNE-lipoplex at 40 microg did not produce overt toxicity or deaths, indicating that the no observable adverse effect level (NOAEL) dose for IM injection was >or=40 microg. Single intravenous (IV) infusion of GNE-lipoplex was lethal in 33% of animals at 100 microg dose, with a small proportion of animals in the 40 microg cohort demonstrating transient toxicity. Thus the NOAEL dose by the IV route was greater than 10 microg and less than or equal to 40 microg. Real-time RT-qPCR analysis demonstrated recombinant human GNE mRNA expression in 100% of muscle tissues that received IM injection of 40 microg GNE-lipoplex, at 2 weeks. These results indicate that GNE-lipoplex gene transfer is safe and can produce durable transgene expression in treated muscles. Our findings support future exploration of the clinical efficacy of GNE-lipoplex for experimental gene therapy of HIBM2.