Rachel E. Johns

RNAi-based Therapeutics Targeting Survivin and PLK1 for Treatment of Bladder Cancer

Harnessing RNA interference (RNAi) to silence aberrant gene expression is an emerging approach in cancer therapy. Selective inhibition of an overexpressed gene via RNAi requires a highly efficacious, target-specific short interfering RNA (siRNA) and a safe and efficient delivery system. We have developed siRNA constructs (UsiRNA) that contain unlocked nucleobase analogs (UNA) targeting survivin and polo-like kinase-1 (PLK1) genes. UsiRNAs were encapsulated into dialkylated amino acid-based liposomes (DiLA(2)) containing a nor-arginine head group, cholesteryl hemisuccinate (CHEMS), cholesterol and 1, 2-dimyristoyl-phosphatidylethanolamine-polyethyleneglycol 2000 (DMPE-PEG2000). In an orthotopic bladder cancer mouse model, intravesical treatment with survivin or PLK1 UsiRNA in DiLA(2) liposomes at 1.0 and 0.5 mg/kg resulted in 90% and 70% inhibition of survivin or PLK1 mRNA, respectively. This correlated with a dose-dependent decrease in tumor volumes which was sustained over a 3-week period. Silencing of survivin and PLK1 mRNA was confirmed to be RNA-induced silencing complex mediated as specific cleavage products were detected in bladder tumors over the duration of the study. This report suggests that intravesical instillation of survivin or PLK1 UsiRNA can serve as a potential therapeutic modality for treatment of bladder cancer.

An Amino Acid-based Amphoteric Liposomal Delivery System for Systemic Administration of siRNA

We demonstrate a systematic and rational approach to create a library of natural and modified, dialkylated amino acids based upon arginine for development of an efficient small interfering RNA (siRNA) delivery system. These amino acids, designated DiLA₂ compounds, in conjunction with other components, demonstrate unique properties for assembly into monodisperse, 100-nm small liposomal particles containing siRNA. We show that DiLA₂-based liposomes undergo a pH-dependent phase transition to an inverted hexagonal phase facilitating efficient siRNA release from endosomes to the cytosol. Using an arginine-based DiLA₂, cationic liposomes were prepared that provide high in vivo siRNA delivery efficiency and are well-tolerated in both cell and animal models. DiLA₂-based liposomes demonstrate a linear dose-response with an ED₅₀ of 0.1 mg/kg against liver-specific target genes in BALB/c mice.

Differential monocyte/macrophage interleukin-1β production due to biomaterial topography requires the β2 integrin signaling pathway

Monocytes/macrophages are crucial mediators of the host response to biomaterials, and their level of activation can be directly affected by material characteristics. Previous work has demonstrated that primary human monocytes cultured on polytetrafluoroethylene materials of varying topography but identical surface chemistry are differentially affected. Monocytes/macrophages on biaxially-expanded polytetrafluoroethylene with an average intranodal distance of 4.4 μm (4.4-ePTFE) produced higher levels of the inflammatory cytokine interleukin-1 beta (IL-1β) compared with monocytes/macrophages on nonporous polytetrafluoroethylene (np-PTFE). The current study provides a mechanistic understanding of this response. Scanning electron microscopy revealed that monocytes/macrophages cultured on np-PTFE were more spread than those on 4.4-ePTFE. In addition, the actin cytoskeleton and intact β2 integrin receptors were necessary for IL-1β production by monocytes/macrophages on 4.4-ePTFE. This IL-1β production also required the transcription factor nuclear factor kappa-B, another component of the β2 integrin signaling pathway, although it may not be the primary transcription factor involved. These studies demonstrate the importance of several β2 integrin signaling components to the monocyte/macrophage response to biomaterial topography.

Mechanistic analysis of macrophage response to IRAK-1 gene knockdown by a smart polymer-antisense oligonucleotide therapeutic.

An excessive inflammatory response is a clinical problem following major infections and severe injury that may lead to Sepsis Syndrome and Multiple Organ Failure (MOF), including the Acute Respiratory Distress Syndrome (ARDS). Management of excessive inflammation may be possible through control of key inflammatory pathways such as those mediated by the important interleukin-1 receptor associated kinase-1 (IRAK-1). In the current study, we report the impact on gene expression induced by lipopolysaccharide (LPS) stimulation of THP-1 cells treated with an antisense oligonucleotide (ASODN) against the IRAK-1 gene using cDNA microarrays and quantitative RT-PCR. The therapeutic ASODN was delivered using a pH-sensitive, membrane-interactive polymer that destabilizes the endosomal membrane to enhance access cytoplasmic delivery in targeted cells. Following LPS stimulation, the anti-inflammatory activity of ASODN against the IRAK-1 gene expression is evidenced by the lower expression of inflammatory chemokines, cytokines and acute-phase proteins compared to control cells. These results provide a larger mechanistic picture of IRAK-1 knockdown by this polymer therapeutic in macrophage-like cells.

Abstract 281: Novel nucleic acid constructs and formulations for treatment of bladder cancer and malignant ascites

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Nucleic acid-based therapeutics offer significant promise in treatment of difficult diseases by providing high sequence specificity against target genes including “non-druggable targets”. Different nucleic acid constructs, single-stranded antisense oligonucleotides, microRNA mimetics, miRNA-specific antagomirs (AntimiR), and double-stranded short-interfering RNAs (siRNAs), provide diverse therapeutic platforms to access targets in treatment of cancer and other diseases. Single-stranded oligonucleotides require additional modifications for increased serum stability, nuclease resistance, and to minimize immune response. Conformationally restricted nucleotide (CRN)-substituted AntimiRs targeting miR-21 and miR-122 demonstrated significant improvement in hybridization affinity with a concomitant increase in potency. A CRN-substituted AntimiR targeting microRNA-122 dosed at up to 50 mg/kg/day over three consecutive days led to a dose-dependent effect on de-repression of downstream liver-specific target genes, Aldolase A (AldoA), Glycogen Synthase I (GYS1) and Solute Carrier Family 7 member 1 (SLC7A1). This CRN-modified AntimiR produced up to a 5-fold increase in AldoA mRNA while demonstrating a good tolerability profile with no body weight changes and normal serum chemistry. Development of therapeutic siRNA constructs can be hampered by off-target effects and cytokine induction. Substitution of unlocked nucleobase analogs (UNAs) at specific positions in the passenger and guide strands of siRNA confers high specificity with minimal to no off-target activity. UsiRNAs were successfully delivered to bladder or abdominal tumors when encapsulated in DiLA2- or SMARTICLES®-derived liposomes. DiLA2−encapsulated polo-like kinase 1 (PLK1) UsiRNA demonstrated 95% mRNA reduction and up to 70-fold tumor growth inhibition in a bladder cancer orthotopic mouse model and 40-70% mRNA inhibition and ∼80% reduction in tumor-specific bioluminescence in a malignant ascites mouse model following intravesical or intraperitoneal dosing, respectively. In an orthotopic liver tumor animal model, liposomal encapsulated miRNA mimetics provided ∼80% reduction in tumors compared to buffer control. Combinations of formulations and construct capitalize on the advantages of nucleic acid-based therapeutics for precise targeting and flexible delivery to challenging drug targets in cancer and other indications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 281. doi:1538-7445.AM2012-281

639. Gene Expression Analysis of LPS Stimulated THP-1 Cells Treated with Antisense IRAK1 Delivered Using a pH-Sensitive, Membrane Disruptive Polymer

Goals and Significance: The inflammatory response, which is initiated upon trauma, infection, or in the presence of a foreign body, is necessary for normal wound healing. However, during chronic inflammation, control of this response becomes clinically advantageous. The interleukin-1 (IL-1) pathway to nuclear transcription factor κB (NF-κB) is a major contributor to the inflammatory response. IL-1 Receptor Associated Kinase-1 (IRAK1) is a central kinase in this pathway and is a target molecule for controlling NF-κB activation and the subsequent production of inflammatory mediators. Decreasing the expression of IRAK1 has been accomplished through the use of an antisense oligonucleotide. By delivering an 18-mer oligodeoxynucleotide (ODN) complimentary to the mRNA sequence of IRAK1, production of this kinase was inhibited. ODN uptake generally occurs through endocytosis, after which molecules are trafficked to the lysosome and degraded, resulting in loss of the therapeutic ODN. In an effort to improve the delivery of ODNs and other biological molecules, a family of pH-sensitive membrane disruptive polymers has been developed in our lab. These polymers are able to enhance cytoplasmic delivery of therapeutics by releasing them from the endosome. The polymer used in this study consisted of a hydrophobic, membrane disruptive backbone linked via acid degradable acetal bonds to hydrophilic poly(ethylene glycol) (PEG). The PEG groups mask the hydrophobic backbone until the acetal bonds degrade at the low pH of the endosome or lysosome, freeing the hydrophobic backbone to disrupt the endosomal membrane and deliver the therapeutic cargo to the cytoplasm of the cell.