1. Aim I. RNA Synthetic Biology for Cancer Gene Therapy
  2. Aim II.  Peptide & Protein Chemical Biology for Cancer Immunotherapy.
  3. References.

Synopsis. My research program is based on the innovation of molecular therapeutics, based on nucleic acids and peptides, for anti-cancer applications. More specifically, my research program revolves around 2 central research aims: Aim I. RNA Synthetic Biology for Cancer Gene Therapy and Aim II.  Peptide & Protein Chemical Biology for Cancer Immunotherapy. Each research aim is multidisciplinary, combining elements of chemical synthesis of synthetic biologicals and their structural mimics, bioanalyses for evaluation of molecular structure and sequence information, molecular cell biology and in vivo testing for screening therapeutic potential and translational biomedical research for establishing clinical utility.  Therefore, my research program within the Department of Chemistry at Carleton University will be broadly related to the development of a successful anti-cancer research program with faculty, students, and leading scientists across our campus and beyond!

Aim I. RNA Synthetic Biology for Cancer Gene Therapy

   Branch & Hyperbranch siRNAs. An important RNA function inside the cell is related to the inhibition of detrimental (mutated) gene expression, also known as gene silencing. This process leads to the loss of protein production and cell death. In cancer, RNA interference (RNAi) uses complementary double-stranded RNA (siRNA) to target an oncogene (tumor promoter), inhibit its expression and trigger cancer cell death.1 Chemically derived siRNAs, containing modifications and/or structural motifs that may increase the potency of the RNAi response are crucial for successful gene therapy applications in cancer. In this pioneering project, new classes of branch and hyperbranch siRNAs have been designed and developed.2 A divergent synthetic approach was developed by solid phase oligonucleotide synthesis to construct the branch and hyperbranch RNA. In annealing buffer, the complementary RNA strands hybridized forming stable branch and hyperbranch siRNAs targeting the Glucose Regulated Protein of 78 kilodalton (GRP78) oncogene. GRP78 has been classified as a clinically relevant tumor biomarker, implicated in tumor proliferation and spread as well as anti-apoptotic signaling which confers tumor treatment resistance.3 Silencing GRP78 expression in HepG2 hepatoblastoma resulted in tumor cell death, to a greater extent relative to linear siRNA controls, underscoring their potential utility in cancer gene therapy (Fig 1a).

   RNA Self-Assembly and RNAi Screening. Building upon this branching siRNA biotechnology in collaboration with MSKCC, an siRNA self-assembly strategy has been designed and developed.4 This de novo RNAi approach was created using the branch and hyperbranch RNA templates previously described2 to self-assemble siRNA nanostructures of multiple sizes and geometric shapes (Fig 1b). The siRNA nanostructures were genetically encoded to target multiple GRPs for assessing their role in cancer biology.5 A lead Y-shape self-assembled siRNA targeting GRP-75, 78 and 94 triggered synergistic knockdown of all three oncogenes which translated into significant tumor cell death across a panel of cell lines, and to a more significant extent relative to control conditions with the linear siRNAs transfected independently and in combination. Interestingly, treatment of a non-tumorigenic control cell line displaying regulated GRP expression resulted in less GRP silencing with diminished toxicity, underlying the importance of GRP function in tumor viability, while also providing valuable insights into tumor treatment specificity. Our so-called ‘RNAi screening approach’ can be used for screening a wide range of oncogene targets to determine their influence on cancer activity, while also potentiating the cancer cell death response by silencing multiple oncogenes in synergy.

   RNA Bioconjugation. To improve RNA functional utility, we have established bioconjugation approaches that have facilitated the ligation and self-assembly of siRNA into multifunctional biomaterials.6 In these selected applications, solid-phase RNA bioconjugation was optimized to ligate the fluorescent fluorescein isothiocyanate (FITC) reporter to track RNA activity in cells. Following RNA ligation, the FITC-labeled RNA was annealed with complementary branch RNA templates to produce the desired multi-labeled siRNA constructs incorporating double and triple FITCs which enhanced fluorescence detection in prostate cancer (PCa) cells relative to the singly labeled linear siRNA control (Fig 1c). The multi-labeled siRNAs retained RNAi activity ultimately resulting in anti-cancer effects.7 This multi-labeling approach may serve to incorporate other fluorescent reporters and probes for enhanced RNA detection in cancer which may have a favorable effect in the preventative diagnosis and treatment of early-stage tumors.8 In order to expand the repertoire of multifunctional RNA therapeutics, we have designed and developed a novel self-assembly approach in which fatty acid functionalized RNA, were annealed onto complementary branch RNA templates to furnish fatty acid functionalized siRNA.9 The higher-order siRNA structures enabled multiple fatty acid incorporations which enhanced siRNA amphiphilicity for cell uptake and RNAi activity relative to the linear siRNA counterpart with a single fatty acid group. Amphiphilic siRNA was applied without an external transfection reagent which is often toxic for scale-up in vivo applications thus enhancing their therapeutic potential and clinical utility.

   RNA molecular and systems biology. GRP78 silencing siRNA were used as molecular probes to interrogate the biological mechanism associated with anti-cancer activity. In collaboration with HMH, multiple myeloma (MM) and PCa cells revealed that siRNA-mediated GRP78 knockdown triggered concomitant suppression of cell adhesion proteins, such as N-/E-cadherin and beta-catenin which resulted in inhibition of cancer cell adhesion and proliferation, suggesting a signaling and/or molecular interplay in between these classes of biomarkers.10 Suppression of GRP78 expression also triggered drastic changes in PC-3 cells’ morphology and decreased their adhesion to osteoblasts (OSB) dependent, in part, to the reduced N-cadherin expression detected (Fig 1d). This observation suggests that GRP78 may be directly associated with the cellular functions of cell adhesion proteins, which raises important mechanistic implications related to adhesion, spread, and proliferation in the tumor microenvironment. To this effect, GRP78 knockdown was also shown to regulate the epithelial to mesenchymal transition (EMT) associated with tumor spread and metastasis, that was likely related to the observed induction in transforming growth factor beta 1 (TGF-β1) expression. Thus, GRP78 silencing triggers potent anti-cancer effects and makes GRP78 a clinically viable biological marker to reduce the adhesive nature of metastatic tumors to the bone niche.

   Cancer-Targeted Gene Therapy. Cancer-targeting peptides (CTPs) own the ability to bind with high affinity and specificity to cancer cell surface protein receptors, while leaving healthy cells unscathed.11 We have developed an effective solid phase peptide synthesis strategy for the production of poly(arginine) derived CTPs.12 Poly(arginine) has the dual ability to bind and release genetic material into cells for activity while the CTP enables targeted delivery into selected tumors. However, in our preliminary studies, polyarginine-derived CTPs condensed GRP-silencing siRNAs but limited cell uptake and RNAi activity, presumably due to large, aggregated particles observed by TEM and DLS.13 In an effort to circumvent this limitation, we developed a simple and efficient method for templating RNA onto gold (Au) surfaces and their self-assembly into small, discrete nanoparticles for multifunctional RNAi activity.14 The Au-functionalized siRNA particles restored cell uptake which effected GRP knockdown and cell death in PC-3 cells in the absence of a transfection vector. The formulation of stable, small and discrete Au-RNA nanoparticles may thus prove to be valuable multifunctional probes in the treatment of cancer cells.

Fig 1.

Aim II.  Peptide & Protein Chemical Biology for Cancer Immunotherapy.

   Immunostimulatory Peptides. The selection of immunostimulatory peptides derived from epitopes displayed on tumor associated antigens (TAAs) have formed the basis for peptide vaccines and related immunotherapy  against cancer.15 In our contribution, a new class of immunostimulatory peptides capable of binding and activating Natural Killer (NK) cells was discovered (Fig 2a).16 The immunostimulatory peptides were derived from B7H6, an activating tumor antigen for the natural cytotoxicity receptor NKp30 found on the surface of NK cells. B7H6 is constitutively expressed on the surface of tumors and serves as an activating TAA for NK-dependent recognition and elimination of B7H6+ tumors.17 However, tumors gain resistance by B7H6 suppression18 and elimination19 resulting in evasion of NK-dependent immunosurveillance. Thus, peptidic ligands of B7H6 may serve to restore NK-dependent immunity in tumors that lack or are deficient in surface B7H6 presentation. Towards this goal, we have designed and developed B7H6 derived peptide ligands based on the B7H6:NKp30 binding site interface.16,23 These short synthetic peptides displayed secondary structure motifs, including a β-turn conformation that was hypothesized to contribute to NKp30 binding according to molecular docking. The synthetic peptides were screened against NK92-MI cells by flow cytometry, and a lead peptidic sequence (TVPLN) demonstrated NKp30-dependent binding in competition with a fluorochrome-labeled anti-NKp30 mAb. According to ELISA, peptide binding triggered NK-dependent immunostimulatory activity by the secretion of TNF-α without any noticeable toxicity to NK cells. This new class of immunostimulatory peptide may thus prove to be a promising lead in the development of NK-dependent immunotherapy, especially against resilient tumors that lack or are deficient in surface B7H6 presentation.

   Synthetic Antibody Mimics. The evolution of synthetic biologics such as mAb, and their related analogs― the antibody-drug conjugates, antibody fragments (Fab and Fc regions) and the multivalent antibodies have taken center stage in the fight against cancer with more than 80 mAb drugs approved for clinical use.21 Although mAb and their related analogs remain at the forefront of cancer immunotherapy applications, novel therapeutics and/or treatment methods that may overcome their production, administration, and pharmacological limitations, including the evolution of treatment resistance are still in widespread demand.22 Thus, the molecular biology of small to intermediate size molecules with the targeting and effector functions of antibodies may represent a novel class of immunotherapeutics that may circumvent these limitations. In this application, cancer-targeting immunostimulatory peptides (CTIPs) have been designed and developed as synthetic antibody mimics.23 A divergent approach enabled the incorporation of two NKp30-targeting motifs (TVPLN) that span the peptide main chain, while being separated by a short Gly-Lys-Gly (GKG) spacer, in which K was assigned the branching residue for the incorporation of the GRP78-targeting peptides. In this manner, Y-branch bifunctional trimeric peptides were designed and developed to bind to GRP78, specifically expressed on the surface of tumors, and NKp30 on the surface of NK cells for immunotoxicity towards the targeted GRP78+/B7H6– tumors in a manner that may mimic the targeting and activating functions of antibodies (Fig 2b). A lead trimeric peptide displayed GRP78 binding on the surface of A549 lung tumor cells as well as NKp30 binding on NK92-MI cells with and without competing fluorochrome-labeled mAbs which confirmed receptor binding specificities by flow cytometry. In co-culture, fluorescence microscopy revealed that the target GFP-expressing A549 cells were visibly associated with the effector NK cells when pre-activated with lead trimeric peptide. Peptide activation of NK cells was screened by Luminex, which indicated the secretion of inflammatory cytokines and chemokines (TNF-α, IFN-ϒ and IL-8) that resulted in tumor-associated immunotoxicity as evidenced by the loss in GFP signal and detection of early-/late-stage apoptosis (Annexin V/7-AAD staining). Significantly, treatment of peptide-activated NK cells in A549-tumor-bearing mice resulted in a consistent decrease in tumor growth when compared to the untreated control group. This novel class of peptides may thus provide important insights into the development of synthetic antibody mimics and peptide vaccines against selected tumor types for cancer immunotherapy applications.

Fig 2

References.

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