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

Overview. The Sabatino Lab (Sab Lab) is committed to the innovation of biomolecular therapeutics, based upon nucleic acids, peptides and proteins, for anti-cancer applications. Our research objectives are focused on: Aim I. RNA Synthetic Biology for Cancer Gene Therapy and Aim II. Peptide and Protein Chemical Biology for Cancer Immunotherapy. Each aim is interdisciplinary and collaborative in nature, uniting research areas, experts and expertise in chemical synthesis, chemical biology, molecular biology and translational biomedical research in the fight against cancer. A brief description of past and current research projects are described below. Please contact Dr. David Sabatino for more info.

Aim I. RNA Synthetic Biology for Cancer Gene Therapy

   Branch & Hyperbranch RNA. In this pioneering project, a new class of synthetic branch and hyperbranch RNA were designed and developed for silencing the oncogenic Glucose Regulated Protein of 78 kilodalton (GRP78) in human HepG2 hepatoblastoma liver cancer cells.1 Silencing GRP78 expression in HepG2 cells triggered cancer cell death, to a greater extent with the branch and hyperbranch silencing (si)RNA relative to linear siRNA controls, underscoring their potential utility in cancer gene therapy applications (Fig. 1). The Sab lab continues to develop chemical synthesis methods to construct new RNA molecular structures for anti-cancer activity.

Fig 1. Branch and Hyperbranch RNA

   RNA Self-Assembly and RNAi Screening. Building upon this branching RNA biotechnology, an assembly strategy was designed in silico to create supramolecular RNA nanostructures of discrete sizes and geometric shapes (Fig. 2).2 A lead Y-shape siRNA nanostructure that was genetically encoded to silence GRP-75, 78 and 94 triggered synergistic knockdown of all three oncogenes, translating into significant cancer cell death across a panel of human cancer cell lines. This novel RNA biotechnology is currently being used to screen against a panel of oncogene targets, to determine their functional roles in cancer biology, while also potentiating the cancer cell death response by silencing multiple oncogenes in synergy.

Fig 2. RNA Nanostructures

   RNA Bioconjugation. To improve RNA functional utility in biological systems, solid-phase bioconjugation has been implemented in our lab,3 that enabled the ligation of fluorescent (FITC) reporters that tracked RNA activity in cells,4 the incorporation of multiple fatty acids for RNA cell uptake activity,5 and the functionalization of RNA onto gold (Au) nanoparticles for cancer theranostic (therapeutic and diagnostic) applications (Fig. 3).6 The Sabatino lab continues to explore bio-orthogonal chemistry for the ligation of biological probes onto synthetic RNA,7 leading to the innovation of multifunctional RNA therapeutic agents.

Fig 3. RNA Bioconjugation

   RNA molecular and systems biology. Silencing RNA continue to be used as molecular probe systems in our research applications, that interrogate the biological mechanisms of anti-cancer activity. For example, suppression of GRP78 produced drastic changes in prostate cancer (PC-3) cells’ morphology, that decreased their adhesion to osteoblast (bone) cells, partly due to the reduced expression of cell adhesion proteins such as N-/E-cadherin and beta-catenin (Fig. 4).8 This outcome suggests that GRP78 may be associated with the cellular functions of adhesion proteins, which raises important mechanistic implications related to cancer cell adhesion, spread and proliferation contributing to metastasis of tumours to the bone niche. Our current research aims to suppress the adhesive nature of metastatic tumours, leading to the innovation of safe and effective intervention strategies for bone metastasis.

Fig 4. RNA Systems Biology

   Cancer-Targeted Gene Therapy. Cancer-targeting and penetrating peptides (CTP and CPPs) are used in our lab to target and internalize therapeutic agents selectively in cancer cells.9-11 In our most recent studies, the amphiphilic cell surface (cs)GRP78 targeting polyarginine peptides adopted an unusual helical-coiled secondary structure, that templated their self-assembly into peptide nanofibers for cell biology applications.12 The peptides condensed genetic (siRNA and pDNA) material into nanoparticles for cancer-targeted gene therapy in csGRP78-presenting cancer cells (Fig. 5).13,14 The Sab lab continues to innovate new and improved peptide-based gene (and drug) delivery systems for precision oncology applications.

Fig 5. Cancer Targeted Gene Therapy

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) forms the basis of our research contributions in the discovery of peptide-based cancer vaccines and related immunotherapy.15 Our research led to the identification of a new class of immunostimulatory peptide epitopes derived from B7H6, an activating TAA for the natural cytotoxicity receptor, NKp30, found on the surface of Natural Killer (NK) cells, and directly implicated in NK-dependent recognition and elimination of B7H6 presenting tumors.16 However, tumors gain resistance towards NK-dependent killing of cancer cells by shedding or eliminating B7H6 from the cancer cell surface, thereby raising the need for peptide epitope discovery to restore NK-dependent killing of B7H6 deficient tumors. Towards this goal, we have designed and developed B7H6 derived peptide ligands of NKp30 using molecular modelling and docking analysis, that revealed short synthetic peptides with privileged β-turn secondary structure motifs engaging in NKp30 binding and activation of NK (NK92-MI) cells, by the secretion of TNF-α, according to ELISA (Fig. 6). This new class of immunostimulatory peptides may serve as promising leads in the advancement of NK-based vaccination and immunotherapy strategies, especially against resilient B7H6 deficient tumors. The latter continues to be a research priority for our group.

Fig 6. Peptide Epitopes

  Multifunctional Peptide Epitopes. The innovation of multifunctional peptide epitopes with expanded biological/immunological activity profiles based on a single molecule platform or a supramolecular assembly that engages multiple receptor and cellular targets for a synergistic therapeutic response is a newly emerging research direction for our group.17 A combination of machine learning tools, high throughput screening and chemical synthesis have been implemented towards the discovery of multifunctional peptide ligands of important immunological receptor targets for applications in cancer immunotherapy (Fig. 7).

Fig 7. Multifunctional Peptide Epitopes

   Synthetic Antibody Mimics. In this application, cancer-targeting immunostimulatory peptides (CTIPs) function as synthetic antibody mimics.18 The bifunctional peptides bind to csGRP78 on tumors, and NKp30 on NK cells for killing GRP78+/B7H6– tumors in a mechanism reminiscent of the targeting and activating functions of antibodies (Fig 8). A lead peptide displayed GRP78 binding on the surface of human lung (A549) cancer cells and NKp30 binding on the NK92-MI cells. In co-culture, peptide activation of NK cells resulted in the secretion of the pro-inflammatory cytokines and chemokines (TNF-α, IFN-ϒ and IL-8) that led to cancer cell death by the detection of early-/late-stage apoptosis. Significantly, treatment of peptide-activated NK cells in A549-tumor-bearing mice resulted in a consistent decrease in tumor growth during the treatment period, making this novel class of multifunctional peptide epitopes applicable to the development of synthetic antibody mimics for cancer immunotherapy applications.

Fig 8. Synthetic Antibody Mimics

References

  1. Maina, A.; Blackman, B.A.; Parronchi, C.J.; Morozko, E.; Bender, M.E.; Blake, A.D.; Sabatino, D. Solid-phase synthesis, characterization and RNAi activity of branch and hyperbranch Bioorg. Med. Chem. 2013; 23, 5270-5274.
  2. Patel, M.R.; Kozuch, S.D.; Cultrara, C.N.; Yadav, R.; Huang, S.; Samuni, U.; Koren, J.; Chiosis, G.; Sabatino, D. RNAi Screening of the Glucose-Regulated Chaperones in Cancer with Self-Assembled siRNA Nanostructures. Nano Lett., 2016; 16, 6099–6108.
  3. Patel, P.L.; Rana, N.; Patel, M.; Kozuch, S.D.; Sabatino, D.Nucleic Acid Bioconjugates in Cancer Detection and Therapy. ChemMedChem. 2016, 11(3), 252-269.
  4. Kozuch, S.D.; Cultrara, C.N.; Beck, A.E.; Heller, C.J.; Shah, S.; Patel, M.R.; Zilberberg, J.; Sabatino, D. Enhanced Cancer Theranostics with Self-Assembled, Multilabeled siRNAs. ACS Omega. 2018; 3, 12975–12984.
  5. Shah, S.S.; Cultrara, C.N.; Kozuch, S.D.; Patel, M.R.; Ramos, J.A.; Samuni, U.; Zilberberg, J.; Sabatino, D. Direct Transfection of Fatty Acid Conjugated siRNAs and Knockdown of the Glucose Regulated Chaperones in Prostate Cancer Cells. Bioconjugate Chem., 2018, 29, 3638–3648.
  6. Shah, S.S.; Cultrara, C.N.; Ramos, J.A.; Samuni, U.; Zilberberg, J.; Sabatino, D. Bifunctional Au-templated RNA nanoparticles enable direct cell uptake detection and GRP75 knockdown in prostate cancer. J Mater Chem B. 2020; 8, 2169-2176.
  7. Cultrara, C.N.; Shah, S.; Kozuch, S.D.; Patel, M.R.; Sabatino, D. Solid Phase Synthesis and Self-Assembly of Higher-Order siRNAs and their Bioconjugates. Biol. Drug Des. 2019; 93, 999-1010.
  8. Cultrara, C.N.; Kozuch, S.D.; Heller, C.J.; Ramasundaram, P.; Shah, S.; Beck, A.E.; Sabatino, D.; Zilberberg, J. GRP78 Modulates Cell Adhesion Markers in Prostate Cancer and Multiple Myeloma Cell Lines. BMC Cancer 2018; 18, 1263.
  9. Shah, S.S.; Casanova, N.; Antuono, G.; Sabatino, D. Polyamide Backbone Modified Cell Targeting and Penetrating Peptides in Cancer Detection and Treatment. Front Chem. 2020; 8, 218.
  10. Joseph, S.C.;Blackman, B.A.; Kelly, M.L.; Phillips, M.; Beaury, M.W.; Martinez, I.; Parronchi, C.J.; Bitsaktsis, C.;  Blake, A.D.; Sabatino, D. Synthesis, Characterization and Biological Activity of Poly(arginine)-derived Cancer-Targeting Peptides in HepG2 Liver Cancer Cells. J. Pept. Sci. 2014, 20, 736-745.
  11. Cultrara, C.N.; Shah, S.S.; Antuono, G.; Heller, C.; Ramos, J.; Samuni, U.; Zilberberg J.; Sabatino, D. Size Matters: Arginine-Derived Peptides Targeting the PSMA Receptor can Efficiently Complex but not Transfect siRNA. Ther. 2019; 18, 863-870.
  12. Daniel, G.; Hilan, G.; Ploeg; L; Sabatino, D. Self-Assembly of Helical-Coiled Amphiphilic Peptide Nanofibers and Inhibition of Fibril Formation by Curcumin. Bioorg. Med. Chem. Lett., 2024; 102, 129682.
  13. Daniel, G.; Collak, F.; Hilan, G.; Robillard, E.; Willmore, W.; McKay, B.; Sabatino, D. Enhancement of Peptide-Based pDNA Gene Transfer with Spermidine and Chloroquine. DNA. 2025, manuscript in review. manuscript ID: dna-3689491, preprint available online.
  14. Hilan, G.; Daniel, G.; Collak, F.; Willmore, W.; McKay, B.; Sabatino, D. Cancer-Targeting Peptides Functionalized with Polyarginine Enables GRP78-Dependent Cell Uptake and siRNA Delivery within the DU145 Prostate Cancer Cells. J. Pept. Sci. 2025; 31(3), e70007.
  15. Sabatino, D. Medicinal Chemistry and Methodological Advances in the Development of Synthetic Peptide Vaccines. Med. Chem. 2020; 63, 14184-14196.
  16. Phillips, M.; Romeo, F.; Bitsaktsis, C.; Sabatino, D. B7H6 Derived Peptides Trigger TNF-a Dependent Immunostimulatory Activity of Lymphocytic NK92-MI Cells. Biopolymers 2016; 106, 658-672.
  17. Nissan, N.; Allen, M.; Sabatino, D.; Biggar, K. Future Perspective: Harnessing the Power of Artificial Intelligence in the Generation of New Peptide Drugs. Biomolecules 2024, 14(10), 1303.
  18. Descalzi-Montoya, D.; Montel, R.A.; Smith, K.; Dziopa, E.; Darwich, A.; Yang, Z.; Bitsaktsis, C.; Korngold, R.; Sabatino, D. Synthetic Antibody Mimics based on Cancer-Targeting Immunostimulatory Peptides. Chembiochem. 2021, 22, 1589-1596.

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