Engineered stabilized peptide-based combination therapy against breast and kidney cancer

Mallick, Argha Mario (2024) Engineered stabilized peptide-based combination therapy against breast and kidney cancer. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Argha Mario Mallick (16RS033)) 16RS033.pdf - Submitted Version Restricted to Repository staff only Download (24MB)

Abstract

The key aim of this work is to develop improved peptide-based therapeutics against aggressive cancers with a special emphasis on breast and kidney cancer. Conventional treatment for metastatic breast and kidney cancer which includes surgery, radiation therapy, chemotherapy, and other current methods, is frequently found to be ineffective with high recurrence rate and exhibiting local and systemic toxicity. Peptides have emerged as a new paradigm for cancer treatment as they can be engineered to be bio-compatible, having optimum stability with minimum non-specific toxicity. Developing next-generation combination therapy with clinically safe biocompatible anti-cancer drugs which will not pose systemic toxicity will make huge impact in cancer treatment. This study also focuses on developing mechanistically inspired rational combination therapy by employing peptides as potential therapeutic drug and siRNA transporters for enabling RNAi technology against metastatic breast and kidney cancer. Chapter 1 summarizes the use peptides in anti-cancer treatment with a special emphasis in designing peptide-based siRNA transporters. This chapter also discusses the rationale for designing an effective combination therapy against cancers. Chapter 2 discusses our initiative to design improved lipopeptide based siRNA transporter and the development of siRNA-based combination therapy against triple negative breast cancer. The biggest challenge in implementing siRNA therapeutics is to deliver siRNA in its functional form to the cytosol of the cell so the it can access the RISC complex. Towards this goal, we have engineered short, non-immunogenic and clinically safe facial lipopeptides with high gene silencing efficacy. The presence of unnatural amino acid sarcosine (N-methylglycine) in between two consecutive arginine residues make the lipopeptide optimum protease stable. These lipopeptides, self-assemble with siRNA and mediate high internalization of siRNA in TNBC cell line (1.8 times higher than HiPerFect) and “hard to transfect” primary cell line, HUVEC (1.53 times higher than HiPerFect). HiPerFect is a gold-standard transfection reagent sold by Qiagen, Germany. Our designed lipopeptide has vitamin E moiety for chemo-preventive role and the long partially constrained alkyl chain of vitamin E moiety ensures that our lipopeptide-siRNA can escape early endosomes, having lower acidic pH (pH – 6.5-6), thus maintaining the functionality of the siRNA. The designed Notch1 silencing nanocomplex exhibited long-term gene silencing efficacy compared to HiPerFect (1.6 times higher gene silencing efficacy at day 7). The designed Notch-1 silencing nanocomplex can reduce metastasis (downregulate MMP-2 and N-cadherin and upregulate E-cadherin) and stemness (upregulated CD24 and downregulated CD44) related markers in TNBC cell line, MDA-MB-231. It also drastically reduced nanobridge formation between cancer cells and endothelial cells, responsible for metastasis. Additionally, we have repurposed, m-TOR inhibiting anti-diabetic drug, metformin and used it along with our engineered Notch-1 silencing bio-molecule as combination therapy and demonstrated its efficacy in in vivo zebrafish model. Chapter 3 reports the designing and use of ionophore gramicidin A inspired short membrane-active hydrophobic peptide and the development of siRNA-based therapeutics against highly metastatic, clear cell renal cell carcinoma (ccRCC) cell line SK-RC-45. Unlike gramicidin A, the engineered serum-stable gramicidin inspired peptide exhibits no haemolysis and is non-toxic to non-cancerous human cell lines at its effective dose. The designed peptide can induce pores/leakage in plasma membrane mimicking GUVs and cause mitochondrial depolarization to cancer cells hinting that they have the ability to disrupt pH regulatory machinery of RCC cells. This designed peptide along with the combination therapy not only induces cell cycle arrest and cause apoptosis to ccRCC cell line SK-RC-45, but also alters the transcription of various oncogenes which are of high therapeutic implications for treating ccRCC. The designed peptide could also upregulate VHL (Von Hippel-Lindau; responsible for degradation of HIF-α) and downregulate HIF-2α (hypoxia inducing factor 2α; responsible for upregulating various oncogenes) protein expression in SK-RC-45 cells. At the end, we have also demonstrated that our designed combination therapy lowers the expression of immunosuppressive proteins like PD-L1 (responsible for preventing T cell activation) and CD47 (responsible for preventing phagocytosis). The designed combination therapy further reduced the gene expression of ganglioside synthase, GM2 synthase on SK-RC-47 cells. These collective phenomena lead to increased phagocytosis of cancer cells by macrophages and reduced apoptosis of T cells induced by SK-RC-45 cells, thus potentiating immunotherapy against ccRCC.

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