iRGD-guided Tumor-penetrating Nanocomplexes for Therapeutic siRNA Delivery to Pancreatic Cancer

Abstract

Pancreatic cancer is one of the leading causes of cancer-related death, with 5-year survival of 8.5%. The lack of significant progress in improving therapy reflects our inability to overcome the desmoplastic stromal barrier in pancreatic ductal adenocarcinoma (PDAC) as well as a paucity of new approaches targeting its genetic underpinnings. RNA interference holds promise in targeting key mutations driving PDAC; however, a nucleic acid delivery vehicle that homes to PDAC and breaches the stroma does not yet exist. Noting that the cyclic peptide iRGD mediates tumor targeting and penetration through interactions with αβ integrins and neuropilin-1, we hypothesized that "tandem" peptides combining a cell-penetrating peptide and iRGD can encapsulate siRNA to form tumor-penetrating nanocomplexes (TPN) capable of delivering siRNA to PDAC. The use of directly conjugated iRGD is justified by receptor expression patterns in human PDAC biopsies. In this work, we optimize iRGD TPNs with polyethylene glycol (PEG)-peptide conjugates for systemic delivery to sites of disease. We show that TPNs effectively knockdown siRNA targets in PDAC cell lines and in an immunocompetent genetically engineered mouse model of PDAC. Furthermore, we validate their tumor-penetrating ability in three-dimensional organoids and autochthonous tumors. In murine therapeutic trials, TPNs delivering anti- siRNA significantly delay tumor growth. Thus, iRGD TPNs hold promise in treating PDAC by not only overcoming physical barriers to therapy, but by leveraging the stroma to achieve knockdown of the gold-standard genetic target. Moreover, the modular construction of this delivery platform allows for facile adaptation to future genetic target candidates in pancreatic cancer..

Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes

“…Although sgRNA/Cas9 is molecularly different and much larger than siRNA, we reasoned that similarly to siRNA (theoretical net charge −40), a Cas9 (theoretical net charge +22)/sgRNA (theoretical net charge −101) RNP complex should also be polyanionic (theoretical net charge −79) and therefore, has the potential to nanocomplex with tandem peptides that have successfully delivered siRNA in vivo. 52,55,57,58 For targeting, we utilized a cyclic peptide, iRGD (sequence: CRGDKGPDC, net charge 0), which mediates tumor targeting and internalization by binding αvβ3/αvβ5 integrins and neuropilin-1. 54,55 In addition, we designed and tested different lipid tails and the palmitoyl-TP-iRGD (pTP-iRGD) was found to be the most optimal candidate (data not shown).…”

“…52,55,57,58 For targeting, we utilized a cyclic peptide, iRGD (sequence: CRGDKGPDC, net charge 0), which mediates tumor targeting and internalization by binding αvβ3/αvβ5 integrins and neuropilin-1. 54,55 In addition, we designed and tested different lipid tails and the palmitoyl-TP-iRGD (pTP-iRGD) was found to be the most optimal candidate (data not shown).…”

“…Based on a tandem peptide nanocomplex system, [52][53][54][55][56][57][58] previously developed by our lab to achieve targeted delivery of siRNA to tumors, we hypothesized a simple and modular delivery approach, CRISPR-GPS (Guiding Peptide Sequences). We discovered that it can co-deliver native or modified sgRNA and Cas9 protein to various cell types in vitro with comparable efficiency as lipofectamine® RNAiMAX, and may also offer the capacity to target specific cell types in vivo.…”

Cell Penetrating Peptides, Novel Vectors for Gene Therapy

“…Tumor extracellular matrix has a high level of αvβ3/5 integrin expression that binds cyclic RGD peptide. Tandem peptides combing a CPP with tumor targeting RGD through a disulfide bond complexed with anti-KRAS siRNA formed nanoparticles that significantly delayed tumor growth in a mouse model of pancreatic cancer [102]. In a similar manner, a peptide targeting epidermal growth factor, overexpressed in oral cancer cells, was conjugated to an endosomal disruptive peptide, to successfully deliver siRNA targeting cancerous inhibitor of protein phosphatase 2A (CIP2A), an oncogene [103].…”

Characterization of iRGD-Ligand Modified Arginine-Histidine-Rich Peptides for Nucleic Acid Therapeutics Delivery to αvβ3 Integrin-Expressing Cancer Cells

“…Application of iRGD as targeting moiety is perspective for peptide-based non-viral vectors; however, to date, a few iRGD-modified peptides have been studied as vehicles for NA therapeutics delivery. Feasibility of this approach was demonstrated recently in RNAi treatment of a pancreatic cancer model and breast cancer cells [ 20 , 21 ].…”