Polyetheylenimine-Polyplexes of Spiegelmer NOX-A50 Directed against Intracellular High Mobility Group Protein A1 (HMGA1) Reduce Tumor Growth in Vivo

Abstract

High mobility group A1 (HMGA1) proteins belong to a group of architectural transcription factors that are overexpressed in a range of human malignancies, including pancreatic adenocarcinoma. They promote anchorage-independent growth and epithelial-mesenchymal transition and are therefore suggested as potential therapeutic targets. Employing in vitro selection techniques against a chosen fragment of HMGA1, we have generated biostable L-RNA oligonucleotides, so-called Spiegelmers, that specifically bind HMGA1b with low nanomolar affinity. We demonstrate that the best binding Spiegelmers, NOX-A50 and NOX-f33, compete HMGA1b from binding to its natural binding partner, AT-rich doublestranded DNA. We describe a formulation method based on polyplex formation with branched polyethylenimine for efficient delivery of polyethylene glycol-modified Spiegelmers and show improved tissue distribution and persistence in mice. In a xenograft mouse study using the pancreatic cancer cell line PSN-1, subcutaneous administration of 2 mg/kg per day NOX-A50 formulated in polyplexes showed an enhanced delivery of NOX-A50 to the tumor and a significant reduction of tumor volume. Our results demonstrate that intracellular targets can be successfully addressed with a Spiegelmer using polyethylenimine-based delivery and underline the importance of HMGA1 as a therapeutic target in pancreatic cancer.The mammalian nonhistone chromatin high mobility group A1 proteins HMGA1a, 3 HMGA1b, and HMGA1c belong to one protein family encoded by the HMGA1 gene. They act as architectural transcription factors by binding via AT hook motifs to the minor groove of AT-rich DNA, leading to modulation of chromatin and nucleosome remodeling. Their ability to regulate the activity of several genes through the recognition and alteration of the DNA double helix and chromatin substrates has been linked to the development of human cancers (1-3). The overexpression of HMGA proteins is associated with different types of tumors, e.g. pancreas, breast, prostate, cervical, gastric, colorectal cancer as well as lymphomas and neuroblastic malignancies (4 -6). The level of expression of HMGA1a/b is low or almost undetectable in most differentiated or nonproliferating normal cells (7-9) but is rapidly up-regulated in response to growth-stimulatory factors (8, 10). Thus, HMGA1 is considered a promising target to treat cancer development, progression, and metastasis.The aim of our study was to evaluate whether the intracellular HMGA1 protein family can be successfully targeted with a Spiegelmer, a large structured molecule that is comparable with a monoclonal antibody in terms of specific target binding. Spiegelmers (German: Spiegel ϭ mirror) are structured biostable L-oligonucleotides that differ from their aptamer counterparts in their sugar moiety, which consists of mirrorimage L-(deoxy)ribose rather than D-(deoxy)ribose and makes Spiegelmers highly resistant to nucleases (11,12). Spiegelmers have already been described to act potently as inhibitors in vivo (13-15) and have ...

Aptamers as Remarkable Diagnostic and Therapeutic Agents in Cancer Treatment

“…These L-oligonucleotides, mirror images of selected D-sequences (called Spiegelmers), specifically target the natural protein and are not degraded in vitro, even after 60 hours incubation in serum [33]. Spiegelmers have already been described to act potently as inhibitors in vivo [14,34,35] and have proven to be exceptionally safe in two Phase I clinical studies.…”

“…Many cancers originate from genetic events leading to dysregulation in the expression of cells molecules or receptors. Therefore targeting these molecules or receptors can aid in the diagnosis and treatment of disease by, for example, helping to improve the sensitivity and/or specificity of diagnostic assay through molecular imaging [6,11,12], inhibiting disease process [13][14][15] or targeting the delivery of drugs to diseased tissue [16][17][18].…”

The high mobility group A1 molecular switch: turning on cancer – can we turn it off?

“…Synthetic oligonucleotide sequences that competitively bind to oncogenic transcription factors represent another promising approach that has been proposed to target HMGA1 [105,106]. Two prior studies tested AT-rich oligonucleotides as decoys or ‘sponges’ for HMGA1 to prevent DNA binding [105,106].…”

Invading target cells: multifunctional polymer conjugates as therapeutic nucleic acid carriers

“…Aptamers' targets range from small molecules like ATP [9] to large macromolecular compounds like cell-surface proteins [8,12]. Therefore they are a promising tool for diagnostic, therapeutic or targeting purposes [11,13]. A polyethylene glycole (PEG) modified aptamer that specifically binds to the vascular endothelial growth factor (VEGF) has been approved by the FDA and has been marketed for the treatment of neovascular agerelated macular degeneration.…”

“…Aptamers are DNA or RNA based single stranded oligonucleotides that bind molecular targets with high affinity and specificity which are comparable to those of antibodies [7][8][9][10][11][12][13]. Aptamers' targets range from small molecules like ATP [9] to large macromolecular compounds like cell-surface proteins [8,12].…”