Development of Novel Class of Therapeutic Oligonucleotides Based on Small Molecule Screening

Highly inefficient transit of oligonucleotides from outside cells to the intracellular compartments where functional activity of oligonucleotides takes place is the most serious limitation to the practical realization of a full potential of oligonucleotide-based therapies. Several classes of oligonucleotide therapeutics (ONT), including antisense oligonucleotides (ASO), hydrophobically modified siRNAs (hsiRNA), GalNAc-conjugated siRNAs, and LNP-formulated siRNAs have validated biological efficacy and are in clinic. In all cases, the fraction of oligonucleotides reaching the intended place of biological function is surprisingly low, with the majority of molecules being trapped in wrong cellular compartments, resulting in low efficiency and clinically limiting toxicity. We have recently completed a cell-based screen using the LOPAC library and identified a panel of small molecules that alter cellular localization and dramatically enhance the efficacy of hydrophobically modified siRNAs (hsiRNAs) developed previously [4] (Navaroli et al 2013). In the presence of top two hits (Guanabenz and Phenamil), we have observed a dose-dependent enhancement of oligonucleotide efficacy, with both a significant increase in cellular uptake and decrease in EC50 values. Use of small molecules as enhancers and modulators of oligonucleotide therapeutic efficacy is a new paradigm in formulation development with wide implications on compounds in clinic and future developments

Guanabenz (Wytensin) selectively enhances uptake and efficacy of hydrophobically modified siRNAs

One of the major obstacles to the pharmaceutical success of oligonucleotide therapeutics (ONTs) is efficient delivery from the point of injection to the intracellular setting where functional gene silencing occurs. In particular, a significant fraction of internalized ONTs are nonproductively sequestered in endo-lysosomal compartments. Here, we describe a two-step, robust assay for high-throughput de novo detection of small bioactive molecules that enhance cellular uptake, endosomal escape, and efficacy of ONTs. Using this assay, we screened the LOPAC (Sigma-Aldrich) Library of Pharmacologically Active Compounds and discovered that Guanabenz acetate (Wytensin), an FDA-approved drug formerly used as an antihypertensive agent, is capable of markedly increasing the cellular internalization and target mRNA silencing of hydrophobically modified siRNAs (hsiRNAs), yielding a approximately 100-fold decrease in hsiRNA IC50 (from 132 nM to 2.4 nM). This is one of the first descriptions of a high-throughput small-molecule screen to identify novel chemistries that specifically enhance siRNA intracellular efficacy, and can be applied toward expansion of the chemical diversity of ONTs

Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways

Efficient delivery of therapeutic RNA beyond the liver is the fundamental obstacle preventing its clinical utility. Lipid conjugation increases plasma half-life and enhances tissue accumulation and cellular uptake of small interfering RNAs (siRNAs). However, the mechanism relating lipid hydrophobicity, structure, and siRNA pharmacokinetics is unclear. Here, using a diverse panel of biologically occurring lipids, we show that lipid conjugation directly modulates siRNA hydrophobicity. When administered in vivo, highly hydrophobic lipid-siRNAs preferentially and spontaneously associate with circulating low-density lipoprotein (LDL), while less lipophilic lipid-siRNAs bind to high-density lipoprotein (HDL). Lipid-siRNAs are targeted to lipoprotein receptor-enriched tissues, eliciting significant mRNA silencing in liver (65%), adrenal gland (37%), ovary (35%), and kidney (78%). Interestingly, siRNA internalization may not be completely driven by lipoprotein endocytosis, but the extent of siRNA phosphorothioate modifications may also be a factor. Although biomimetic lipoprotein nanoparticles have been explored for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation

A non-canonical DNA structure is a binding motif for the transcription factor SP1 in vitro

SP1 is a ubiquitous transcription factor that is involved in the regulation of various house-keeping genes. It is known that it acts by binding to a double-stranded consensus motif. Here, we have discovered that SP1 binds also to a non-canonical DNA structure, a G-quadruplex, with high affinity. In particular, we have studied the SP1 binding site within the promoter region of the c-KIT oncogene and found that this site can fold into an anti-parallel two-tetrad G-quadruplex. SP1 pull-down experiments from cellular extracts, together with biophysical binding assays revealed that SP1 has a comparable binding affinity for this G-quadruplex structure and the canonical SP1 duplex sequence. Using SP1 ChIP-on-chip data sets, we have also found that 87% of SP1 binding sites overlap with G-quadruplex forming sequences. Furthermore, while many of these immuoprecipitated sequences (36%) even lack the minimal SP1 consensus motif, 5′-GGGCGG-3′, we have shown that 77% of them are putative G-quadruplexes. Collectively, these data suggest that SP1 is able to bind both, canonical SP1 duplex DNA as well as G-quadruplex structures in vitro and we hypothesize that both types of interactions may occur in cells

Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo

Abstract Small interfering RNA (siRNA)-based drugs require chemical modifications or formulation to promote stability, minimize innate immunity, and enable delivery to target tissues. Partially modified siRNAs (up to 70% of the nucleotides) provide significant stabilization in vitro and are commercially available; thus are commonly used to evaluate efficacy of bio-conjugates for in vivo delivery. In contrast, most clinically-advanced non-formulated compounds, using conjugation as a delivery strategy, are fully chemically modified (100% of nucleotides). Here, we compare partially and fully chemically modified siRNAs in conjugate mediated delivery. We show that fully modified siRNAs are retained at 100x greater levels in various tissues, independently of the nature of the conjugate or siRNA sequence, and support productive mRNA silencing. Thus, fully chemically stabilized siRNAs may provide a better platform to identify novel moieties (peptides, aptamers, small molecules) for targeted RNAi delivery

Small-molecule-induced DNA damage identifies alternative DNA structures in human genes.

Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here we show that the G-quadruplex-interacting drug pyridostatin promotes growth arrest in human cancer cells by inducing replication- and transcription-dependent DNA damage. A chromatin immunoprecipitation sequencing analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein abundance and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target of this drug. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions

Template-assembled synthetic G-quartets (TASQS)

Fabrication of functional supramolecular structures requires a certain degree of control which may not be achieved by relying solely on noncovalent interactions. The current study aims to investigate the effect of a rigid cavitand template on morphology, function and stability of lipophilic G-quadruplexes. The first Chapter of this thesis introduces different aspects of G quadruplex chemistry and explains how these structures are particularly suited for the creation of supramolecular architectures. The second Chapter of this thesis presents the synthesis and self-assembly of a new class of supramolecular architectures composed of four guanosines attached to a rigid cavitand template. These structures, named template-assembled synthetic G-quartets (TASQs), were synthesized via the “click” reaction and manifest an ordered topology dictated by the template. The lipophilic TASQs were found to self-associate spontaneously to form a singular basket-like structure in chloroform. Moreover, it was found that TASQs form cation-free G-quartets which exhibit remarkable stability under this condition. The third Chapter of this thesis describes the preparation, characterization and solution study of the cation-bound complexes TASQNa⁺, TASQK⁺, TASQCs⁺, and TASQSr²⁺. Cations play a major role in controlling the morphology and stability of G-quadruplexes. The analysis of the cation-specific structures of TASQs reveals the formation of a monomeric G quartet for Na⁺ and Sr²⁺,a dimeric system for Cs⁺ and a mixture of monomers and dimers for K⁺. The factors governing the formation of these structures were evaluated, the selectivities of TASQs for cations were determined, and the cation-dependent structural transformations were studied. The fourth Chapter describes the efforts towards synthesizing a hydrophilic TASQ via the “click” reaction. The following steps have been taken: 1) a water-soluble cavitand has been successfully synthesized and characterized, which can potentially serve as a hydrophilic template, and 2) two oligonucleotides have been appropriately functionalized and preliminary coupling reactions were attempted. The next phases of this research along with potential future directions are discussed in Chapter five.Science, Faculty ofChemistry, Department ofGraduat

Abstract 3180: Suppression of the CPEB3 ribozyme modulates the progression of glioblastoma

Abstract: Glioblastoma multiforme (GBM) is the most aggressive primary malignant brain tumor in adults, with a poor prognosis that highlights a dire clinical need for innovative therapeutic interventions. Despite significant advances in diagnoses and multimodality therapies, the overall prognosis for patients with GBM remains poor, with a median survival time of 15-18 months. Therefore, there is an unmet medical need to develop alternative treatment strategies to improve clinical outcomes. Dysregulation of post-transcriptional control and translational machinery have been implicated in malignant tumor development. Cytoplasmic polyadenylation element binding proteins (CPEB1-CPEB4) are RNA-binding proteins that regulate poly(A) tail elongation of target mRNAs and subsequently contribute to phenotypic changes in cancer cells. Notably, a self-cleaving ribozyme was identified in the CPEB3 gene, but its role in cancer is wholly unexplored. Considering the role of CPEB3 as a tumor suppressor gene and the promotion of cancer progression through the downregulation of CPEB3, our hypothesis is that the CPEB3 ribozyme regulates CPEB3 expression, and its activity contributes to the progression of tumors. Using antisense oligonucleotides (ASOs) as an approach, we demonstrated that inhibition of CPEB3 ribozyme resulted in an increase of CPEB3 mRNA and protein expression. Blocking the CPEB3 ribozyme led to a significant reduction in cell proliferation, migration, and invasion in GBM cell lines. Gene set enrichment analysis (GSEA) revealed the downregulation of epithelial-mesenchymal transition (EMT), angiogenesis, and hypoxia gene sets in GBM cells treated with ASO compared to Ctrl-ASO. We further measured VEGFA mRNA and protein expression and found that ASO-treated GBM cells secreted significantly less VEGF in conditioned media. Inhibition of the CPEB3 ribozyme also mitigated the EMT process in GBM cells. Subsequently, ASO strategies were applied to patient-derived glioma stem cells (GSCs), representing a clinically relevant model for pre-clinical therapeutic intervention. We found that treatment of CPEB3 ribozyme ASO up-regulated CPEB3 mRNA and inhibited cell proliferation in GSCs. Furthermore, the combination of ASO and temozolomide chemotherapy exhibited a more pronounced decrease in GSCs proliferation compared to individual treatment alone. Collectively, this study highlights the significance of the CPEB3 ribozyme in GBM and explores therapeutic approaches focused on targeting CPEB3 in cancer. Citation Format: Claire Chen, Eric Wang, Lily Tong, Mehran Nikan, Daniela A. Bota, Claudia Benavente, Andrej Luptak. Suppression of the CPEB3 ribozyme modulates the progression of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3180

S-Acyl-2-Thioethyl: A Convenient Base-Labile Protecting Group for the Synthesis of siRNAs Containing 5′-Vinylphosphonate

We recently reported that (E)-5′-vinylphosphonate (5′-VP) is a metabolically-stable phosphate mimic for siRNA and demonstrated that 5′-VP improves the potency of the fully modified siRNAs in vivo. Here, we report an alternative synthesis of 5′-VP modified guide strand using S-pivaloyl-2-thioethyl (tBu-SATE) protecting group. The tBu-SATE group is readily removed during the final cleavage of the oligonucleotide from the solid support and providing a more convenient route for the synthesis of siRNA guide strand carrying a 5′-vinylphosphonate