Antisense Oligonucleotide in LNA-Gapmer Design Targeting TGFBR2—A Key Single Gene Target for Safe and Effective Inhibition of TGFβ Signaling

Abstract

Antisense Oligonucleotides (ASOs) are an emerging drug class in gene modification. In our study we developed a safe, stable, and effective ASO drug candidate in locked nucleic acid (LNA)-gapmer design, targeting TGFβ receptor II (TGFBR2) mRNA. Discovery was performed as a process using state-of-the-art library development and screening. We intended to identify a drug candidate optimized for clinical development, therefore human specificity and gymnotic delivery were favored by design. A staggered process was implemented spanning in-silico-design, in-vitro transfection, and in-vitro gymnotic delivery of small batch syntheses. Primary in-vitro and in-vivo toxicity studies and modification of pre-lead candidates were also part of this selection process. The resulting lead compound NVP-13 unites human specificity and highest efficacy with lowest toxicity. We particularly focused at attenuation of TGFβ signaling, addressing both safety and efficacy. Hence, developing a treatment to potentially recondition numerous pathological processes mediated by elevated TGFβ signaling, we have chosen to create our data in human lung cell lines and human neuronal stem cell lines, each representative for prospective drug developments in pulmonary fibrosis and neurodegeneration. We show that TGFBR2 mRNA as a single gene target for NVP-13 responds well, and that it bears great potential to be safe and efficient in TGFβ signaling related disorders.

Reconditioning the Neurogenic Niche of Adult Non-human Primates by Antisense Oligonucleotide-Mediated Attenuation of TGFβ Signaling

“…In vitro NVP-13 treatment could efficiently downregulate target mRNA and target protein alone, or in presence of the ligand TGF-β1, similar to the clinical situation in a neurodegenerative process. We confirmed our biochemical data by immunocytochemistry showing effective downregulation of the target protein TGFβ-RII within neuronal precursor cells, a finding we described earlier in more detail [31]. TGFβ-RII antagonization also reverted upregulated markers for fibrotic scarring and extracellular matrix changes (Fig.…”

“…1A). Data have been published recently in detail [31] showing that NVP-13 significantly downregulates TGFβ-RII mRNA (qPCR) and protein (WB) expression in vitro, which was confirmed by immunocytochemistry under physiological and also disease-simulating conditions. To mimic the neuroinflammatory process characterized by increased TGFβ-system activity, TGFβ coincubation was always investigated in parallel.…”

“…To follow up the hypothesis of a dysregulated TGFβsystem underlying the detrimental disease course of neurodegenerative disorders like ALS, we developed a novel antisense oligonucleotide (NVP-13) in a locked nucleic acid (LNA)-gapmer design, targeting TGFβ-RII mRNA. With NVP-13 treatment, reducing the ligand binding TGF-RII protein, we aimed to reverse various pathological hallmarks of neurodegeneration, including reduced neurogenesis, in a highly efficient and safe manner [31] ready for clinical translation. The goals of the current study were (i) to determine the efficacy of NVP-13 on TGFβ mRNA and protein downregulation in vivo, (ii) to further characterize changes in downstream phosphorylation of intracellular signaling cascades, (iii) to investigate the regulation of neurogenic niche marker expression both in vitro (human neural precursor cells) and in vivo (male and female cynomolgus monkeys), and (iv) to evaluate potential clinical relevance of NVP-13 treatment.…”

Beyond ribose and phosphate: Selected nucleic acid modifications for structure–function investigations and therapeutic applications

“…In combination with the high selectivity for RNA sequences, this makes LNA-modified oligonucleotides well suited for use as antisense therapeutics. Recent publications have used LNA’s high affinity for RNA sequences in gapmer-designed antisense oligonucleotides for successful targeting of a key gene involved in TGFβ inhibition [ 142 ]. The inclusion of LNA nucleosides within a larger single-stranded DNA oligonucleotide has also allowed for subtle gene modifications to be implemented while evading mismatch repair (MMR) [ 143 ].…”

Transforming Growth Factor-β and Long Non-coding RNA in Renal Inflammation and Fibrosis

“…Nevertheless, new technologies such as CRISPR/Cas9 editing ( Wang et al, 2019 ; Horlbeck et al, 2020 ), Gapmer antisense oligonucleotide-mediated lncRNA silencing ( Castanotto et al, 2015 ; Kuespert et al, 2020 ), plasmid/vector-delivery short hairpin RNAs (shRNAs) ( Zhu et al, 2019 ; Yao et al, 2020 ) and ultrasound-mediated gene transfer method ( Zhou et al, 2015a ; Feng et al, 2018 ; Sun et al, 2018 ; Zhang et al, 2019c ) may represent the novel strategies to modulate the expression and function of lncRNA in kidney diseases in the future.…”