No abstract

Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases.

Antisense oligonucleotides (ASOs) hold promise for therapeutic splice switching correction for genetic diseases, in particular for Duchenne muscular dystrophy (DMD), for which ASO-exon skipping represents one of the most advanced therapeutic strategies. We have previously reported the therapeutic potential of tricyclo-DNA (tcDNA) in mouse models of DMD, highlighting the unique pharmaceutical properties and unprecedented uptake in many tissues after systemic delivery, including the heart and central nervous system. TcDNA-ASOs demonstrate an encouraging safety profile and no particular class-related toxicity, however, when administered in high doses for several months, mild renal toxicity is observed secondary to predictable phosphorothioate (PS)-ASO accumulation in kidneys. In this study, we investigate the influence of the relative content of PS linkages in tcDNA-ASOs on exon skipping efficacy. Mdx mice were injected intravenously once weekly for 4 weeks with tcDNA carrying various amounts of PS linkages (0%, 25%, 33%, 50%, 67%, 83%, and 100%). The results indicate that levels of exon-23 skipping and dystrophin rescue increase with the number of PS linkages in most skeletal muscles except in the heart. As expected, plasma coagulation times are shortened with decreasing PS content, and tcDNA-protein binding in serum directly correlates with the number of PS linkages on the tcDNA backbone. Altogether, these data contribute in establishing the appropriate sulfur content within the tcDNA backbone for maximal efficacy and minimal toxicity of the oligonucleotide.

The use of lipid-based nanoparticles for the delivery of biomacromolecules has attracted considerable attention due to the current interest in protein-based therapeutics. Cubosomes protect the incorporated therapeutics, which are susceptible to degradation by enzymes, thereby improving their bioavailability, and concomitantly enhance cellular uptake. The cubosome nanoparticles presented herein were loaded with bovine serum albumin (BSA) and characterized by small-angle X-ray scattering and dynamic light scattering techniques, while the BSA encapsulation and its release were evaluated in vitro. The ability of this formulation to increase the cellular uptake of albumin by 2-fold was tested on various types of renal tubular cells and confirmed by in vivo renal uptake experiments in mice. The obtained results show that cubosomes are able to deliver BSA inside the cell through distinct uptake and intracellular routing. These data were substantiated, with evidence of a high cubosome-mediated uptake of BSA in Clcn5 knockout mice characterized by defective receptor-mediated endocytosis. The use of cubosomes as a delivery system thus represents a promising approach to overcome the low endocytic uptake in diseased epithelial cells and to treat dysfunctions of the kidney proximal tubule.

Cockayne syndrome complementation group B (CSB) protein is engaged in transcription-coupled repair (TCR) of UV induced DNA damage and its deficiency leads to progressive multisystem degeneration and premature aging. Here, we show that human CSB-deficient cells are hypersensitive to physiological concentrations (1–10 µM) of a lipid peroxidation product, trans-4-hydroxy-2-nonenal (HNE), and in response to HNE they develop a higher level of sister chromatid exchanges (SCEs) in comparison to the wild-type cells. HNE-DNA adducts block in vitro transcription by T7 RNA polymerase, as well as by HeLa cell-free extracts. Treatment of wild-type cells with 1–20 µM HNE causes dephosphorylation of the CSB protein, which stimulates its ATPase activity necessary for TCR. However, high HNE concentrations (100–200 µM) inhibit in vitro CSB ATPase activity as well as the transcription machinery in HeLa cell-free extracts. Cell lines expressing CSB protein mutated in different ATPase domains exhibit different sensitivities to HNE. The motif II mutant, which binds ATP, but is defective in ATP hydrolysis was as sensitive to HNE as CSB-null cells. In contrast, motif V mutant cells were as sensitive to HNE as were the cells bearing wild-type protein, while motif VI mutant cells showed intermediate sensitivity to HNE. These mutants exhibit decreased ATP binding, but retain residual ATPase activity. Homology modeling suggested that amino acids mutated in motifs II and VI are localized closer to the ATP binding site than amino acids mutated in ATPase motif V. These results suggest that HNE-DNA adducts are extremely toxic endogenous DNA lesion, and that their processing involves CSB. When these lesions are not removed from the transcribed DNA strand due to CSB gene mutation or CSB protein inactivation by high, pathological HNE concentrations, they may contribute to accelerated aging.