Integrating Display and Delivery Functionality with a Cell Penetrating Peptide Mimic as a Scaffold for Intracellular Multivalent Multitargeting

Abstract

The construction of a multivalent ligand is an effective way to increase affinity and selectivity toward biomolecular targets with multiple-ligand binding sites. Adopting this strategy, we used a known cell-penetrating peptide (CPP) mimic as a scaffold to develop a series of multivalent ligand constructs that bind to the expanded dCTG (CTG(exp)) and rCUG nucleotide repeats (CUG(exp)) known to cause myotonic dystrophy type I (DM1), an incurable neuromuscular disease. By assembling this polyvalent construct, the hydrophobic ligands are solubilized and delivered into cell nuclei, and their enhanced binding affinity leads to the inhibition of ribonuclear foci formation and a reversal of splicing defects, all at low concentrations. Some of the multivalent ligands are shown to inhibit selectively the in vitro transcription of (CTG·CAG)74, to reduce the concentration of the toxic CUG RNA in DM1 model cells, and to show phenotypic improvement in vivo in a Drosophila model of DM1. This strategy may be useful in drug design for other trinucleotide repeat disorders and more broadly for intracellular multivalent targeting.

Myotonic Dystrophies: State of the Art of New Therapeutic Developments for the CNS

“…The past few years, most efforts have focused on mutant DMPK RNA in order to eliminate the toxic RNAs or to release the sequestrated mediators such as MBNL proteins in muscles (reviewed in Klein et al, 2015). Antisense oligonucleotides (ASOs, with various chemistry and/or linked with peptides or nanoparticles) targeting the expanded repeat or specific DMPK sequences, small molecules, conjugated cell penetrating peptides (CPPs), miRNA “sponges” and viral-based gene therapeutic approaches have proven to be efficient in cell models and to some extent in animal models especially for muscles (Gao and Cooper, 2013; Klein et al, 2015; Bai et al, 2016; Cerro-Herreros et al, 2016). Twenty-five years after discovery of the DM1 mutation and with these validated proofs of concept, the first phase 1/2a clinical trial was launched in December 2014 using gapmer-ASOs aiming at destroying DMPK transcripts in muscles (trial NCT02312011).…”

“…For gene therapy, including CRISPR-Cas and TALE nucleases, viral approaches especially adenovirus associated virus (AAV) vectors can be considered although progress has to be made for better targeting and to reduce immune response (Swiech et al, 2015; de Solis et al, 2016; Lee et al, 2016; Ma et al, 2016; Murlidharan et al, 2016). Nanotechnologies as well as cell-penetrated peptides are very promising tools and might help to enable transport across the BBB via intracranial, systemic, or intranasal administration (Krupa et al, 2014; McGowan et al, 2015, 2016; Bai et al, 2016; Kristensen et al, 2016; Sharma et al, 2016; Zhang et al, 2016). Finally, the possibility to modify stem cells open a possible route toward autologous cell transfer (Xia et al, 2015; Gao et al, 2016).…”

Supramolecular Recognition and Selective Protein Uptake by Peptide Hybrids

“…[10,15,[36][37][38][39][40][41] Helical polyglutamates have been developed as antimicrobial agents [42] that were subsequently adapted for nucleotide delivery. [43] Balanced polymeric glycopeptide hybrids for gene delivery [44,45] and nanocapsules for protein and lectin delivery [46,47] have been recently developed. However,e fforts to equip short penetrating peptides with glycan moieties have met with limited success.…”

Intrinsically cell-penetrating multivalent and multitargeting ligands for myotonic dystrophy type 1

“…However, rather than using an optimized dimeric linkage, multivalent ligands are often prepared by covalent attachment of ligands to readily available extrinsic scaffolds, such as a PNA or dendrimer. In these cases, the ease of synthesis often comes with loss of tunability and spatial optimization (7)(8)(9). A separate consideration comes into play when the target is intracellular.…”