Cyclic Peptides to Improve Delivery and Exon Skipping of Antisense Oligonucleotides in a Mouse Model for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle wasting disorder caused by reading frame disrupting mutations in the DMD gene. Exon skipping is a therapeutic approach for DMD. It employs antisense oligonucleotides (AONs) to restore the disrupted open reading frame, allowing the production of shorter, but partly functional dystrophin protein as seen in less severely affected Becker muscular dystrophy patients. To be effective, AONs need to be delivered and effectively taken up by the target cells, which can be accomplished by the conjugation of tissue-homing peptides. We performed phage display screens using a cyclic peptide library combined with next generation sequencing analyses to identify candidate muscle-homing peptides. Conjugation of the lead peptide to 2'-O-methyl phosphorothioate AONs enabled a significant, 2-fold increase in delivery and exon skipping in all analyzed skeletal and cardiac muscle of mdx mice and appeared well tolerated. While selected as a muscle-homing peptide, uptake was increased in liver and kidney as well. The homing capacity of the peptide may have been overruled by the natural biodistribution of the AON. Nonetheless, our results suggest that the identified peptide has the potential to facilitate delivery of AONs and perhaps other compounds to skeletal and cardiac muscle.

Next Generation Exon 51 Skipping Antisense Oligonucleotides for Duchenne Muscular Dystrophy.

In the last two decades, antisense oligonucleotides (AONs) that induce corrective exon skipping have matured as promising therapies aimed at tackling the dystrophin deficiency that underlies the severe and progressive muscle fiber degeneration in Duchenne muscular dystrophy (DMD) patients. Pioneering first generation exon 51 skipping AONs like drisapersen and eteplirsen have more recently been followed up by AONs for exons 53 and 45, with, to date, a total of four exon skipping AON drugs having reached (conditional) regulatory US Food and Drug Administration (FDA) approval for DMD. Nonetheless, considering the limited efficacy of these drugs, there is room for improvement. The aim of this study was to develop more efficient [2'-O-methyl-modified phosphorothioate (2'OMePS) RNA] AONs for DMD exon 51 skipping by implementing precision chemistry as well as identifying a more potent target binding site. More than a hundred AONs were screened in muscle cell cultures, followed by a selective comparison in the hDMD and hDMDdel52/mdx mouse models. Incorporation of 5-methylcytosine and position-specific locked nucleic acids in AONs targeting the drisapersen/eteplirsen binding site resulted in 15-fold higher exon 51 skipping levels compared to drisapersen in hDMDdel52/mdx mice. However, with similarly modified AONs targeting an alternative site in exon 51, 65-fold higher skipping levels were obtained, restoring dystrophin up to 30% of healthy control. Targeting both sites in exon 51 with a single AON further increased exon skipping (100-fold over drisapersen) and dystrophin (up to 40%) levels. These dystrophin levels allowed for normalization of creatine kinase (CK) and lactate dehydrogenase (LDH) levels, and improved motor function in hDMDdel52/mdx mice. As no major safety observation was obtained, the improved therapeutic index of these next generation AONs is encouraging for further (pre)clinical development.

Feasibility of SPECT-CT Imaging to Study the Pharmacokinetics of Antisense Oligonucleotides in a Mouse Model of Duchenne Muscular Dystrophy.

Antisense oligonucleotides (AONs) are promising candidates for treatment of Duchenne muscular dystrophy (DMD), a severe and progressive disease resulting in premature death. However, more knowledge on the pharmacokinetics of new AON drug candidates is desired for effective application in the clinic. We assessed the feasibility of using noninvasive single-photon emission computed tomography-computed tomography (SPECT-CT) imaging to determine AON pharmacokinetics in vivo. To this end, a 2'-O-methyl phosphorothioate AON was radiolabeled with 123I or 111In, and administered to mdx mice, a rodent model of DMD. SPECT-CT imaging was performed to determine AON tissue levels, and the results were compared to data obtained with invasive analysis methods (scintillation counting and a ligation-hybridization assay). We found that SPECT-CT data obtained with 123I-AON and 111In-AON were qualitatively comparable to data derived from invasive analytical methods, confirming the feasibility of using SPECT-CT analysis to study AON pharmacokinetics. Notably, also AON uptake in skeletal muscle, the target tissue in DMD, could be readily quantified using SPECT-CT imaging, which was considered a particular challenge in mice, due to their small size. In conclusion, our results demonstrate that SPECT-CT imaging allows for noninvasive characterization of biodistribution and pharmacokinetics of AONs, thereby enabling quantitative comparisons between different radiolabeled AON drug candidates and qualitative conclusions about the corresponding unmodified parent AONs. This technology may contribute to improved (pre)clinical drug development, leading to drug candidates with optimized characteristics in vivo.

Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated Myotubes In Vitro.

Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.

Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON.

Liquid chromatography-mass spectrometry study towards the pH and temperature-induced N-acyl migration in polymyxin B.

In the course of the synthesis and purification of new polymyxins and analogues, formation of a by-product with identical mass was observed and it was believed that this might be the result of acyl migration from the Nalpha- to the Ngamma-position of residue alpha,-gamma-diaminobutyric acid 1 (Dab1) under acidic conditions. Therefore, a LC-MS/MS study was initiated to establish the stability of polymyxin B3 in aqueous solution at room temperature and 60 degrees C, as well as different pH values (i.e. 1.4, 4.4 and 7.4). It was shown that the by-product, which is actually formed in the course of the purification of polymyxin B3 after evaporation in acidic medium, has a retention time similar to Ngamma-polymyxin B3. Acyl-migration occurred most rapidly at 60 degrees C and pH 7.4. Furthermore, it was established that migration of the acyl from the Nalpha- to the Ngamma-position of residue Dab1 is reversible and that the equilibrium seems to be in favor of the Nalpha-acylated compound.

Peptide conjugation of 2'-O-methyl phosphorothioate antisense oligonucleotides enhances cardiac uptake and exon skipping in mdx mice.

Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy that is currently being tested in various clinical trials. This approach is based on restoring the open reading frame of dystrophin transcripts resulting in shorter but partially functional dystrophin proteins as found in patients with Becker muscular dystrophy. After systemic administration, a large proportion of AONs ends up in the liver and kidneys. Therefore, enhancing AON uptake by skeletal and cardiac muscle would improve the AONs' therapeutic effect. For phosphorodiamidate morpholino oligomer, AONs use nonspecific positively charged cell penetrating peptides to enhance efficacy. However, this is challenging for negatively charged 2'-O-methyl phosphorothioate oligomer. Therefore, we screened a 7-mer phage display peptide library to identify muscle and heart homing peptides in vivo in the mdx mouse model and found a promising candidate peptide capable of binding muscle cells in vitro and in vivo. Upon systemic administration in dystrophic mdx mice, conjugation of a 2'-O-methyl phosphorothioate AON to this peptide indeed improved uptake in skeletal and cardiac muscle, and resulted in higher exon skipping levels with a significant difference in heart and diaphragm. Based on these results, peptide conjugation represents an interesting strategy to enhance the therapeutic effect of exon skipping with 2'-O-methyl phosphorothioate AONs for Duchenne muscular dystrophy.

Evaluation of the antibacterial spectrum of drosocin analogues.

Drosocin is a 19-mer, cationic antimicrobial peptide from Drosophila melanogaster. The aim of the study was to examine the antibacterial spectrum of unglycosylated drosocin analogues. Furthermore, the amino acid sequence of DnaK, drosocin's intracellular target, from susceptible species was aligned and studied for sequence homology. From this a panel of 31 bacterial strains, including Salmonella strains with truncated lipopolysaccharide structures, was tested for susceptibility towards three drosocin analogues. Available bacterial DnaK amino acid sequences were retrieved from the ExPASy proteomics server of the Swiss Institute of Bioinformatics studied for sequence homology. Seventeen of the 31 strains tested were susceptible for the drosocin analogues. Minimal inhibitory concentration values against mainly Gram-negative bacteria ranged from 3.1 to 100 microm. With the exception of Micrococcus luteus and Xanthomonas campestris all drosocin analogue-susceptible strains were Enterobacteriaceae showing a high DnaK amino acid sequence homology.

Biological evaluation of Tyr6 and Ser7 modified drosocin analogues.

An array of analogues of the cationic antimicrobial peptide drosocin was synthesized containing substitutions of Tyr6 and Ser7 in order to increase the proteolytic stability. Stabilizing the N-terminus with unnatural amino acids increased the serum stability of analogues by almost a factor 30 over an 8 h period.

Solid-phase synthesis of new saphenamycin analogues with antimicrobial activity.

An array of 12 new saphenamycin analogues modified at the benzoate moiety was synthesized on solid support. Synthesis commenced with a chemoselective anchoring of saphenic acid through the carboxyl group to a 2-chlorotrityl functionalized polystyrene resin. The secondary alcohol was acylated in parallel with a series of differently substituted benzoic acid derivatives. Treatment with TFA-CH(2)Cl(2) (5:995) released the expected saphenamycin analogues into solution. These new analogues were purified, characterized and screened for antimicrobial activity against Bacillus subtilis and Proteus mirabilis. Eight analogues exhibited MIC values against B. subtilis ranging from 0.07 to 3.93 microg/mL, comparable to the activities of previously reported saphenamycin analogues.