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.

Phage display screening without repetitious selection rounds.

Phage display screenings are frequently employed to identify high-affinity peptides or antibodies. Although successful, phage display is a laborious technology and is notorious for identification of false positive hits. To accelerate and improve the selection process, we have employed Illumina next generation sequencing to deeply characterize the Ph.D.-7 M13 peptide phage display library before and after several rounds of biopanning on KS483 osteoblast cells. Sequencing of the naive library after one round of amplification in bacteria identifies propagation advantage as an important source of false positive hits. Most important, our data show that deep sequencing of the phage pool after a first round of biopanning is already sufficient to identify positive phages. Whereas traditional sequencing of a limited number of clones after one or two rounds of selection is uninformative, the required additional rounds of biopanning are associated with the risk of losing promising clones propagating slower than nonbinding phages. Confocal and live cell imaging confirms that our screen successfully selected a peptide with very high binding and uptake in osteoblasts. We conclude that next generation sequencing can significantly empower phage display screenings by accelerating the finding of specific binders and restraining the number of false positive hits.

Antimicrobial Peptide Omiganan Enhances Interferon Responses to Endosomal Toll-Like Receptor Ligands in Human Peripheral Blood Mononuclear Cells.

LL-37 is a cationic antimicrobial peptide and the sole human member of cathelicidins. Besides its bactericidal properties, LL-37 is known to have direct immunomodulatory effects, among which enhancement of antiviral responses via endosomal toll-like receptors (TLRs). Omiganan pentahydrochloride is a synthetic cationic peptide in clinical development. Previously, omiganan was primarily known for its direct bactericidal and antifungal properties. We investigated whether omiganan enhances endosomal TLR responses, similar to LL-37. Human peripheral blood mononuclear cells were treated with endosomal TLR3, -7, -8, and -9 ligands in the presence of omiganan. Omiganan enhanced TLR-mediated interferon-α release. Subsequent experiments with TLR9 ligands showed that plasmacytoid dendritic cells were main contributors to omiganan-enhanced IFN production. Based on this type I interferon-enhancing effect, omiganan may qualify as potential treatment modality for virus-driven diseases. The molecular mechanism by which omiganan enhances endosomal TLR responses remains to be elucidated.

Host genetics and tumor environment determine the functional impact of neutrophils in mouse tumor models.

BACKGROUND: Neutrophils have been reported to have protumor, antitumor or neutral effects in cancer progression. The underlying causes for this functional variability are not clear. METHODS: We studied the role of neutrophils in six different mouse tumor models by intratumoral injection of antimicrobial peptides or vaccination. Changes in systemic and intratumoral immune cells were analyzed by flow-cytometry and mass-cytometry. The role of neutrophils was studied by antibody-mediated neutrophil depletion. Neutrophils from different mouse strains were compared by RNA sequencing. RESULTS: The antimicrobial peptide Omiganan reduced the growth of TC-1 tumors in BL/6 mice and CT26 tumors in BALB/c mice. No significant effects were observed in B16F10, MC38 and 4T1 tumors. Growth delay was associated with increased abundance of neutrophils in TC-1 but not CT26 tumors. Systemic neutrophil depletion abrogated Omiganan efficacy in TC-1 but further reduced growth of CT26, indicating that neutrophils were required for the antitumor effect in TC-1 but suppressed tumor control in CT26. Neutrophils were also required for a therapeutic vaccine-induced T-cell mediated control of RMA tumors in BL/6 mice. Clearly, the circulating and intratumoral neutrophils differed in the expression of Ly6G and CD62L, between TC-1 and CT26 and between blood neutrophils of tumor-naïve BL/6 and BALB/c mice. RNA-sequencing revealed that neutrophils from BL/6 mice but not BALB/c mice displayed a robust profile of immune activation, matching their opposing roles in TC-1 and RMA versus CT26. CONCLUSIONS: Neutrophil functionality differs strongly between mouse strains and tumor types, with consequences for tumor progression and therapy.

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.

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.