The Usefulness of Determining Plasma and Tissue Concentrations of Phosphorodiamidate Morpholino Oligonucleotides to Estimate Their Efficacy in Duchenne Muscular Dystrophy Patients.

Currently, four kinds of phosphorodiamidate morpholino oligomers (PMOs), such as viltolarsen, have been approved for the treatment of Duchenne muscular dystrophy (DMD); however, it is unclear whether human efficacy can be estimated using plasma concentrations. This study summarizes the tissue distribution of viltolarsen in mice and cynomolgus monkeys and evaluates the relationship between exposure and efficacy based on exon skipping. In the tissue distribution studies, all muscles in DMD-model mice showed higher concentrations of viltolarsen than those in wild-type mice and cynomolgus monkeys, and the concentrations in skeletal muscle were correlated with the exon-skipping efficiency in mice and cynomolgus monkeys. In addition, a highly sensitive bioanalytical method using liquid chromatography with tandem mass spectrometry shows promise for determining plasma concentrations up to a later time point, and the tissue (muscle)/plasma concentration ratio (Kp) in DMD-model mice was shown to be useful for predicting changes in pharmacodynamic (PD) markers in humans. Our results suggest that pharmacokinetic (PK)/PD analysis can be conducted by using the human PK profile or Kp values and skipping efficiency in DMD-model mice. This information will be useful for the efficient and effective development of PMOs as therapeutic agents. SIGNIFICANCE STATEMENT: We evaluated the relationship between the plasma or tissue concentrations and the efficiency of exon skipping for viltolarsen as an example phosphorodiamidate morpholino oligomers in the skeletal and cardiac muscle of mice and cynomolgus monkeys for pharmacokinetic/pharmacodynamic (PK/PD) analysis. The results suggest that PK/PD analysis can be conducted by using the human PK profile or tissue (muscle)/plasma concentration ratios and skipping efficiency in DMD-model mice.

Cell fate conversion by conditionally switching the signal-transducing domain of signalobodies.

Conditionally and strictly controlling cell fates is important for biomedical applications including cell therapies. Although previous studies have been based on regulating the expression or activation of signaling molecules, the techniques therein require improvement in terms of reducing leakiness and complexity. In this study, we propose a novel cell fate converting system using our previously developed antibody/receptor chimeras named "signalobodies" in combination with a Cre/loxP recombination system. We designed a "switch vector" where a growth signalobody gene was flanked by two loxP sites and a death signalobody gene was placed downstream of the floxed cassette. Cells transduced with the switch vector showed superior growth activity in the presence of a specific antigen. Subsequent expression of Cre induced the death signalobody, leading to conditional cell death. This technology could be applicable for other cell fate conversion systems including differentiation and migration, by using appropriate signal-transducing domains.

Exon 44 skipping in Duchenne muscular dystrophy: NS-089/NCNP-02, a dual-targeting antisense oligonucleotide.

Exon-skipping therapy mediated by antisense oligonucleotides is expected to provide a therapeutic option for Duchenne muscular dystrophy. Antisense oligonucleotides for exon skipping reported so far target a single continuous sequence in or around the target exon. In the present study, we investigated antisense oligonucleotides for exon 44 skipping (applicable to approximately 6% of all Duchenne muscular dystrophy patients) to improve activity by using a novel antisense oligonucleotide design incorporating two connected sequences. Phosphorodiamidate morpholino oligomers targeting two separate sequences in exon 44 were created to target two splicing regulators in exon 44 simultaneously, and their exon 44 skipping was measured. NS-089/NCNP-02 showed the highest skipping activity among the oligomers. NS-089/NCNP-02 also induced exon 44 skipping and dystrophin protein expression in cells from a Duchenne muscular dystrophy patient to whom exon 44 skipping is applicable. We also assessed the in vivo activity of NS-089/NCNP-02 by intravenous administration to cynomolgus monkeys. NS-089/NCNP-02 induced exon 44 skipping in skeletal and cardiac muscle of cynomolgus monkeys. In conclusion, NS-089/NCNP-02, an antisense oligonucleotide with a novel connected-sequence design, showed highly efficient exon skipping both in vitro and in vivo.

Immortalized Canine Dystrophic Myoblast Cell Lines for Development of Peptide-Conjugated Splice-Switching Oligonucleotides.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.

Death signalobody: inducing conditional cell death in response to a specific antigen.

As the possibility of tumorigenesis and undesirable immune responses in patients cannot be completely excluded in gene and cell therapies, a conditional death switch to eliminate the therapeutic cells would be a valuable tool to enhance the safety of these therapies. A few ligand-receptor conditional death switches have already been developed; however, they cannot be used if patients exhibit side effects upon administration of the ligand. Here we demonstrate a death-inducing chimeric antibody named "death signalobody," in which the antigen-antibody system, having virtually infinite ligand-receptor combinations, is utilized for the activation of death signaling. We designed a death signalobody named "SFas," which has an antifluorescein single-chain variable fragment and the cytoplasmic domain of Fas. SFas efficiently induced conditional apoptosis in murine pro-B Ba/F3 cells in response to fluorescein-conjugated bovine serum albumin. Moreover, SFas was also able to induce antigen-dependent conditional apoptosis in human cancer cell lines. The death signalobody technique will be a valuable tool for the conditional elimination of cells of interest in multiple therapeutic applications.