Exon-skipping oligonucleotides are a well-researched therapeutic strategy for Duchenne's muscular dystrophy (DMD). Despite remarkable successes in animal models with intramuscular and intravenous delivery of unmodified oligonucleotides, the ability to specifically target both normal and dystrophic muscle with a simple peptide ligand could decrease the therapeutic dose required and reduce the potential for toxicity. Thus, 3 rounds of in vivo phage display utilizing a 12-mer peptide library were performed with mdx mice and a peptide motif with potential for targeting to muscle but not liver was identified. This motif was shown to have enhanced binding affinity to C2C12 myoblasts over a scrambled control peptide and in vivo application of a fluorescein-labeled peptide containing the identified motif resulted in increased specificity for the heart and quadriceps muscle after tail-vein administration in C57BL/6 mice. This work has many potential applications for oligonucleotide or drug delivery to muscle for myopathies.
"In line with the above observations, no enhanced exon-skipping activity could be detected when a nonmuscle targeting control peptide, derived from our phage display screen as a negative control and showing no binding affinity with muscle and heart16 (Table 1), was inserted into the chimeric peptide, in place of MSP. Consistently, compared with B-PMO as we reported earlier,14 this nonmuscle targeting control chimeric peptide-PMO conjugate was less effective than B-PMO in either orientation (Supplementary Figure 1, online), suggesting that the addition of nonmuscle targeting peptides partially negated the cell-penetrating property of the B peptide, which is likely due to the steric hindrance caused by the additional nonmuscle targeting peptide by preventing effective membrane contact of the CPP moiety. "
[Show abstract][Hide abstract] ABSTRACT: We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.
"These researchers recently reported long-term prevention (7 months) of cardiac dysfunction after early treatment with a CPP–PMO.139 Other CPPs for enhanced PMO delivery have been reported,141,150,151 and this promises to be a most active area of research. Although many CPPs examined to date greatly enhance PMO uptake, concerns remain regarding the clinical applicability of these compounds due to potential adverse effects. "
[Show abstract][Hide abstract] ABSTRACT: The identification of dystrophin and the causative role of mutations in this gene in Duchenne and Becker muscular dystrophies (D/BMD) was expected to lead to timely development of effective therapies. Despite over 20 years of research, corticosteroids remain the only available pharmacological treatment for DMD, although significant benefits and extended life have resulted from advances in the clinical care and management of DMD individuals. Effective treatment of DMD will require dystrophin restitution in skeletal, cardiac, and smooth muscles and nonmuscle tissues; however, modulation of muscle loss and regeneration has the potential to play an important role in altering the natural history of DMD, particularly in combination with other treatments. Emerging biological, molecular, and small molecule therapeutics are showing promise in ameliorating this devastating disease, and it is anticipated that regulatory environments will need to display some flexibility in order to accommodate the new treatment paradigms.
The Application of Clinical Genetics 03/2011; 4:29-44. DOI:10.2147/TACG.S8762
[Show abstract][Hide abstract] ABSTRACT: Most cancer chemotherapies cause severe side effects due mainly to the toxicity of the drugs to normal healthy cells. Conventional drugs lack satisfactory specificity towards cancer cells, and have difficulties overcoming biological barriers; this highlights the importance of developing safe and effective delivery systems. Opportunities for the design of intelligent drug delivery vehicles are provided by the unique physiological properties of tumor tissues and the overexpression of specific receptors in tumor cells and neovasculature. These delivery vehicles include peptides, polymers and lipids. Targeting ligands, such as cell targeting peptides, have been used to decorate delivery vehicles in order to guide the therapeutic or diagnostic agents to tumor sites and enhance their efficacy. Recently, attention has been drawn to bacteria-mediated cancer therapy. This approach targets the tumor environment by taking advantage of the wide flexibility of bacterial genetics. This chapter focuses on recent progress indrug delivery approaches in cancer therapy.
Biomedical Materials and Diagnostic Devices, Edited by Ashutosh Tiwari, Murugan Ramalingam, Hisatoshi Kobayashi, Anthony P.F. Turner, 10/2012: chapter 15: pages 477-513; Scrivener publishing LLC., ISBN: 978-1-1180-3014-1
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