Feature Collection in Peptide Therapeutics: Current Applications and Future Directions.

The use of peptides in medicine began long ago with peptidic hormones [...

Oligonucleotides-Based Therapeutics.

This Special Issue of aims to outline nucleic-acid-based strategies that have emerged as tools to regulate specific gene expression and, more recently, as a new class of medicines [...

Anti-apoptotic peptide for long term cardioprotection in a mouse model of myocardial ischemia-reperfusion injury.

Reperfusion therapy during myocardial infarction (MI) leads to side effects called ischemia-reperfusion (IR) injury for which no treatment exists. While most studies have targeted the intrinsic apoptotic pathway to prevent IR injury with no successful clinical translation, we evidenced recently the potent cardioprotective effect of the anti-apoptotic Tat-DAXXp (TD) peptide targeting the FAS-dependent extrinsic pathway. The aim of the present study was to evaluate TD long term cardioprotective effects against IR injury in a MI mouse model.

In Vitro Assays to Assess Exon Skipping in Duchenne Muscular Dystrophy.

Cell-penetrating peptide (CPP)-mediated delivery of phosphorodiamidate morpholino oligomers (PMO) results in efficient exon skipping and has shown great promise as a potential therapy for Duchenne muscular dystrophy (DMD). However, large differences in efficiency have been observed between CPPs and in delivery to different tissues. Cellular trafficking has appeared to be an important determinant of activity.

Delivery of therapeutic oligonucleotides with cell penetrating peptides.

Oligonucleotide-based drugs have received considerable attention for their capacity to modulate gene expression very specifically and as a consequence they have found applications in the treatment of many human acquired or genetic diseases. Clinical translation has been often hampered by poor biodistribution, however. Cell-penetrating peptides (CPPs) appear as a possibility to increase the cellular delivery of non-permeant biomolecules such as nucleic acids.

Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells.

Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes.

Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency.

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.

Systemic delivery of BH4 anti-apoptotic peptide using CPPs prevents cardiac ischemia-reperfusion injuries in vivo.

There is an obvious need to develop pharmacological strategies to protect the heart in patients suffering from acute myocardial infarction. Apoptosis was evidenced as a main contributor of myocardial ischemia-reperfusion (IR) injury. Our cardioprotective strategy was based on the use of four cell penetrating peptides (CPP: Tat, (RXR)4, Bpep and Pip2b) which were conjugated to the BH4-peptide, derived from the BH4 domain of the Bcl-xL anti-apoptotic protein.

Cell-penetrating peptides-based strategies for the delivery of splice redirecting antisense oligonucleotides.

Progress in our understanding of the molecular pathogenesis of human malignancies has provided therapeutic targets amenable to oligonucleotide (ON)-based strategies. Antisense ON-mediated splicing regulation in particular offers promising prospects since the majority of human genes undergo alternative splicing and since splicing defects have been found in many diseases. However, their implementation has been hampered so far by the poor bioavailability of nucleic acids-based drugs.

Insights into the cellular trafficking of splice redirecting oligonucleotides complexed with chemically modified cell-penetrating peptides.

Conjugates of cell-penetrating peptides (CPP) and splice redirecting oligonucleotides (ON) display clinical potential as attested by in vivo experimentation in murine models of Duchenne muscular dystrophy. However, micromolar concentrations of these conjugates are required to obtain biologically relevant responses as a consequence of extensive endosomal sequestration following endocytosis. Recent work from our group has demonstrated that appending stearic acid to CPPs increases their efficiency and that the inclusion of pH titrable entities leads to further improvement.

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo.

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation.

Splice redirection as a convenient assay to monitor CPP-ON efficiency and mechanism.

Several strategies based on synthetic oligonucleotides (ON) have been proposed to control gene expression. As for most biomolecules, however, delivery has remained a major roadblock for in vivo applications. Conjugation of steric-block neutral DNA mimics, such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligonucleotides (PMO), to cell-penetrating peptides (CPP) has recently been proposed as a new delivery strategy.

Synthesis and splice-redirecting activity of branched, arginine-rich peptide dendrimer conjugates of peptide nucleic acid oligonucleotides.

Arginine-rich cell-penetrating peptides have found excellent utility in cell and in vivo models for enhancement of delivery of attached charge-neutral PNA or PMO oligonucleotides. We report the synthesis of dendrimeric peptides containing 2- or 4-branched arms each having one or more R-Ahx-R motifs and their disulfide conjugation to a PNA705 splice-redirecting oligonucleotide. Conjugates were assayed in a HeLa pLuc705 cell assay for luciferase up-regulation and splicing redirection.

A non-covalent strategy combining cationic lipids and CPPs to enhance the delivery of splice correcting oligonucleotides.

Modulation of pre-mRNA splicing by steric-block oligonucleotides constitutes a promising strategy for the treatment of many diseases, but requires efficient delivery to cell nuclei. In the present study, we evaluated the efficacy of a non-covalent strategy that combines a cell penetrating peptide with a lipoplex-based formulation to mediate the delivery of splice-switching oligonucleotides. The splice correcting ability of these new formulations was assessed using splice-switching oligonucleotides targeted towards the mutated splicing site of human beta-globin pre-mRNA in the HeLa pLuc/705 splice correction model.

Cell penetrating peptides: overview and applications to the delivery of oligonucleotides.

Crossing biological barriers represents a major limitation for clinical applications of biomolecules such as nucleic acids, peptides or proteins. Cell penetrating peptides (CPP), also named protein transduction domains, comprise short and usually basic amino acids-rich peptides originating from proteins able to cross biological barriers, such as the viral Tat protein, or are rationally designed. They have emerged as a new class of non-viral vectors allowing the delivery of various biomolecules across biological barriers from low molecular weight drugs to nanosized particles.

Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy.

In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery.

A peptide-based dendrimer that enhances the splice-redirecting activity of PNA conjugates in cells.

The full therapeutic potential of oligonucleotide (ON)-based agents has been hampered by cellular delivery challenges. Cell-penetrating peptides (CPP) represent promising delivery vectors for nucleic acids, and their potential has recently been evaluated using a functional splicing redirection assay, which capitalizes on the nuclear delivery of splice-correcting steric-block ON analogues such as peptide nucleic acids (PNA). Despite encouraging in vitro and in vivo data with arginine-rich CPP-steric block conjugates, mechanistic studies have shown that entrapment within the endosome/lysosome compartment after endocytosis remains a limiting factor.

Peptide-based delivery of steric-block PNA oligonucleotides.

Several strategies based on synthetic oligonucleotides (ON) have been proposed to control gene expression. As for most biomolecules, however, delivery has remained a major roadblock for in vivo applications. Conjugation of steric-block neutral DNA mimics such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligonucleotides (PMO) to cell penetrating peptides (CPP) has recently been proposed as a new delivery strategy.

Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle.

Steric blocking peptide nucleic acid (PNA) oligonucleotides have been used increasingly for redirecting RNA splicing particularly in therapeutic applications such as Duchenne muscular dystrophy (DMD). Covalent attachment of a cell-penetrating peptide helps to improve cell delivery of PNA. We have used a HeLa pLuc705 cell splicing redirection assay to develop a series of PNA internalization peptides (Pip) conjugated to an 18-mer PNA705 model oligonucleotide with higher activity compared to a PNA705 conjugate with a leading cell-penetrating peptide being developed for therapeutic use, (R-Ahx-R)(4).

Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies.

Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)(4) (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)(4)-PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy.

PNA-peptide conjugates as intracellular gene control agents.

Serum-stabilized PNA-internalization peptides (Pip) conjugated to PNA complementary to the 705 aberrant beta-globin splice site are able to correct splicing and increase luciferase production in Hela pLuc705 cells with sub microM EC(50) in the absence of a transfection agent. Inhibition of microRNA-122 in liver cells is achieved by treatment with complementary PNA containing just a few attached Lys residues, again without need of a transfection agent.