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. Whereas 8-Arg branched peptide−PNA conjugates showed poor activity compared to a linear (R-Ahx-R)4−PNA conjugate, 2-branched and some 4-branched 12 and 16 Arg peptide−PNA conjugates showed activity similar to that of the corresponding linear peptide−PNA conjugates. Many of the 12- and 16-Arg conjugates retained significant activity in the presence of serum. Evidence showed that biological activity in HeLa pLuc705 cells of the PNA conjugates of branched and linear (R-Ahx-R) peptides is associated with an energy-dependent uptake pathway, predominantly clathrin-dependent, but also with some caveolae dependence

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. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure–activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs

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. We show that Pip–PNA705 conjugates are internalized in HeLa cells by an energy-dependent mechanism and that the predominant pathway of cell uptake of biologically active conjugate seems to be via clathrin-dependent endocytosis. In a mouse model of DMD, serum-stabilized Pip2a or Pip2b peptides conjugated to a 20-mer PNA (PNADMD) targeting the exon 23 mutation in the dystrophin gene showed strong exon-skipping activity in differentiated mdx mouse myotubes in culture in the absence of an added transfection agent at concentrations where naked PNADMD was inactive. Injection of Pip2a-PNADMD or Pip2b-PNADMD into the tibealis anterior muscles of mdx mice resulted in ∼3-fold higher numbers of dystrophin-positive fibres compared to naked PNADMD or (R-Ahx-R)4-PNADMD

Peptidic vectors for the delivery of oligonucleotides : design, mechanism of cellular internalization and applications to regulate splicing.

L'utilisation des oligonucléotides antisens PMO ou PNA, pour corriger les erreurs d'épissage par blocage stérique, constitue une nouvelle stratégie prometteuse pour réguler l'expression génétique. Ces ON peuvent mener au traitement de maladies comme la β-thalassémie, la dystrophie musculaire de Duchenne (DMD) ou les cancers. Cependant leur développement clinique requiert un système de délivrance efficace. Les peptides cationiques (CPPs) sont caractérisés par leur capacité à s'internaliser dans les cellules eucaryotes. Cependant leur efficacité à promouvoir la délivrance cytoplasmique et nucléaire des ON est limitée par leur séquestration dans des vésicules d'endocytose, ce qui est à l'origine de la dégradation du matériel internalisé. Nous avons contribué à l'étude du trafic intracellulaire et de l'activité dans un essai de correction d'épissage de plusieurs familles de CPPs capables de délivrer efficacement des analogues d'ON à des doses non toxiques et en absence d'agents endosomolytiques. Nos études mécanistiques indiquent que ces constructions (covalentes ou non covalentes) CPP-ON sont endocytées par la voie clathrine, que la ségrégation dans les endosomes reste une limitation et qu'il existe une bonne corrélation entre leur activité biologique et leur capacité à déstabiliser les membranes endosomales.The use of antisense oligonucleotides PMO or PNA to correct splicing errors by steric- block represents a new promising therapeutic strategy. These ONs lead to the treatment of diseases such as β-thalassemia, Duchenne muscular dystrophy (DMD) or cancers. However their functional success requires efficient delivery. Cationic cell penetrating peptides (CPPs) are characterized by their ability to be internalized in eukaryotic cells. However their efficiency in promoting cytoplasmic and nuclear delivery of ON has been hampered by endocytic sequestration and subsequent degradation of internalized material in endocytic vesicles, which is responsible for the degradation of internalized material. We have contributed to the study of intracellular trafficking and activity (using splicing correction assay) of several families of CPPs capable of delivering effective analogs ON at nontoxic doses and in the absence of agents endosomolytic. Our mechanistic studies indicate that these constructs (covalent or noncovalent) CPP-ON are internalized through clathrin, that segregation in endosomes remains a limitation and that there is good correlation between biological activity and their ability to destabilize endosomal membranes

Vecteurs peptidiques pour la délivrance d'oligonucléotides (conception, mécanisme d'internalisation cellulaire et applications à la régulation de l'épissage.)

L'utilisation des oligonucléotides antisens PMO ou PNA, pour corriger les erreurs d'épissage par blocage stérique, constitue une nouvelle stratégie prometteuse pour réguler l'expression génétique. Ces ON peuvent mener au traitement de maladies comme la b-thalassémie, la dystrophie musculaire de Duchenne (DMD) ou les cancers. Cependant leur développement clinique requiert un système de délivrance efficace. Les peptides cationiques (CPPs) sont caractérisés par leur capacité à s'internaliser dans les cellules eucaryotes. Cependant leur efficacité à promouvoir la délivrance cytoplasmique et nucléaire des ON est limitée par leur séquestration dans des vésicules d'endocytose, ce qui est à l'origine de la dégradation du matériel internalisé. Nous avons contribué à l'étude du trafic intracellulaire et de l'activité dans un essai de correction d'épissage de plusieurs familles de CPPs capables de délivrer efficacement des analogues d'ON à des doses non toxiques et en absence d'agents endosomolytiques. Nos études mécanistiques indiquent que ces constructions (covalentes ou non covalentes) CPP-ON sont endocytées par la voie clathrine, que la ségrégation dans les endosomes reste une limitation et qu'il existe une bonne corrélation entre leur activité biologique et leur capacité à déstabiliser les membranes endosomales.The use of antisense oligonucleotides PMO or PNA to correct splicing errors by steric- block represents a new promising therapeutic strategy. These ONs lead to the treatment of diseases such as b-thalassemia, Duchenne muscular dystrophy (DMD) or cancers. However their functional success requires efficient delivery. Cationic cell penetrating peptides (CPPs) are characterized by their ability to be internalized in eukaryotic cells. However their efficiency in promoting cytoplasmic and nuclear delivery of ON has been hampered by endocytic sequestration and subsequent degradation of internalized material in endocytic vesicles, which is responsible for the degradation of internalized material. We have contributed to the study of intracellular trafficking and activity (using splicing correction assay) of several families of CPPs capable of delivering effective analogs ON at nontoxic doses and in the absence of agents endosomolytic. Our mechanistic studies indicate that these constructs (covalent or noncovalent) CPP-ON are internalized through clathrin, that segregation in endosomes remains a limitation and that there is good correlation between biological activity and their ability to destabilize endosomal membranes.MONTPELLIER-BU Pharmacie (341722105) / SudocSudocFranceF

Cell penetrating peptide conjugates of steric block oligonucleotides.

Charge neutral steric block oligonucleotide analogues, such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO), have promising biological and pharmacological properties for antisense applications, such as for example in mRNA splicing redirection. However, cellular uptake of free oligomers is poor and the utility of conjugates of PNA or PMO to cell penetrating peptides (CPP), such as Tat or Penetratin, is limited by endosomal sequestration. Two new families of arginine-rich CPPs named (R-Ahx-R)(4) AhxB and R(6)Pen allow efficient nuclear delivery of splice correcting PNA and PMO at micromolar concentrations in the absence of endosomolytic agents. The in vivo efficacy of (R-Ahx-R)(4) AhxB PMO conjugates has been demonstrated in mouse models of Duchenne muscular dystrophy and in various viral infections

PNA-peptide conjugates as intracellular gene control agents.

International audienceSerum-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

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. Whereas 8-Arg branched peptide−PNA conjugates showed poor activity compared to a linear (R-Ahx-R)₄−PNA conjugate, 2-branched and some 4-branched 12 and 16 Arg peptide−PNA conjugates showed activity similar to that of the corresponding linear peptide−PNA conjugates. Many of the 12- and 16-Arg conjugates retained significant activity in the presence of serum. Evidence showed that biological activity in HeLa pLuc705 cells of the PNA conjugates of branched and linear (R-Ahx-R) peptides is associated with an energy-dependent uptake pathway, predominantly clathrin-dependent, but also with some caveolae dependence