Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells

The trans-activation response (TAR) RNA stem–loop that occurs at the 5′ end of HIV RNA transcripts is an important antiviral target and is the site of interaction of the HIV-1 Tat protein together with host cellular factors. Oligonucleotides and their analogues targeted to TAR are potential antiviral candidates. We have investigated a range of cell penetrating peptide (CPP) conjugates of a 16mer peptide nucleic acid (PNA) analogue targeted to the apical stem–loop of TAR and show that disulfide-linked PNA conjugates of two types of CPP (Transportan or a novel chimeric peptide R(6)-Penetratin) exhibit dose-dependent inhibition of Tat-dependent trans-activation in a HeLa cell assay when incubated for 24 h. Activity is reached within 6 h if the lysosomotropic reagent chloroquine is co-administered. Fluorescein-labelled stably-linked conjugates of Tat, Transportan or Transportan TP10 with PNA were inactive when delivered alone, but attained trans-activation inhibition in the presence of chloroquine. Confocal microscopy showed that such fluorescently labelled CPP–PNA conjugates were sequestered in endosomal or membrane-bound compartments of HeLa cells, which varied in appearance depending on the CPP type. Co-administration of chloroquine was seen in some cases to release fluorescence from such compartments into the nucleus, but with different patterns depending on the CPP. The results show that CPP–PNA conjugates of different types can inhibit Tat-dependent trans-activation in HeLa cells and have potential for development as antiviral agents. Endosomal or membrane release is a major factor limiting nuclear delivery and trans-activation inhibition

Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide

Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5–10 μM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 μM concentration of the R6Pen–PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen–PNA705 structure–function relationship have also been evaluated

Optimisation des vecteurs peptidiques : application à la délivrance d'analogues d'oligonucléotides à visée thérapeutique (PNA et PMO)

Antisense oligonucleotides have a large therapeutic potential. However, the low effectiveness with which they cross biological membranes limits their clinical development. Many delivery strategies were proposed to circumvent this problem but the majority remain inadapted to an in vivo use. During this last decade, several peptides able to cross the plasma membrane were characterized. Gathered under the name of Cell Penetrating Peptides, these peptides are polycationic and sometimes amphipatic. Our work, using luciferase splice correction cells model, indicates that these CPPs and their conjugates to PNA or PMO remain blocked in endocytic vesicles. Endosomolytic agents, like chloroquine, promote the endosomal escape and improves the splice correcting efficency. It is now admitted that the development of new peptides vectors with an intrinsic endosomolytic property would constitute a major step in the field of the delivery. Two conjugates (R-Ahx-R)4-PMO and R6Pen-PNA, effectively correct splicing without addition of chloroquine. The mechanistic studies indicate that these conjugates are internalised in the cells by an endocytotic mechanism. The structure-activiy studies indicate a correlation between the affinity of CCP-ON to the heparan sulphate as well as theirs hydrophobicities and the effectiveness of correction. Work on the animals models showed a broad biodisponibility of (R-Ahx-R)4-RMO.Les oligonucléotides antisens possèdent un immense potentiel thérapeutique. Cependant, la faible efficacité avec laquelle ils traversent les membranes biologiques limite leur utilisation. De nombeuses stratégies de délivrances ont été proposées pour contourner ce problème mais la plupart restent peu adaptées à une utilisation in vivo. Durant cette dernière décennie, plusieurs peptides capables de traverser la membrane plasmique ont été caractérisés. Regroupés sour le nom de Cell Penetrating Peptide, ces peptides sont polycationiques et parfois amphipatiques. Nos travaux d'évaluation de ces CPPs dans le modèle cellulaire de correction d'épissage indiquent que ces vecteurs, couplé à des PNA ou PMO, restent bloqués dans les vésicules d'endocytose. L'utilisation d'agents endosomolytiques comme la chloroquine, libère ces conjugués améliorant ainsi l'efficacité de la correction d'épissage. D'une manière générale il est admis que le développement de nouveaux peptides vecteurs présentant une propriété endosomolytique intrinsèque constituerait une avancée majeure dans le domaine de la délivrance. Deux conjugués (R-Ahx-R)4-PMO et R6Pen-PNA corrigent efficacement l'épissage sans addition de chloroquine. Ces conjugués sont internalisés dans les cellules par un mécanisme endocytotique. Les études de structure activité ont indiqué une corrélation entre l'affinité des conjugués aux héparanes sulfates ainsi que de leur hydrophobicité et l'efficacité de correction. Les travaux sur les modèles animaux ont montré une large biodisponibilité du conjugé (R-Ahx-R)4-PMO. Nos collaborations continuent pour améliorer ces deux peptides de délivrance

Chemical modifications to improve the cellular uptake of oligonucleotides.

Specific control of gene expression by synthetic oligonucleotides (ON) is now widely used for target validation but clinical applications are limited by ON bioavailability. Moreover, most currently used strategies for physical and chemical delivery cannot be easily implemented in vivo. This article reviews new strategies which appear promising for ON delivery. The first part deals with ON chemical modifications aiming at improving cellular uptake as for instance the grafting of cationic groups on the ON backbone. The second part concerns ON conjugation to cell penetrating peptides

Lipoplex and peptide-based strategies for the delivery of steric-block oligonucleotides.

Synthetic oligonucleotides offer interesting prospects for the control of gene expression but clinical applications have been severely limited by their poor bioavailability. Cationic lipids have been widely used for the delivery of charged oligonucleotide (ON) analogues but most of the commercial formulations are toxic and poorly stable in the presence of serum proteins. We have developed a DOGS/DOPE liposome formulation named DLS (for delivery liposomal system), that allows for the efficient nuclear delivery of negatively charged antisense ON analogues as monitored by fluorescence microscopy and by their ability to correct deficient pre-mRNA splicing, even in serum-supplemented cell culture. Uncharged DNA mimics such as peptide nucleic acids (PNA), or phosphorodiamidate morpholino (PMO) ON are particularly interesting for their high metabolic stability and affinity for complementary RNA targets but they cannot be delivered with cationic lipids. Cell penetrating peptides (CPP), such as Tat or penetratin, have been used widely as conjugates for the delivery of various biomolecules and might be appropriate for neutral ON analogues. However, entrapment within endocytic vesicles severely limits the efficiency of PNA delivery by CPPs in the absence of endosomolytic drugs, such as chloroquine. The conjugation of new arginine-rich CPPs to PNA allows efficient nuclear delivery in the absence of chloroquine as monitored in a splicing correction assay. Both strategies have their advantages but DLS-mediated delivery remains more efficient than CPP delivery for the nuclear targeting of splice correcting ON analogues in vitro

Lipoplex and peptide-based strategies for the delivery of steric-block oligonucleotides.

Synthetic oligonucleotides offer interesting prospects for the control of gene expression but clinical applications have been severely limited by their poor bioavailability. Cationic lipids have been widely used for the delivery of charged oligonucleotide (ON) analogues but most of the commercial formulations are toxic and poorly stable in the presence of serum proteins. We have developed a DOGS/DOPE liposome formulation named DLS (for delivery liposomal system), that allows for the efficient nuclear delivery of negatively charged antisense ON analogues as monitored by fluorescence microscopy and by their ability to correct deficient pre-mRNA splicing, even in serum-supplemented cell culture. Uncharged DNA mimics such as peptide nucleic acids (PNA), or phosphorodiamidate morpholino (PMO) ON are particularly interesting for their high metabolic stability and affinity for complementary RNA targets but they cannot be delivered with cationic lipids. Cell penetrating peptides (CPP), such as Tat or penetratin, have been used widely as conjugates for the delivery of various biomolecules and might be appropriate for neutral ON analogues. However, entrapment within endocytic vesicles severely limits the efficiency of PNA delivery by CPPs in the absence of endosomolytic drugs, such as chloroquine. The conjugation of new arginine-rich CPPs to PNA allows efficient nuclear delivery in the absence of chloroquine as monitored in a splicing correction assay. Both strategies have their advantages but DLS-mediated delivery remains more efficient than CPP delivery for the nuclear targeting of splice correcting ON analogues in vitro

-activation inhibitory effects of disulfide-linked CPP–PNA conjugates – in the HeLa cell reporter assay with 6 h delivery () or 24 h ()

<p><b>Copyright information:</b></p><p>Taken from "Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent -activation in cells"</p><p>Nucleic Acids Research 2005;33(21):6837-6849.</p><p>Published online 30 Nov 2005</p><p>PMCID:PMC1301599.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Firefly luciferase activity represents Tat-TAR dependent expression whilst luciferase activity represents control constitutive expression. Bars (left to right) in each case represent 2.5, 1.25, 0.625, 0.312 and 0 µM CPP–PNA concentrations

Confocal microscopy images of the uptake of fluorescein (FAM)-labelled CPP–PNA conjugates when incubated for 5

<p><b>Copyright information:</b></p><p>Taken from "Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent -activation in cells"</p><p>Nucleic Acids Research 2005;33(21):6837-6849.</p><p>Published online 30 Nov 2005</p><p>PMCID:PMC1301599.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p>5 h with unfixed HeLa cells. Nuclei are stained red with hydroethidine. ( and ) Orange filter to view both the red colour of hydroethidine and the fluorescein fluorescence. ( and ) Green filter to view only the fluorescein fluorescence. (A and B) Show incubations in the absence of chloroquine, (C and D) Show incubations in the presence of 100 µM chloroquine. First line Tat–PNA (); second line Transportan–PNA (); third line NLS–PNA–Tat (); fourth line K–PNA (). In (C), second line, yellow dots are marked with arrows showing co-localization of hydroethidine dye and fluorescein fluorescence on the inner wall of the nucleus in several nuclei