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Target specific delivery systems for gene therapy based on cell penetrating peptides

Leistungen

To understand the role of RNA-binding proteins (RBPs) in the regulation of gene expression, methods are needed for the in vivo identification of RNA-protein interactions. We have developed the peptide nucleic acid (PNA)-assisted identification of RBP technology to enable the identification of proteins that complex with a target RNA in vivo. Specific regions of the 3' and 5' UTRs of ankylosis mRNA were targeted by anti-sense PNAs transported into cortical neurons by the cell-penetrating peptide transportan 10. An array of proteins was isolated in complex with or near the targeted regions of the ankylosis mRNA through UV-induced cross-linking of the annealed PNA-RNA-RBP complex. The first evidence for pharmacological modulation of these specific protein-RNA associations was observed. These data show that the PNA-assisted identification of the RBP technique is a reliable method to rapidly identify proteins interacting in vivo with the target RNA.
We examined a number of cell penetrating peptides (CPPs) for their ability to deliver functional biological cargo (short peptides and siRNA anti-sense) in vitro in a range of different cell types. In addition, in order to assess the suitability of different CPPs for biological studies we also looked at cellular toxicity. In all we examined the following CPPs -TAT, D-TAT, TAT(+ K), antennapedia, transportan and polyarginine. With the attachment of a short peptide cargo we examined both the uptake and cytotoxicity in HeLa, A549 and CHO K1 cells. Based on uptake and cytotoxicity our results showed that both TAT and antennapedia were the "best" CPPs for peptide delivery and that rate and amount of uptake was largely independent of cell type. Using TAT and antennapedia CPPs we were also able to deliver functional siRNA anti-sense material both in vitro and in vivo. Firstly, in vitro using TAT-siRNA conjugates directed against p38 MAPK we achieved 80% and 50% knockdown in human and mouse cell lines respectively. Both TAT and antennapedia siRNA conjugates also showed functional activity in vivo, lowering the endogenous level of p38 in the mouse lung by 40% following intra-tracheal administration. The demonstration of the application of CPPs for the cellular delivery of biological cargo is potentially of great interest for the future utility of alternative non-viral delivery systems both for in vitro and in vivo target validation studies and future therapeutics.
The localisation of CPPs and transduced cargoes in cells after internalisation has not been unambiguously defined despite of numerous studies by fluorescence techniques. We characterised the cellular whereabouts of CPPs and delivered cargo protein molecules in the cell interior by using electron (and also fluorescence) microscopy. The transport peptides concentrate on the plasma membrane and typically to microvillar or filopodial structures that seem to mediate first association. In cells interior CPPs show wide distribution, localising to endocytotic vesicles but also in cytoplasm, Golgi region and to some extent in nuclei and mitochondria. Protein molecules coupled to transport peptide non-covalently accumulate on the cell surface before entering cells. In analogy with CPPs, the first association of delivered protein occurs often at cell-surface extensions. CPP-protein complexes are taken up by cells mostly in vesicular structures that differ by size and morphology. CPP-protein constructs at high concentration induce formation of large endosomes reminiscent macropinosomes and constructs are confined to vesicular structures mostly. At lower CPP-protein constructs concentrations small vesicles pinch off from the plasma membrane into cells and the complexes are detected in the cytoplasm and Golgi region outside vesicles.
Cell-penetrating peptides (CPP) have served as efficient vectors for translocation of hydrophilic molecules into living cells. The assays for selection of peptides with cell-penetrating ability have been laborious and we optimised these for using in HTS. Translocation of fluorescein-labelled peptides into cells was screened by the emission signal of cells in 96-well plates after quenching the extra-cellular fluorescence of not-internalised peptides. For the fast screening of unlabelled peptides we designed and optimised an assay based on the changes in the cellular uptake of protein-binding fluorescent dyes upon the internalisation of CPPs. The peptides with CPP properties were further characterised for the ability to carry into cell interior cargo molecules using similar assays. The cargoes were coupled to transport peptide non-covalently, by forming a complex between biotin-tagged CPP and labelled avidin/streptavidin. The protein was quantified in cells by the label of protein, a fluorescent dye or enzyme activity, and all studied CPP showed protein delivery activity. Few CPPs were even capable of forming a stable complex with cargo (protein, oligonucleotide, plasmid DNA) without using biotin-avidin interaction, and subsequent cellular delivery. The specificity of CPP-mediated delivery in vivo could be further characterised by using Cre-recombinase (coupled to peptides covalently or non-covalently) in respective reporter mice.
Cell-penetrating peptides (CPPs) have proven themselves as valuable vectors for intracellular delivery. Relatively little is known about the frequency of cell-penetrating sequences in native proteins and their functional role. By computational comparison of peptide sequences, we predicted that intracellular loops of G-protein coupled receptors (GPCR) have high probability for occurrence of cell-penetrating motifs. Since the loops are also receptor and G-protein interaction sites, we postulated that the short CPPs, derived from GPCR, when applied extra-cellularly can pass the membrane and modulate G-protein activity similarly to parent receptor proteins. Two model systems were analysed as proofs of the principle. A peptide called M511, based on the C-terminal intracellular sequence of the rat angiotensin receptor (AT1AR) internalized into live cells and elicited blood vessel contraction even in the presence of AT1AR antagonist. M511 interacted with the same selectivity towards G-protein subtypes as agonist-activated AT1AR and blockade of phospholipase C abolished its effect. Another CPP, G53-2, derived from human glucagon-like peptide receptor (GLP-1R) induced insulin release from isolated pancreatic islets. The mechanism was again found to be shared with the original GLP-1R, namely G11-mediated inositol 1,4,5-triphosphate release pathway. These data reveal a novel possibility to mimic the effects of signalling transmembrane proteins by application of shorter peptide fragments.
Cellular epithelia are major barriers restricting the diffusion of molecules throughout the body. The present result describes a peptide that uses unconventional mechanism of transfer across an epithelium. The potential application is to allow the transfer of biologically active molecules linked to this peptide across an epithelium.
Cell penetrating peptides (CPP) have been intensively studied as delivery vectors for various bio-molecules. However, their cellular internalisation mainly occurs through endocytosis and leads to sequestration within endocytic vesicles. It is now recognized, largely though our own studies, that escape from endosomes is the limiting factor for the efficient delivery of bio-molecules as peptides or nucleic acids. This is an important issue knowing the potential applications of these bio-molecules in bio-technomogy. In keeping with this hypothesis, endosomolytic agents as chloroquine largely increase the nuclear delivery of oligonucleotide analogs (2OMe,PNA,PMO) cojugated to CPPs. Ongoing studies in collaboration with the groups of Dr.M.Gait in this consortium and Dr.H.Moulton (AVI Biopharma) are focusing on new CPPs which allow the nuclear delivery of conjugated oligonucleotides in the absence of endosomolytic agents. We have identified at least two such CPP derivatives, which are now the object of studies in terms of mechanism of cell internalisation and are subject to SAR studies.
In order to gain insight into structural requirements for CPPs, we synthesized peptides showing systematically altered structural properties, charge and size, derived from the -helical model peptide KLALK LALKA LKAAL KLA-NH2 (KLA). Moreover, a set of modified ApoE-derived peptides has been synthesized and studied with regard to cellular uptake. Beside the synthesis of intracellularly cleavable disulfide-linked CPP-cargo conjugates, several CPP-PNA conjugates were synthesised by a novel enzyme-mediated ligation strategy, resulting in stable amide bond links between CPP and cargo. This newly developed methodology has been proved to be very useful in the preparation of highly pure and well-characterized conjugates. Uptake studies using different cell cultures and different analytical tools demonstrated that most of the cargo-CPP conjugates possess a significant intracellular accumulation. However, the intracellular concentration is seemingly strongly influenced not only by the uptake behaviour, but also by the export, which was found to be significant e. g. for acidic peptides, whereas strongly basic and amphipathic peptides are retained inside the cells, probably by binding to intracellular proteins and/or oligonucleotides. For assessing the influence of novel CPP-conjugations on the biological activity of PNAs, we measured the influences of the pre-treatment of spontaneously beating neonatal rat cardiomyocytes (CM) with PNA-derivatives, complementary to bases 12-23 of the translated region of the nociceptin/orphanin FQ receptor, on the positive chronotropic effect exerted by the neuropeptide nociceptin. In the second case the splice-correction assay developed by Kole et al. was used in collaboration with Lebleu's group. In both assays we achieved anti-sense effects. Corresponding conjugates with scrambled PNA-sequences proved inactive, thus supporting the anticipated anti-sense mechanism.
Cell Penetrating peptides are small peptidic sequences that, upon fusion, allow the cellular uptake of cargo molecules bearing intracellular biological activity. The characterization of a growing number of peptides with potential CPP activity needs to develop quantitative procedures to assess their actual CPP activity. The present result is a screening procedure to evaluate the efficiency of Cell Penetrating Peptides (CPPs) as vectors for intracellular protein delivery. Intracellular delivery is monitored by the intracellular recombination activity of exogenously added CRE recombinase (the cargo) fused to the tested CPP (the vector). In a dedicated engineered stable cell line, CRE-dependent recombination induces the permanent expression of a reporter protein (Beta-Galactosidase). The screen first consists in a 96 well format procedure allowing the accurate quantification of the intracellular delivery of CRE:CPP fusion protein in the reporter cell line. It is completed by a more physiologically relevant model based on embryonic brain explant culture from transgenic mice, to address the cellular tropism of the CRE:CPP fusion proteins. The screening has been validated with different CRE:CPP fusion proteins and will be improved with small scale production of recombinant proteins.
New chemistry methods were developed for the conjugation of CPPs to a range of to gene modulating steric blocking anti-sense oligonucleotides, PNA and short interference RNA, aimed at obviating the need for cell transfection agents. CPP conjugation often enhanced uptake of such cargoes into cultured cells but uptake amount varied depending on the types of CPP and cargo. Conjugates were found predominantly in endosomal or membrane bound compartments. Steric block cargoes were tested in a model HIV-1 Tat-dependent trans-activation system, where activity is attained only if the cargo enters the cell nucleus and binds an RNA target (TAR). Activity was found only for certain disulfide linked PNA-CPP conjugates delivered for 24h at µM concentrations, demonstrating that release into cytosol and nucleus is extremely difficult and structure-dependent. Activities could often be enhanced by co-administration of the endosomal release reagent chloroquine. CPPs disulfide-linked to siRNA in some cases were able to elicit reductions in expression of target p38 MAP kinase when incubated at µM concentrations with cells. The results are not yet advanced enough for commercial or medical application, but will guide future research into a) PNA-peptide conjugates for gene correction of splicing and as potential antiviral agents, and b) cell and mouse lung delivery of siRNA.
We reassessed the highly controversial issue of direct permeation of strongly cationic peptides across negatively charged lipid membranes. The microscopic studies of rhodamine-labeled giant vesicles incubated with carboxyfluorescein-labeled penetratin yielded no evidence of transbilayer movement. We have also exploited isothermal titration calorimetry and intrinsic tryptophan fluorescence spectroscopy to study the thermodynamics of penetratin partitioning into and translocation across unilamellar-vesicle membranes. A combination of uptake, release, and dilution experiments consistently demonstrated that, at low peptide concentrations, penetratin is not able to cross phospholipid bilayers, thus suggesting that transmembrane diffusion cannot be responsible for its cellular internalisation. Novel dialysis experiments and isothermal titration calorimetry were found to be fast, reliable, and versatile tools for assessing membrane translocation also of other charged compounds, thereby eliminating the need for specific reporter groups.
We introduced and characterised the novel chimerical lipopeptide P2A2, which is a highly promising candidate for drug delivery from micellar and liposomal nanoparticles owing to its straightforward synthesis, its unique physicochemical properties and its adaptability to a broad spectrum of targets. P2A2 consists of a strongly cationic, ApoE-based peptide, which is able to overcome cell membranes, and two palmitoyl chains promote detergent-like aggregation and partitioning into lipid bilayers. Biophysical experiments revealed that the lipopeptide self-assembled into small micelles comprising about 20 monomers. P2A2-lipid interactions were analysed and yielded a phase diagram permitting mastery of the colloidal state and thus rendering both micellar as well as liposomal formulations available for drug delivery.