The fundamental element of life known to man is the gene. The information contained in genes regulates all cellular functions, in health and disease. The ability to selectively alter genes or their transcript intermediates with designed molecular tools, as synthetic oligonucleotides, represents a paradigm shift in human medicine. The full potential of oligonucleotide therapeutics is however dependent on the development of efficient delivery vectors, due to their intrinsic characteristics, as size, charge and low bioavailability. Cell-penetrating peptides are short sequences of amino acids that are capable of mediating the transport of most types of oligonucleotide therapeutics to the cell interior. It is the interaction of cell-penetrating peptides with oligonucleotides and the transport of their non-covalently formed complexes across the cellular membrane, that constitutes the main subject of this thesis. In Paper I we studied the effects of different types of oligonucleotide cargo in the capacity of cationic and amphipathic peptides to interact with lipid membranes. We found that indeed the cargo sequesters some of the peptide’s capacity to interact with membranes. In Paper II we revealed the simultaneous interaction of different molecular and supramolecular peptide and peptide/oligonucleotide species in equilibrium, with the cellular membrane. In Paper III we developed a series of peptides with improved affinity for oligonucleotide cargo as well as enhanced endosomal release and consequently better delivery capacity. In Paper IV we investigated the effect of saturated fatty acid modifications to a cationic cell-penetrating peptide. The varying amphipathicity of the peptide correlated with the complex physicochemical properties and with its delivery efficiency. This thesis contributes to the field with a set of characterized mechanisms and physicochemical properties for the components of the ternary system – cell-penetrating peptide, oligonucleotide and cell membrane – that should be considered for the future development of gene therapy. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Glioblastoma multiforme is the most aggressive form of malignant brain tumor with poor prognosis. The efficacy of brain cancer treatment by chemotherapeutics is limited by the blood-brain barrier (BBB) which allows less than 2% of the small molecules and blocks almost all the macromolecules to transport into the brain. Delivery of the large molecules such as proteins and nucleic acids across the BBB is a great challenge for brain-targeted drug delivery. To overcome this obstacle, cell-penetrating peptides (CPPs) were used as vectors for delivery of nucleic acids across the BBB targeting glioblastomas. The CPPs have shown such promising carriers to deliver various cargoes ranging from small molecules to large molecules into the cells. This thesis is focused on the development of glioblastoma-targeting vectors based on modifications of the CPPs and the targeting peptides. The peptide-based vectors were developed to improve the transport of the nucleic acids across the BBB and specifically target glioblastomas. In this thesis, a series of peptide-based vectors targeting glioblastomas were synthesized and modified with targeting peptides by either covalent conjugation or non-covalent complex formation. The delivery of plasmid DNA (pDNA) in the complex with the peptide-based vectors was studied in the in vitro model of the BBB. The role of receptors expressed on the BBB was investigated. Scavenger receptors class A and B were found to be expressed on the BBB, and they were involved in the delivery of the pDNA across the BBB model. Moreover, various targeting peptides were modified with hexaglutamate to form non-covalent complexes with the CPPs for small interfering RNA (siRNA) delivery to glioblastoma cells. The non-covalent complex of the CPP and the targeting peptide showed greater gene-silencing efficiency than the consecutively covalent conjugation of the CPP and the targeting peptide for siRNA delivery to glioblastoma cells. Lastly, a number of novel, amphipathic peptides were developed based on the model amphipathic peptide. The prediction of the biological effect of the designed peptides using quantitative structure-activity relationship model showed a correlation with the experimental data. Finally, the CPP-based nucleic acid delivery vectors with homing peptide strategy have a potential for the BBB shuttle and the future use as a glioblastoma-targeted drug carrier in the in vivo studies and the clinical applications. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Cancer testis antigens (CTA) represent attractive targets for targeted radiotherapy and imaging as their expression is restricted to cancer and germ cells. NY-ESO-1, a member of the CTA family, is highly immunogenic and expressed in multiple tumor types including carcinoma of bladder, liver lung. The aim of this study was to develop radioimmunoconjugates (RIC) to target NY-ESO-1 protein in cancer cells. Anti-NY-ESO-1 antibodies were modified by addition of DTPA for 111In-labelling or, in the presence of Iodogen, were 123I-labelled. Delivery of radiolabeled immunoconjugates across the cell membrane was achieved using a protein transfection (PT) reagent (SAINT-PhD) and by chemical linkage with the cell-penetrating and nuclear-localizing peptide, TAT (YGRKKRRQRRR). Cellular internalization, distribution and efflux of 111In-DTPA-anti-NY-ESO-1-TAT-PT and 123I-anti-NY-ESO-1-TAT-PT were investigated in cell fractionation and retention assays. It was shown that protein transfection reagent has promoted the cellular uptake of RICs into SK-MEL-37 and both of 111In-DTPA-anti-NY-ESO-1-TAT-PT and 123I-anti-NY-ESO-1-TAT-PT was retained longer in SK-MEL-37 cells in comparison to their isotope control RIC. In clonogenic assays, 111In-DTPA-anti-NY-ESO-1-TAT-PT significantly reduced surviving fraction of SK-MEL-37 cells. Cytotoxicity was inversely proportional to specific activity and the concentration of cells exposed to 111In-DTPA-anti-NY-ESO-1-TAT-PT. siRNA knock down of NY-ESO-1 resulted in partial reversal of 111In-DTPA-anti-NY-ESO-1-TAT-PT associated cytotoxicity. These promising results obtained from the in vitro study has brought the probe further into in vivo study. In preliminary biodistribution studies in SK-MEL-37 xenograft-bearing mice, tumour:muscle ratio for 111In-DTPA-anti-NY-ESO-1-TAT-PT was statistically significant compared to the control RIC 48 h post injection. This clearly indicated that the probe can be delivered into tumour in in vivo model and the successful uptake of radioactivity increased the chance of causing cytotoxicity to tumour cells through DNA damage. All of these findings have suggested that intracellular cancer associated antigen NY-ESO-1 can be reached by protein transfection reagent and cell penetrating peptide and initiates DNA damage through radio-isotope mediated cytotoxicity. Therefore, it represents a novel approach to the treatment of CTA-expressing cancers.

Membrane active peptides with the ability to cross the plasma membrane represent a promising class of therapeutic compounds. However, translocation efficacy and membrane toxicity of these peptides appear correlated and a better understanding of their mechanisms of action is needed to achieve the desired effect. Here, a range of coarse grain molecular dynamics simulations have been performed to systematically investigate the interactions of such cell-penetrating peptides (CPPs) with biologically relevant membranes. Challenges associated to the development of a suitable asymmetric mammalian membrane model demonstrated the importance of lipid species distribution on the bilayer mechanical properties, as well as the effect of coarse graining on its electrostatic properties. However, simulations successfully discriminated between two CPPs, penetratin and transportan, and were consistent with the experimental data available for these. The results obtained suggest that the ability of transportan peptides to aggregate into flexible, dynamic, transmembrane bundles is responsible for their relative membrane toxicity. The stability and structure of these aggregates, as well as the extent of the bilayer perturbations they induced, were shown to depend on the membrane composition and asymmetry, thus providing a molecular basis to explain how the toxicity of CPPs is modulated by membranes. In particular, bilayer destabilisation was enhanced by the presence of anionic lipids and hampered by that of cholesterol. Transportan aggregates were also observed to trigger lipid flip-flops above a certain size and a new pathway for such events, not relying on the formation of water defects, was characterised.

La maurocalcine (MCa) est une toxine de 33 acides aminés issus du venin de scorpion Scorpio maurus palmatus. Ce peptide a initialement été étudié pour son activité pharmacologique en tant qu'activateur du récepteur à la ryanodine (RyR1) des muscles squelettiques. En étudiant comment cette toxine pouvait atteindre le RyR qui est localisé à l'intérieur des cellules, il a été montré que la maurocalcine pouvait être classé dans la liste croissante des peptides de pénétration cellulaire. Depuis la découverte que la maurocalcine peut servir de vecteur à la délivrance intracellulaire de streptavidine fluorescente, les données se sont accumulées pour illustrer l'incroyable valeur biotechnologique de cette toxine. Plusieurs nouveaux analogues ont été produits qui séparent les propriétés pharmacologiques et de pénétration cellulaire du peptide comme une maurocalcine sans ponts disulfulres, synthétisée en remplaçant les résidus cystéine par des acides aminobutyriques, ou en remplaçant tous les acides aminés par leur isomère de conformation D. La maurocalcine s'est avérée efficace pour la délivrance cellulaire de nanoparticules ouvrant ainsi une myriade possible d'applications high-tech. Enfin, la maurocalcine a été couplé à la doxorubicine, un agent anti-tumoral, pour rendre chimio-sensibles des cellules cancéreuses devenues chimio-résistantes. Il semble donc que la maurocalcine débute sa carrière comme outil biotechnologique, mais aussi que cette toxine s'avèrera utile pour déchiffrer finement les détails mécanistiques du fonctionnement du récepteur à la ryanodine. / Maurocalcine (MCa) is a 33 mer toxin initially identified from a tunisian scorpion venom, scorpio maurus palmatus. This peptide initially triggered our interest for its pharmacological activity on Ryanodine Receptor type 1 (RyR1) of skeletal muscles. In studying how this toxin reaches the intracellular RyR1, it has been shown that MCa could be placed in the growing family of cell penetrating peptides. Since the discovery that MCa can act as a transport agent for the intracellular delivery of fluorescent streptavidine, data have accumulated to illustrate the amazing biotechnological properties of this toxin. Several new analogs have been produced that keep cell penetration properties and lose pharmacological activity of the native molecule. This is the case for a linear analog of MCa synthesized by replacing internal cysteine residues by aminobutyric acid, or by the synthesis of a MCa analog with all its amino acid in D conformation. MCa proved efficient for the intracellular delivery of nanoparticles leading to a myriad of hi-tech applications. Finally, MCa has been grafted on an anti-tumor agent, doxorubicin, to made chemo-resistant tumor cells chemo-sensitive. So it seems that MCa begins its career as a biotechnological tool, and that this toxin will be helpful to see the light on the mechanistic aspects of RyR function.

Peptides are emerging as promising therapeutics due to their inhibitory affinity towards protein-protein interactions (PPI). However, peptides have been limited mainly by their poor bio-stability and lack of cell permeability. Efforts to generate drug-like peptides have led to the development of macrocyclic peptides, which exhibit improved stability. Yet, most macrocyclic peptides still require the assistance of a cell penetrating peptide (CPP) for cellular entry. High throughput technologies have been exceptional tools for the discovery of peptides to interrupt PPIs. This work details the recent advancements we have made to improve our high throughput technique, mRNA display, to yield more therapeutically relevant peptides to inhibit PPIs. Our advancements are focused on cell permeability, protease stability, and secondary structure for enhanced affinity. Here we develop and optimize a cyclic CPP that can be included in future mRNA display libraries. We also tested the ability of our CPP to deliver an impermeable peptide cargo into cells. We rationally designed and tested linear and cyclic peptides to improve affinity to the BRCA1 protein. We used computational work to complement our experimental results for our CPPs and BRCA1 inhibitors. We examined peptides that arose from a library containing a mix of linear, monocyclic, and bicyclic peptides constructed using orthogonal cyclization chemistries. We rationally designed cyclic peptides and tested their affinity against Hsp70. We proposed a novel selection strategy to find optimal CPP motifs.

In the work presented here we have used a function-based approach to isolate 12 novel cell-penetrating peptides from a 10,000+ member peptide library of rational design. Our unique high-throughput screen differentiates non-membranolytic from membranolytic translocation of peptides across lipid bilayers, thus allowing the selection of potential cell-penetrating peptides over potential antimicrobial peptides or peptide toxins. The 12 residue framework of the peptide library, designed with translocation in mind, is a series of 9 combinatorial sites followed by a C-terminal α-1-chymotrypsin cleavage site that is integral to the screen. The resulting residue in each of the combinatorial sites is one of 2 - 4 variable amino acids, with a hydrophobic or cationic residue available in each position. The sequences of nonpore-forming translocating peptides pulled from the screen have a 3 residue motif, Leu-Leu-Arg (p=10-5), and an overall under representation of basic residues in favor of hydrophobic amino acids. Upon characterization, these novel peptides were shown to behave akin to known cell-penetrating peptides found in nature. Ex vivo studies, in mammalian tissue cultures, revealed that the peptides translocate across the cell membrane without toxicity to the cell. In addition, structural studies showed a lack of convergence regarding a structure- function relationship, a continued trend seen among membrane-active peptides. In the course of the screen and the ex vivo studies, the peptides carried small polar molecules across lipid bilayers and biological membranes respectively; suggesting that, in addition to being cell-penetrating peptides, they could be put to use as effective therapeutic agents. The discovery of these novel cell-penetrating peptides by use of our screen supports function-based screening of peptide libraries as the best way to arrive at de novo membrane-active peptides with specific functions of interest. / acase@tulane.edu

Les peptides vecteurs (CPP) présentent un grand intérêt pour l'internalisation de principes actifs dans les cellules. Les mécanismes permettant aux peptides de traverser la membrane restent sujets à discussion. Mieux comprendre leurs interactions avec la membrane pourrait permettre d'améliorer leur efficacité. L'organisation des lipides après interaction avec le peptide pénétratine a été étudiée par DSC et par fluorescence du Laurdan, sur des membranes modèles composées de lipides naturels. La pénétratine a induit de l'hétérogénéité dans la membrane, ce qui pourrait être un élément important pour déstabiliser la membrane durant son internalisation dans la cellule. En outre, le cholestérol est un régulateur parmi les plus importants des domaines membranaires. En raison de son affinité pour les lipides saturés, il peut former des domaines ordonnés. Grâce au cholestérol-pyrène, une sonde fluorescente, nous avons étudié les domaines liquides ordonnés (Lo) et désordonnés (Ld) de la membrane. Nous avons, par analyse statistique en composante principale, déterminé les longueurs d'onde d'émission caractéristiques des domaines Lo et Ld. Les peptides pénétratine, R9 et RW9 diminuent l'assemblage du cholestérol et RW9 augmente la fluidité de la membrane. RW9 a été le seul peptide capable de traverser des membranes (Ld) sur de vésicules lipidiques dans nos conditions expérimentales. Nous pouvons ainsi en déduire que la distribution des lipides dans la membrane est un factor important pour le passage des CPP. Notamment, l'interface entre les différents domaines semble jouer un rôle prépondérant pour l'internalisation. / Cell penetrating peptides are promising vectors for molecular drug delivery in eukaryotic cells. Despite of their discovery 20 years ago, the mechanisms of peptide membrane crossing are still controversial. Understanding then how they modify the membrane will allow later on a more efficient internalisation into the cell. Lipid organisation after penetratin interaction was studied by DSC and Laurdan fluorescence. Penetratin was able to induce membrane heterogeneity, which could be important for membrane destabilisation during cell internalisation. Furthermore, cholesterol is one of the most important regulators of membrane domains. Due to its strong affinity with saturated lipids, cholesterol presents the ability to form “rafts” (ordered domains). By cholesterol-pyrene, which is a probe that mimics cholesterol, we studied the liquid ordered (Lo) and liquid disordered (Ld) domains of the membrane. Firstly, we determined the wavelengths that characterise each of these domains by multivariable analysis and then, we verify the peptide effect (R9, RW9 and penetratin) in the distribution of these domains. RW9 were the only CPP able to cross the membrane (Ld). We can deduce that lipid distribution in the membrane is important for the peptide internalisation and the interfaces between these domains may play an important role during this process.

<p>Our genome operates by sending instructions, conveyed by mRNA, for the manufacture of proteins from chromosomal DNA in the nucleus of the cell to the protein synthesizing machinery in the cytoplasm. Alternative splicing is a natural process in which a single gene can encode multiple related proteins. During RNA splicing, introns are selectively removed resulting in alternatively spliced gene products. Alternatively spliced protein products can have very different biological effects, such that one protein isoform is disease-related while another isoform is desirable. Splice switching opens the door to new drug targets, and antisense oligonucleotides (asONs), designed to switch splicing, are effective drug candidates. Cellular uptake of oligonucleotides(ONs) is poor, therefore utilization of cell-penetrating peptides (CPPs), well recognized for intracellular cargo delivery, is a promising approach to overcome this essential issue. Most CPPs are internalized by endocytosis, although the mechanisms involved remain controversial.</p><p>Here, evaluation of CPP-mediated ON delivery over cellular membranes has been performed. A protocol that allows for convenient assessment of CPP-mediated cellular uptake and characterization of corresponding internalization routes is established. The protocol is based on both fluorometric uptake measurements and a functional splice-switching assay, which in itself is based on biological activity of conveyed ONs. Additionally, splice switching ONs (SSOs) have been optimized for high efficiency and specificity. Data suggest that SSO activity is improved for chimeric phosphorothioate SSOs containing locked nucleic acid (LNA) monomers. It is striking that the LNA monomers in such chimeric constructs give rise to low mismatch discrimination of target pre-mRNA, which highlight the necessity to optimize sequences to minimize risk for off-target effects.</p><p>The results are important for up-coming work aimed at developing compounds consisting of peptides and novel synthetic nucleic acids, making these entities winning allies in the competition to develop therapeutics regulating protein expression patterns.</p>

<p>Alzheimer’s disease is the most common form of dementia. It is a neurodegenerative disorder characterized by extracellular senile plaques and intracellular neurofibrillary tangles. The main constituent of the senile plaques is the neurotoxic β-amyloid peptide. Surrounding the senile plaques are activated astrocytes and microglia, believed to contribute to neurotoxicity through secretion of proinflammatory cytokines, like interleukin-1β and interleukin-6. For many inflammatory actions, including the cytokine induction in glial cells, the transcription factor NFκB plays a key role. This suggests that therapeutical strategies aimed to control the development of Alzheimer’s disease could include administration of drugs that hinder NFκB activation.</p><p>The major aim of this thesis was to examine the effects of β-amyloid together with interleukin-1β on cytokine expression as well as NFκB activation in glial cells. The possibility to block NFκB activation, and downstream effects like interleukin-6 expression, by using an NFκB decoy was investigated. The possibility to improve the cellular uptake of the decoy by linking it to a cell-penetrating peptide was also investigated.</p><p>The results obtained provide supportive evidence that inflammatory cytokines are induced by β-amyloid, and that they can indeed potentiate its effects. The results further demonstrate that by blocking NFκB activation, the induction of interleukin-6 expression can be inhibited. By using an improved cellular delivery system, the uptake of the NFκB decoy and hence the downstream cytokine inhibition could be increased. In conclusion, these results demonstrate the possibility to decrease the inflammatory reactions taken place in Alzheimer’s disease brains, which may ultimately lead to a possible way of controlling this disorder.</p>