The autosomal recessive disorder spinal muscular atrophy (SMA) causes motor neuron degeneration and muscle wasting, progressing to paralysis and death in severe cases. The disease is caused by deficiency of survival motor neuron protein (SMN) due to deletion or mutation of the SMN1 gene. We seek to develop a protein-based therapy for SMA using an adenoviral vector which encodes a secretable form of SMN fused to a protein transduction domain (PTD) derived from the trans-acting activator of transcription (TAT) from HIV. We generated secretable GFP proteins using transient transfection in mammalian cells and determined that the secretory peptide was inefficient when paired with the native PTD. We generated TAT-GFP proteins in bacteria and observed that the variant TAT3 most reliably tranduced cells in vitro. We did not observe uptake of the therapeutic protein following infection with an adenoviral vector and subsequent secretion of the protein from infected cells.

The autosomal recessive disorder spinal muscular atrophy (SMA) causes motor neuron degeneration and muscle wasting, progressing to paralysis and death in severe cases. The disease is caused by deficiency of survival motor neuron protein (SMN) due to deletion or mutation of the SMN1 gene. We seek to develop a protein-based therapy for SMA using an adenoviral vector which encodes a secretable form of SMN fused to a protein transduction domain (PTD) derived from the trans-acting activator of transcription (TAT) from HIV. We generated secretable GFP proteins using transient transfection in mammalian cells and determined that the secretory peptide was inefficient when paired with the native PTD. We generated TAT-GFP proteins in bacteria and observed that the variant TAT3 most reliably tranduced cells in vitro. We did not observe uptake of the therapeutic protein following infection with an adenoviral vector and subsequent secretion of the protein from infected cells.

La sélénoprotéine T (SelT) est une protéine thiorédoxine-like abondamment exprimée au cours du développement embryonnaire chez le rat, mais son expression tend à disparaître après la naissance, notamment dans le coeur, suggérant un rôle limité de la SelT à l’âge adulte. Néanmoins, nous avons pu montrer que la SelT est réexprimée au niveau cardiaque suite à une ligature de l’artère coronaire (LC), suggérant le rôle potentiellement protecteur de cette protéine au cours des pathologies cardiovasculaires. Le but de notre projet fut donc d’évaluer les effets cardiaques d’une thérapie par la SelT au cours de l’insuffisance cardiaque, moyennant soit une thérapie protéique, soit une thérapie génique visant à surexprimer la SelT au niveau cardiaque ou au niveau systémique. La supplémentation en SelT (15μg/kg/jour, minipompes ip) a permis d’améliorer significativement le débit cardiaque et la fraction de raccourcissement du VG, mais également d’améliorer les pressions télé-systoliques et télé-diastoliques du ventricule gauche ainsi que la perfusion coronaire. Ces changements sont associés à une diminution du stress oxydant cardiaque ainsi qu’à une répression des mécanismes inflammatoires cardiaques. L’ensemble de ces améliorations a été observé sans modification de la taille d’infarctus. En parallèle, nous avons pu montrer qu’une injection intraveineuse d’un rAAV9-SelT (1.1011vg) une semaine après la LC permettait de diminuer significativement la dilatation ventriculaire gauche 3 mois après la LC. De manière concomitante, la thérapie génique par la SelT améliore le débit cardiaque ainsi que la perfusion cardiaque. Ces changements sont associés à une amélioration de la compliance et de l’élastance cardiaque. Par ailleurs, l’injection intramusculaire d’un rAAV8-SelT suivant le même protocole que précédemment. Nous avons pu montrer que le traitement par cet AAV permettait de diminuer significativement la dilatation du VG et d’améliorer la fraction de raccourcissement. De plus, la thérapie génique a permis d’améliorer la perfusion cardiaque ainsi que la relaxation coronaire endothélium-dépendante. Nous avons également pu montrer que l’ensemble des effets de la SelT sont médiés par le résidu Sec, dès lors que la modification de ce résidu par une alanine, annihile totalement l’ensemble des effets positifs observés au cours de notre étude. Ainsi, nos résultats ont permis de montrer clairement que le rôle bénéfique d’un traitement par la SelT au cours de l’ICC, et ce, grâce à un mécanisme sélénocystéine-dépendant. La SelT semble donc être une cible thérapeutique prometteuse pour le traitement de cette pathologie. / Selenoprotein T (SelT) is a thioredoxin-like protein, which is abundantly but transiently expressed in the heart during the embryonic development, suggesting that SelT plays a limited role during adulthood. However, data from our laboratory show that cardiac SelT expression increases after myocardial infarction. This suggests that SelT may play a yet unrevealed role in cardiovascular diseases but SelT’s potential protective role is unknown. Thus, we sought to investigate the cardiac effects of a SelT-mediated therapy in chronic heart failure (CHF) using either a protein or gene therapy through either a protein supplementation, or rAAV encoding for different forms of SelT. SelT supplementation (15μg/kg/day, IP, administered for 1 month starting 7 days after MI) resulted in a restoration of cardiac output and LV fractional shortening (sham: 178,1±14,8; MI: 161,1±7,7; MI+SelT; 177,6 ±8,0 and sham: 44,5±5,1; MI: 17,1±0,8; MI+SelT: 25,6±2,4, respectively), in association with an improvement of LV end-diastolic and end-systolic pressures as well as LV tissue perfusion. These changes were associated with a lower oxidative status and with a decrease in inflammation pathways (-32,7% vs MI for oxidative stress and - 27,2% and - 31,4% for inflammation, measured by electron paramagnetic resonance and western blotting analyses of IL1ß and IL6 expressions, respectively). All these effects were observed at identical infarct sizes. In parallel, a single intravenous injection of rAAV9-SelT (1.1011 virus - genome copies) one week after MI resulted in an increased cardiac SelT expression 3 weeks after injection (+150%, p<0.05). This SelT - overexpression reduced HF-induced increase in left ventricular diameters in both systole and diastole (at 1 and 3 months post-MI). Simultaneously, SelT improved stroke volume and cardiac output, without change in heart rate or body weight. Moreover, cardiac perfusion was improved by rAAV9-SelT in both interventricular septum and in the border zone of the infarct. These changes were associated with an improvement in cardiac compliance and elastance parameters assessed by invasive pressure/volume curves (compliance: sham: 18.2 ±1.5; HF: 11.0±1.0; HF+rAAV9-SelT: 15.3±0.5 and elastance: sham: 1.3±0.2; HF: 2.8±0.2; HF+rAAV9-SelT: 1.4±0.2, respectively; p<0.05 vs. HF). The third part of this project consisted in a single intramuscular injection of rAAV8-SelT (1.1011 virus-genome copies, 7 days after CAL) of either the normal form (rAAV8-SelTSec), either the modified form in which the Sec residue is replaced by an Ala (rAAV8-SelTAla). rAAV8-SelTSec administration resulted in a significant increase in cardiac SelT levels as soon as 3 weeks post-administration. After 3 months, SelTSec reduced LV dilation and restored cardiac output. Simultaneously SelT improved both LV elastance and compliance. In contrast, administration of the rAAV8-SelTAla did not modify the CHF-related cardiac dysfunction, suggesting that the selenocysteine residue is essential to the normal protein function. Our results clearly show that increasing SelT to supra-normal levels reduces CHF-induced cardiac dysfunction through a selenium-dependent pathway. These results suggest that SelT might be a promising therapeutic option in the treatment of CHF.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by reduced levels of the survival motor neuron (SMN) protein. SMA results in degeneration of motor neurons, progressive muscle atrophy, and death in severe forms of the disease. Currently, there is a lack of inexpensive, readily accessible, accurate biomarkers to study the disease. Furthermore, the current FDA approved therapeutic is neither 100 % effective nor accessible for all patients, thus more research is required. Tiny cell derived vesicles known as exosomes have been evaluated in an attempt to identify novel biomarkers for many disease states and have also shown therapeutic promise through their ability to deliver protein and nucleic acid to recipient cells. The research presented herein investigates whether (1) the level of SMN protein in exosomes isolated from the medium of cells, and serum from animal models and patients of SMA is indicative of disease, to serve as a biomarker for monitoring disease progression and therapeutic efficacy; (2) SMN-protein loaded exosomes can be utilized to deliver SMN protein to SMN-deficient cells; (3) adenoviral vectors are effective at creating SMN protein-loaded exosomes in situ for body wide distribution of SMN protein. This research has shown SMN protein is naturally released in extracellular vesicles, and the level of exosomal SMN protein is reflective of the disease state. Exosomes can also be modified to hold enhanced levels of SMN protein and deliver them to both the cytoplasm and nucleus of SMN-deficient cells. Furthermore, adenoviral vectors expressing luciferase-tagged SMN1 cDNA, targeted to the liver, results in SMN protein-loaded exosomes and detectable luciferase activity, body-wide. Thus, exosomes present as an effective biomarker and potentially a novel approach to treat SMA.

Protein therapeutics offer numerous advantages over small molecule drugs and are rapidly becoming one of the most prominent classes of therapeutics. Unfortunately, they are delivered almost exclusively by injection due to biological obstacles preventing high bioavailability via the oral route. In this work, numerous approaches to overcoming these barriers are explored. PH-Responsive poly(itaconic acid-co-N-vinylpyrrolidone) (P(IA-co-NVP)) hydrogels were synthesized, and the effects of monomer ratios, crosslinking density, microparticle size, protein size, and loading conditions were systematically evaluated using in vitro tests. P(IA-co-NVP) hydrogels demonstrated up to 69% greater equilibrium swelling at neutral conditions than previously-studied poly(methacrylic acid-co-N-vinylpyrrolidone) hydrogels and a 10-fold improvement in time-sensitive swelling experiments. Furthermore, P(IA-co-NVP) hydrogel microparticles demonstrated up to a 2.7-fold improvement in delivery of salmon calcitonin (sCT) compared to methacrylic acid-based systems, with a formulation comprised of a 1:2 ratio of itaconic acid to N-vinylpyrrolidone demonstrating the greatest delivery capability. Vast improvement in delivery capability was achieved using reduced ionic strength conditions during drug loading. Use of a 1.50 mM PBS buffer during loading yielded an 83-fold improvement in delivery of sCT compared to a standard 150 mM buffer. With this improvement, a daily dose of sCT could be provided using P(IA-co-NVP) microparticles in one standard-sized gel capsule. P(IA-co-NVP) was also tested with larger proteins urokinase and Rituxan. Crosslinking density provided a facile method for tuning hydrogels to accommodate a wide range of protein sizes. The effects of protein PEGylation were also explored. PEGylated sCT displayed lower release from P(IA-co-NVP) microparticles, but displayed increased apparent permeability across a Caco-2 monolayer by two orders of magnitude. Therefore, PEG-containing systems could yield high bioavailability of orally delivered proteins. Finally, a modified SELEX protocol for cellular selection of transcellular transport-initiating aptamers was developed and used to identify aptamer sequences showing enhanced intestinal perfusion. Over three selection cycles, the selected aptamer library showed significant increases in absorption, and from an initial library of 1.1 trillion sequences, 5-10 sequences were selected that demonstrated up to 10-fold amplification compared to the naïve library. These sequences could provide a means of overcoming the significant final barrier of intestinal absorption. / text

La compréhension des mécanismes moléculaires de différentes pathologies a permis la caractérisation de gènes et de protéines impliqués dans la pathogénèse et l'identification de cibles thérapeutiques intracellulaires. La nature hydrophobique de la membrane cellulaire empêche le passage des médicaments dans les cellules. Les Cell-Penetrating Peptides (CPP) ou domaines de transduction protéiques (PTD) sont des peptides qui permettent l'internalisation de macromolécules hydrophiles in cellulo et in vivo. Un nouveau peptide issu du facteur de transcription ZEBRA du virus Epstein-Barr, et qui possède des propriétés de transduction a été caractérisé récemment dans notre laboratoire. Des études par mutagénèse de délétion de la protéine ZEBRA ont permis d'identifier la région d'acides aminés (nommé ainsi MD) impliquée dans la pénétration cellulaire. Ce peptide traverse les membranes des cellules de mammifères par un mécanisme de translocation directe, même lorsqu'il est fusionné à des molécules telles que la protéine reportrice eGFP. Le mécanisme de pénétration directe représente un grand avantage pour les applications thérapeutiques: les molécules cargos peuvent être internalisées directement dans le cytoplasme cellulaire sans dégradation et sous une forme biologiquement active. L'objectif de cette thèse est d'étudier les propriétés de pénétration cellulaire du peptide MD et d'évaluer ses applications thérapeutiques comme système de vectorisation des protéines. Ce travail est structuré en trois parties. La première partie porte sur l'étude de l'optimisation de la séquence peptidique MD par réduction de taille et l'évaluation du rôle de sa composition en acides aminés dans le processus de translocation à travers la membrane cellulaire. Cette étude a conduit à l'identification d'une séquence plus courte MD (MD11) possédant une efficacité et un mécanisme de translocation inchangés. La deuxième partie décrit une approche thérapeutique basée sur MD11 visant à la complémentation protéique d'un dysfonctionnement identifiée dans la plupart des cancers. Les cellules tumorales présentent des altérations dans la machinerie de traduction résultant dans une prolifération cellulaire incontrôlée. Parmi les différents facteurs intervenant dans la régulation de ce processus, le facteur eucaryote d'initiation 3 (eIF3) contribue à l'oncogenèse et au maintien de l'état cancéreux. Ce complexe est composé de 13 sous-unités, désignées eIF3 a-m. L'expression de certaines sous-unités est altérée dans plusieurs cancers, et en particulier la sous-unité f (eIF3f) est significativement diminuée dans le mélanome, les cancers du pancréas, de la vulve, du sein, de l'intestin et de l'ovaire. L'expression ectopique par transfection transitoire du gène eIF3f inhibe la synthèse protéique et induit l'apoptose dans le mélanome et dans les cellules cancéreuses pancréatiques. A partir de ces observations, nous avons développé une approche thérapeutique innovante pour le traitement des cancers dans lesquels la protéine manquante eIF3f est produite sous forme recombinante fusionnée à la séquence de MD11, et ensuite internalisée dans les cellules cibles tumorales. Ces résultats démontrent que le système de transfert de eIF3f basé sur MD11 représente une stratégie efficace pour supprimer la prolifération des cellules tumorales. La dernière partie de cette thèse explore la propriété de pénétration de MD11 dans les cellules de levure, et en particulier dans le champignon pathogène Candida albicans. Les résultats obtenus démontrent la polyvalence de MD11, qui fonctionne comme vecteur de protéines à activité biologique aussi bien dans la levure que dans les cellules de mammifères. Le potentiel de MD11 comme système de transport et de relargage des protéines a donc été établis, toutefois certaines améliorations en ce qui concerne la formulation des protéines de fusion et des études in vivo doivent être réalisées afin de valider son efficacité thérapeutique. / In recent years, the understanding of disease molecular mechanisms has led to the identification of genes and proteins that are altered in disease state and many therapeutic targets have been found located within cells. The protective and hydrophobic nature of plasma membrane prevents therapeutic drugs from entering cells. Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have emerged as a group of non-invasive delivery vectors for various hydrophilic macromolecules, and several in vitro and in vivo applications as pharmaceutical carriers have been reported. A novel cell-penetrating peptide deriving from the Epstein-Barr virus ZEBRA transcription factor has been recently characterized in our laboratory. A reductionist study of full-length ZEBRA protein has allowed to identify the amino acid region (named as Minimal Domain, MD) implicated in cellular uptake. This peptide is able to cross the mammalian cell membranes via a direct translocation mechanism even when fused to cargo molecules such as eGFP reporter protein. The direct penetration mechanism represents a great advantage for therapeutic applications as the cargo molecules can be directly delivered into cells cytoplasm in a biological active form. The aim of this thesis is to explore the cell-penetrating properties of the MD peptide and evaluate its applications as therapeutic protein delivery system. This work is structured in three parts.The first part describes the study on the optimization of MD peptide sequence by size-reduction and the evaluation of its amino acid composition role in the translocation process across the cell membrane. This study has led to the identification of a shorter MD sequence (MD11) with unvaried mechanism of translocation. The second section describes a MD11-based therapeutic approach aiming at repair a dysfunction of the protein synthesis identified in most cancers. The regulation of the protein synthesis has a crucial role in governing the eukaryotic cell growth and subtle defects in the translational machinery can alter the cellular physiology and lead to cell malignancy. Among the different factors intervening in the regulation of this process, the eukaryotic initiation factor 3 (eIF3) contributes to oncogenesis and maintenance of the cancer state. This complex is composed of 13 subunits (designated eIF3 a-m). The expression of eIF3 subunits is altered in several cancers, and in particular the f subunit (eIF3f) is significantly down-regulated in pancreas, vulva, breast, melanoma, ovary and small intestine tumors. The eIF3f ectopic expression by transient gene transfection inhibits cellular protein synthesis and induces apoptosis in melanoma and pancreatic cancer cells. Starting from these observations, we developed an innovative therapeutic approach for cancer treatment in which the missing eIF3f protein is produced in vitro in fusion to MD11, and delivered to cells. These results have demonstrated that the MD11- based eIF3f transfer system may represent a powerful strategy to suppress the tumor-cell proliferation. The last part of this thesis explores the cell-penetrating property of MD11 in yeast cells, and in particular in the pathogenic fungus Candida albicans. The presented results demonstrate the versatility of MD11, functioning as vectors in both yeast and mammalian cells and as carrier for proteins with biological activity.The MD11 potential as protein delivery system is evident; however some improvements regarding the fusion protein formulation and in vivo studies should be realized to validate the effectiveness of its therapeutic application.