Immunological understanding of immune response stimulation by synthetic molecules : therapeutic applications

Le développent des biothérapies a connu un essor sans précédent ces dernières années, permettant l'émergence de nombreuses solutions thérapeutiques répondant à de multiples défis en santé humaine et animale afin de lutter contre des maladies héréditaires ou acquises. Parmi ces stratégies, le transfert de gênes revêt un intérêttout particulier, permettant à la fois de réparer des cellules défectueuses en leur apportant une copie saine du gène, ou bien d'activer efficacement le système immunitaire par l'expression d'un antigène d'intérêt thérapeutique. Néanmoins, malgré le développement de nombreux outils de thérapie génique ou d'immunothérapie, de nombreuses limitations persistent à ce jour. Au cours de cette thèse, les résultats obtenus ont permis le développement et l'optimisation d'outils afin d'outrepasser ces limites. Tout d'abord, nous avons pu élucider les mécanismes d'actions du copolymère à blocs 704, une nouvelle classe de vecteurs qui permet d'induire une réponse vaccinale optimale en délivrant de manière directe des acides nucléiques au sein des cellules musculaires et en stimulant des récepteurs à ADN intracellulaires. De plus, ces travaux ont permis de démontrer qu'une nouvelle classe de molécules adjuvantes, les Iipoaminoglycosides, permet de stimuler efficacement le système immunitaire en plus de conduire à la régression de la croissance de tumeurs mammaires murines. Enfin, ces résultats ont pu montrer l'importance de l'assemblage supramoléculaire des lipoplexes sur l'efficacité de transfection afin de développer un vecteur universel pour la délivrance de différentes classes d'acides nucléiques. Dès lors, l'optimisation de la délivrance de macromolécules au sein de cellules et la compréhension de l'interaction entre le système immunitaire! le vecteur I les acides nucléiques nous ont permis d'établir des stratégies afin de développer des formulations spécifiques à chaque application biothérapeutique.The development 01 biotherapies experienced an unprecedented growth in recent years, allowing the emergence al many therapeutic solutions to many challenges in human and animal health in arder to fight against hereditary or acquired diseases. Among these strategies, gene transfer is of particular interest, making it possible either to repair defective cells by providing them a healthy copy 01 the gene, ar to effectively activate the immune system by expressing an antigen of therapeutic interest. Nevertheless, despite the development of numerous tools for gene therapy or immunotherapy, many limitations persist. During this thesis, the results obtained allowed the development and optimization of tools in order to go beyond these limits. First, we have been able to elucidate the mechanism of action 01 the block copolymer 704, a new class 01 vectors which allows to induce an optimal vaccine response by directly delivering nucleic acids into muscle cells and by stimulating intracellular DNA sensors. Moreover, this work has demonstrated that a new class of adjuvant molecules, lipoaminoglycosides, can stimulate efficiently the immune system in addition to lead to the regression of murine mammary tumors growth. Finally, these results showed the importance of the supramolecular assembly of lipoplexes on the \ransfection efficiency in order to develop a universal vector for the delivery of different classes of nucleic acids. Therefore, the optimization of macromolecules delivery within cells and the understanding of the interaction between the immune system i vector I nucleic acids allowed us to establish strategies in order to develop specific formulations tor each biotherapeutic applications

704/DNA vaccines leverage cytoplasmic DNA stimulation to promote anti-HIV neutralizing antibody production in mice and strong immune response against alpha-fetoprotein in non-human primates

Genetic immunization is an attractive approach for prophylactic and therapeutic vaccination using synthetic vectors to deliver antigen-encoding nucleic acids. Recently, DNA delivered by a physical means or RNA by liposomes consisting of four different lipids demonstrated good protection in human phase III clinical trials and received Drugs Controller General of India and US FDA approval to protect against COVID-19, respectively. However, the development of a system allowing for efficient and simple delivery of nucleic acids while improving immune response priming has the potential to unleash the full therapeutic potential of genetic immunization. DNA-based gene therapies and vaccines have the potential for rapid development, as exemplified by the recent approval of Collategene, a gene therapy to treat human critical limb ischemia, and ZyCoV, a DNA vaccine delivered by spring-powered jet injector to protect against SARS-CoV2 infection. Recently, we reported amphiphilic block copolymer 704 as a promising synthetic vector for DNA vaccination in various models of human diseases. This vector allows dose sparing of antigen-encoding plasmid DNA. Here, we report the capacity of 704-mediated HIV and anti-hepatocellular carcinoma DNA vaccines to induce the production of specific antibodies against gp120 HIV envelope proteins in mice and against alpha-fetoprotein antigen in non-human primates, respectively. An investigation of the underlying mechanisms showed that 704-mediated vaccination did trigger a strong immune response by (1) allowing a direct DNA delivery into the cytosol, (2) promoting an intracytoplasmic DNA sensing leading to both interferon and NF-κB cascade stimulation, and (3) inducing antigen expression by muscle cells and presentation by antigen-presenting cells, leading to the induction of a robust adaptive response. Overall, our findings suggest that the 704-mediated DNA vaccination platform is an attractive method to develop both prophylactic and therapeutic vaccines

Design of Ionizable Lipids To Overcome the Limiting Step of Endosomal Escape: Application in the Intracellular Delivery of mRNA, DNA, and siRNA

International audienceThe intracellular delivery of nucleic acid molecules is a complex process involving several distinct steps; among these the endosomal escape appeared to be of particular importance for an efficient protein production (or inhibition) into host cells. In the present study, a new series of ionizable vectors, derived from naturally occurring aminoglycoside tobramycin, was prepared using improved synthetic procedures that allow structural variations on the linker and hydrophobic domain levels. Complexes formed between the new ionizable lipids and mRNA, DNA, or siRNA were characterized by cryo-TEM experiments and their transfection potency was evaluated using different cell types. We demonstrated that lead molecule 30, bearing a biodegradable diester linker, formed small complexes with nucleic acids and provided very high transfection efficiency with all nucleic acids and cell types tested. The obtained results suggested that the improved and "universal" delivery properties of 30 resulted from an optimized endosomal escape, through the lipid-mixing mechanism

Injectable Hyaluronic Acid-co-Gelatin Cryogels for Tissue-Engineering Applications

Polymeric scaffolds such as hydrogels can be engineered to restore, maintain, or improve impaired tissues and organs. However, most hydrogels require surgical implantation that can cause several complications such as infection and damage to adjacent tissues. Therefore, developing minimally invasive strategies is of critical importance for these purposes. Herein, we developed several injectable cryogels made out of hyaluronic acid and gelatin for tissue-engineering applications. The physicochemical properties of hyaluronic acid combined with the intrinsic cell-adhesion properties of gelatin can provide suitable physical support for the attachment, survival, and spreading of cells. The physical characteristics of pure gelatin cryogels, such as mechanics and injectability, were enhanced once copolymerized with hyaluronic acid. Reciprocally, the adhesion of 3T3 cells cultured in hyaluronic acid cryogels was enhanced when formulated with gelatin. Furthermore, cryogels had a minimal effect on bone marrow dendritic cell activation, suggesting their cytocompatibility. Finally, in vitro studies revealed that copolymerizing gelatin with hyaluronic acid did not significantly alter their respective intrinsic biological properties. These findings suggest that hyaluronic acid-co-gelatin cryogels combined the favorable inherent properties of each biopolymer, providing a mechanically robust, cell-responsive, macroporous, and injectable platform for tissue-engineering applications

Hypoxia-inducing cryogels uncover key cancer-immune cell interactions in an oxygen-deficient tumor microenvironment

Hypoxia is a major factor shaping the immune landscape, and several cancer models have been developed to emulate hypoxic tumors. However, to date, they still have several limitations, such as the lack of reproducibility, inadequate biophysical cues, limited immune cell infiltration, and poor oxygen (O2) control, leading to non-pathophysiological tumor responses. Therefore, it is essential to develop better cancer models that mimic key features of the tumor extracellular matrix and recreate tumor-associated hypoxia while allowing cell infiltration and cancer-immune cell interactions. Herein, hypoxia-inducing cryogels (HICs) have been engineered using hyaluronic acid (HA) to fabricate three-dimensional microtissues and model a hypoxic tumor microenvironment. Specifically, tumor cell-laden HICs have been designed to deplete O2 locally and induce long-standing hypoxia. HICs promoted changes in hypoxia-responsive gene expression and phenotype, a metabolic adaptation to anaerobic glycolysis, and chemotherapy resistance. Additionally, HIC-supported tumor models induced dendritic cell (DC) inhibition, revealing a phenotypic change in the plasmacytoid DC (pDC) subset and an impaired conventional DC (cDC) response in hypoxia. Lastly, our HIC-based melanoma model induced CD8+ T cell inhibition, a condition associated with the downregulation of pro-inflammatory cytokine secretion, increased expression of immunomodulatory factors, and decreased degranulation and cytotoxic capacity of T cells. Overall, these data suggest that HICs can be used as a tool to model solid-like tumor microenvironments and has great potential to deepen our understanding of cancer-immune cell relationship in low O2 conditions and may pave the way for developing more effective therapies

Liposome-based Formulation for Intracellular Delivery of Functional Proteins

International audienceThe intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal) enzyme and the anti-cytokeratin8 (K8) antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP)-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies

Important role of phosphoramido linkage in imidazole-based dioleyl helper lipids for liposome stability and primary cell transfection

Background To optimize synthetic gene delivery systems, there is a need to develop more efficient lipid formulations. Most cationic lipid formulations contain 'helper' neutral lipids because of their ability to increase DNA delivery, in particular by improving endosomal escape of DNA molecules via the pH-buffering effect of protonatable groups and/or fusion with the lipid bilayer of endosomes. Methods We evaluated the influence of the linker structure between the two oleyl chains in the helper lipid on transfection efficiency in cell lines, as well as in primary cells (hepatocytes/cardiomyocytes). We reported the synthesis of two new pH-buffering imidazole helper lipids characterized by a polar headgroup containing one (compound 6) or two (compound 5) imidazole groups and two oleyl chains linked by an amide group. We studied their association with the aminoglycoside lipidic derivative dioleylsuccinylparomomycin (DOSP), which contains two oleyl chains linked to the aminoglycoside polar headgroup via an amide function. We compared the morphology and transfection properties of such binary liposomes of DOSP/5 and DOSP/6 with those of liposomes combining DOSP with another imidazole-based dioleyl helper lipid (MM27) in which a phosphoramido group acts as a linker between the two oleyl chains and imidazole function. Results The phosphoramido linker in the helper lipid induces a major difference in terms of morphology and resistance to decomplexation at physical pH for DOSP/helper lipid complexes. Conclusions This hybrid dioleyl linker composition of DOSP/MM27 led to higher transfection efficiency in cell lines and in primary cells compared to complexes with homogeneous dioleyl linker. Copyright (C) 2015 John Wiley & Sons, Ltd

Design of Ionizable Lipids To Overcome the Limiting Step of Endosomal Escape: Application in the Intracellular Delivery of mRNA, DNA, and siRNA

The intracellular delivery of nucleic acid molecules is a complex process involving several distinct steps; among these the endosomal escape appeared to be of particular importance for an efficient protein production (or inhibition) into host cells. In the present study, a new series of ionizable vectors, derived from naturally occurring aminoglycoside tobramycin, was prepared using improved synthetic procedures that allow structural variations on the linker and hydrophobic domain levels. Complexes formed between the new ionizable lipids and mRNA, DNA, or siRNA were characterized by cryo-TEM experiments and their transfection potency was evaluated using different cell types. We demonstrated that lead molecule <b>30</b>, bearing a biodegradable diester linker, formed small complexes with nucleic acids and provided very high transfection efficiency with all nucleic acids and cell types tested. The obtained results suggested that the improved and “universal” delivery properties of <b>30</b> resulted from an optimized endosomal escape, through the lipid-mixing mechanism

Evaluation of tetrafunctional block copolymers as synthetic vectors for lung gene transfer

International audienceIn the present study, we evaluated, in mice, the efficacy of the tetrafunctional block copolymer 704 as a nonviral gene delivery vector to the lungs. SPECT/CT molecular imaging of gene expression, biochemical assays, and immunohistochemistry were used. Our dataset shows that the formulation 704 resulted in higher levels of reporter gene expression than the GL67A formulation currently being used in a clinical trial in cystic fibrosis patients. The inflammatory response associated with this gene transfer was lower than that induced by the GL67A formulation, and the 704 formulation was amenable to repeated administrations. The cell types transfected by the 704 formulation were type I and type II pneumocytes, and transgene expression could not be detected in macrophages. These results emphasize the relevance of the 704 formulation as a nonviral gene delivery vector for lung gene therapy. Further studies will be required to validate this vector in larger animals, in which the lungs are more similar to human lungs