Abstract
In Vivo Delivery of an Adenovirus Encoding Estrogen Receptor Beta Impairs Ovarian Cancer Development
Ovarian cancer is the gynecological cancer exhibiting the highest morbidity and improvement of treatments is still under progress and requires in vivo models. The goal of the work was to develop a gene therapy approach to determine if Estrogen Receptor Beta (ERbeta), a potential tumor suppressor, could impair tumor development. We used a novel preclinical model of ovarian cancer based on luminescent orthotopic xenograft in athymic nude mice. BG-1 ovarian cancer cells, were stably transfected with luciferase gene, and then infected with a backbone Ad5 virus or an adenovirus encoding ERbeta (Ad-ERbeta). Ad5 or Ad-ERbeta infected BG1 cells were then implanted under the bursal membrane of the ovary. Tumor formation as well as metastasis were monitored using a CCD camera. Our results show that implantation of control Ad5 BG1 cells, into ovaries of athymic nude mice allows for a rapid tumor formation. Metastases were observed in the peritoneal cavity, kidney, liver, spleen and lungs, which is close to the clinical situation. On the other hand, Ad-ERbeta infected BG1 cells formed smaller tumors with a strong delay compared to control cells. Metastasis was also strongly reduced in the all metastatic sites, along with a decreased number of circulating cells. We have investigated the potential mechanisms of action of ERbeta. In summary, the use of a gene therapy approach based on ERbeta delivery appears as a promising track for the treatment of ovarian cancer.
New Therapeutic Approaches of Osteosarcoma by RNA Interference
Osteosarcoma, the most frequent malignant primary bone tumor is strongly associated to bone resorption. The osteolysis is mainly regulated by the Receptor Activator of Nuclear Factor kappa B Ligand (RANKL), responsible of osteoclast activity. Preclinical studies have demonstrated that RANKL blockade by soluble receptors is an effective strategy to prevent osteolytic lesions leading to osteosarcoma inhibition. A new therapeutic option could be to directly inhibit RANKL expression by RNA interference.
First, we developed luciferase expressing osteosarcoma cells (LucF-POS-1) in order to allow in vivo bioluminescence imaging (BLI). The BLI allowed us an earlier and more sensitive detection of osteosarcoma tumors than the measure of the tumor volume itself. Moreover, during chemotherapy tumor volume measure showed a strong stagnation whereas BLI indicated a cell death process (fibrotic necrosis).
Second, this model was used to test a protocol for in vivo administration of small interfering RNA complexed with a cationic liposome (RPR209120/DOPE). Treatment with siRNA directed against luciferase mRNA has no effect on the tumor volume evolution as compared to a control siRNA, whereas the number of detected photons was inhibited by at least 50% showing a specific luciferase inhibition.
Thus, our results indicate that siRNA could be delivered using liposomes and could thereby inhibit gene expression in a specific manner in mouse osteosarcoma tumors. This protocol will be applied to target the pro-resorptive molecule RANKL in osteolytic POS-1 osteosarcoma bearing mice in order to counteract the tumor development.
Preclinical Evidence of Using TRAIL in Ewing's Sarcoma Therapy
Ewing's sarcoma is a high-grade neoplasm typically arising in the bones of children and adolescents. Despite improvement in therapy, survival rate decreases from greater than 50% to 20% after 5 years for patients not responding to treatment or presenting pulmonary metastases at diagnosis. Among new therapeutic approaches, TNF-related apoptosis-inducing ligand (TRAIL), with its strong antitumoral activity and minimal toxicity to most normal cells and tissues is a promising candidate. The sensitivity to TRAIL of two human Ewing's sarcoma cell lines, A673 and TC-71 was investigated in terms of receptor expression, proliferation and apoptosis. Its potential preclinical application was then studied in the corresponding models developed in nude mice.
RT-qPCR analysis revealed that both cell lines express TRAIL activator receptors DR4 and DR5. In vitro proliferation assays showed a high sensitivity to TRAIL for both cell lines with a time and dose dependant effect. The inhibition of tumor cell proliferation appeared to be caused by cell death induced by caspase 3 and caspase 8 activations. Using a model of Ewing's sarcoma induced by intra-muscular injection of human A673 cells in nude mice, TRAIL administered by nonviral gene therapy inhibits the primary bone tumor growth (− 87 %) leading to a 2-fold increase of animal survival. TRAIL also exerts a beneficial effect on mice survival in a model of lung nodule dissemination developed from intravenous injection of bone tumor cells.
These results suggest that TRAIL may represent a good candidate for the development of new therapeutic strategies in Ewing's sarcoma.
Mesenchymal Stem Cells and Osteosarcoma: Case Report and Preclinical Data
A patient in complete remission of an humerus telangiectasic osteosarcoma since ten years consulted for a cosmetic correction by lipofilling. Three injections of autologous fat grafts harvested by the Coleman's technique were realized and few months after the last one a local recurrence of osteosarcoma occurred thirteen years after the initial treatment. Such late local recurrence after complete remission of an osteosarcoma is an unexpected event. Then we may ask whether this recurrence is a coincidence or a fact linked to the lipofilling.
Using a mouse model (human osteosarcoma in athymic mice), we showed that osteosarcoma growth is significantly increased by fat injection, whereas fat alone produced only small and stable volumes at injection sites in naive mice. Because of these clinical and pre-clinical data, clinician must be aware that cellular therapy could have a disastrous role in late local relapse of tumors.
Several hypotheses could explain these observations. First we addressed the role of mesenchymal stem cells contained in adipose grafts. Co-injections of mesenchymal stem cells and osteosarcoma cells were tested in immunocompetent and immunodeficient mice and pro-tumoral and/or pro-metastatic effects were observed. MSC secreted factors and phenotype changes were revealed and may be implicated in pro-tumoral effects.
Better knowledge is needed to understand the mechanisms regulating the interactions between graft, tissue recipient and tumor cells and finally to allow safe clinical use of fat and MSC in cancer.
Real-Time Monitoring of Cell Transplantation in Mouse Dystrophic Muscles by a Secreted Alkaline Phosphatase Reporter Gene
Transplantation of muscle precursor cells (MPCs) is a promising approach for the treatment of muscular dystrophies. However, preclinical and clinical results have shown that the technology is not yet efficient enough for most therapeutic applications. Among the problems that remain unsolved are low cellular survival, poor proliferation and lack of migration of the transplanted cells. One major technical hurdle for the optimization of transplantation protocols is how to follow precisely the fate of the cells after transplantation. In this study, we examined the use of a secreted form of the mouse alkaline phosphatase (mSeAP) enzyme as the reporter system transduced into MPCs using a retroviral vector. We show that circulating mSeAP could be detected in the serum of the transplanted mice at different time points after MPC transplantation. We also found that the level of circulating mSeAP is highly correlated with the number of transplanted cells and that mSeAP is an excellent histological marker. Further, studying the levels of circulating mSeAP compared with the number of muscle fibers positive to mSeAP and to dystrophin, enabled detailed analyses of bottleneck steps for successful transplantation. Taken together, our results show that mSeAP is an excellent quantitative ‘real-time’ reporter gene for cell therapy preclinical studies.
Inserm U844, Montpellier, France
Pharmacologically Active Microcarriers (PAM) are poly(D,L lactide-co-glycolide) biocompatible and biodegradable microparticles, that may convey cells on their surface to provide an adequate 3D microenvironment and deliver a growth factor to induce or maintain tissue specific differentiation. Our objective was to implement the PAM for tissue engineering of human mesenchymal stem cells (MSC) to stimulate chondrogenesis.
The most efficient molecule for the biomimetic surface of the PAMs was fibronectin (FN) both for cell adhesion and survival of MSC in vitro. We then set-up a new formulation of PAMs enabling the continuous release of TGF-β3 from the microparticles. Indeed, the in vitro release of TGF-β3 reached 22% of the total amount of encapsulated protein by the first week and a plateau was observed by the first month. Importantly, more than 85% of the released TGF-β3 was functionally active. Chondrogenesis was induced by culture of 1.5 × 105 MSC with 0.5 mg PAMs in chondrogenic medium on low attachment culture plates. Cells rapidly adhered onto the PAMs and progressively aggregated to form a unique pellet-like structure from day 7 to day 21. In PAM-TGF-β3-induced aggregates, high expression of chondrogenic markers occurred in a time-dependent manner whereas expression of osteogenic and adipogenic markers was absent. Intra-articular injection of MSC mixed with PAM-TGF-β3 will confirm their capacity to differentiate in vivo into cartilage tissue.
In summary, the combination of PAMs with TGF-β3 allows MSC to preferentially differentiate into chondrocytes. Indeed, PAMs represent a promising strategy for delivering bioactive molecules that may be useful for tissue engineering.
Nuclear Delivery of DNA/Polymer Complexes: A Real-Time Investigation by Confocal Microscopy
Efficient transfection of cells by pDNA/cationic polymer complexes supposes that the delivery of pDNA in the nucleus requires the capture of polyplexes and their internalization, the escape of pDNA from endosomes and its nuclear import. Histidine-rich polymers containing both (i) primary amines to condense pDNA and protect it against nuclease digestion, and (ii) protonable imidazole groups in acidic medium to favour the endosome escape of pDNA, are remarkable carriers to deliver the nucleic acid into the cytosol. Additionally, a specific DNA-binding sequence (termed 3NF) that is strongly recognized by NFκB upon transfection, has been inserted in pDNA, to favour its nuclear import. The intracellular trafficking of polyplexes has been carried out by real time confocal microscopy. Cells expressing GFP-tagged proteins, specific of endosomes and caveolae, were used to describe the uptake pathway of polyplexes. A quantification of the pDNA copies number in the nucleus of transfected cells indicated that ∼1500 copies of p3NF-luc3NF versus 250 copies of 3NF-free pDNA were imported in the nucleus after 5h transfection. The quantity of p3NF-luc-3NF dropped dramatically in the presence of the BAY 11-7085, an inhibitor of NFκB activation. These data strongly support the remarkable nuclear import of p3NF-luc-3NF, mediated by NFκB. FRET experiments revealed that most of nuclear pDNA is still condensed with the polymer. By using cells expressing GFP-tagged proteins specific of the nuclear pore complex, we visualized for the first time in live cells, the passage of pDNA through the nuclear pore.
INSERM U758, Lyon, F-69007, France ; Ecole Normale Supérieure de Lyon, Lyon F-69007, France ; IFR128 BioSciences Lyon-Gerland Lyon, F-69007, France ; Université de Lyon , UCB-Lyon 1, Lyon F-69003, France .
The human parvovirus Adeno-Associated Virus (AAV) type 2 can only replicate in cells co-infected with a helper virus, such as Adenovirus or Herpes Simplex Virus type 1 (HSV-1), it otherwise establishes a latent infection. Previous studies demonstrated that the HSV-1 helicase/primase (HP) complex (UL5/8/52) and the single-stranded DNA-Binding Protein (ICP8) were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, the HSV-1 ICP0 protein was able to activate rep gene expression from AAV-2 latently infected cells. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and constructs encoding various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, of ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4 and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42) was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Current studies are performed to determine whether this set of HSV-1 genes is sufficient to achieve high levels of rAAV particles production.
Centre de Génétique Moléculaire et Cellulaire (CGMC)–CRNS UMR5534, Université Claude Bernard , Villeurbanne, France
Hepatocellular carcinoma (HCC) is the fifth most prevailing malignancy in the world. Prognosis of advanced HCC remains poor, with a median life expectancy of six months from the time of diagnosis. Oncolytic vectors, which selectively replicate in and kill cancer cells, are very interesting tools to develop novel therapeutic strategies. The overall purpose of this study is to identify and test human HCC-specific promoters that retain their selectivity when expressed from the HSV-1 genome, in order to use them to target expression and multiplication of oncolytic HSV-1 only in HCC cells. We have identified the promoters of alpha-foetoprotein (AFP), angiopoietin-like 3 (ANGPTL3), and apolipoprotein B (ApoB) as HCC-specific using a transcriptomal approach and have shown that they remain active in liver cancer cells following HSV-1 infection. These promoters were cloned from genomic DNA and were introduced into the HSV-1 genome, where they drive Luciferase expression. Other cellular promoters, like E2F1 or the hypoxia-sensitive prolactin-9XHRE promoters, were used as controls. We have studied the infectivity of these viruses and their ability to drive Luciferase expression in cells derived from HCC, from other types of cancers, or in non-cancer cells. To compare more precisely the activity of the HCC-specific cellular promoters we have performed kinetic studies at different multiplicities of infection. Our results indicate that, when inserted into the HSV-1 genome, the AFP and ANGPTL3 promoters were the only that drove Luciferase expression exclusively in HCC cell lines, suggesting that they are excellent candidates to target HSV-1 multiplication
Institut Charles Gerhardt Montpellier , UMR 5253 CNRS-ENSCM-UM2-UM1, 8 rue de l'Ecole Normale F-34296 Montpellier, France
Dendritic cells (DCs) are key cells in immunology, able to stimulate or inhibit the immune response. RNA interference has appeared of great interest to modulate the expression of immunogenic or tolerogenic molecules. In our study, pH-sensitive polyion complex micelles based on a double-hydrophilic block copolymer and poly-
Intra-Articular Electrotransfer of Plasmid Encoding Soluble TNF Receptor in Collagen-Induced Arthritis: Efficiency and Mode of Action
Rheumatoid arthritis (RA) is a disease that causes chronic inflammation of the joints. TNF-α is a main actor in RA and gene therapy is an alternative for the continuous delivery of soluble TNF-alpha receptor (sTNFR) to block TNF-α. We already demonstrated the interest of a non viral gene transfer strategy, namely electrotransfer, to administrate sTNFRI by a systemic way and to cure collagen-induced arthritis in mice (CIA). However, intra-articular nonviral gene therapy using electrotranfer is also of interest in joint-targeted diseases such as RA.
CIA was induced in DBA/1 mice. The plasmid pVax1 mTNFR-Is/mIgG1 encoding the soluble receptor was injected into both knees at time of clinical symptoms onset. After plasmid injection, an electric field was applied. Then, mTNFR-Is joint secretion was evaluated in knee-conditioned media by ELISA. IL-10, IL-17 and TNF-α mRNA expression in the joints were measured by real time quantitative RT-PCR.
Treatment of CIA with pVax mTNFR/mIgG1 electrotransfer induced a local, but not a systemic, effect on clinical arthritis development as compared to controls (NaCl and empty pVax1). More interestingly, we observed that destruction and inflammation in ankles and knees were lower in pVax1mTNFR-Is injected joints compared to controls. Increased IL-17, TNF-α and IL-10 mRNA synovial expressions were shown 15 days after ET in pVax1 mTNFR-Is/mIgG1 ET mice, as compared to NaCl. These results demonstrate the feasibility and the effectiveness of local non viral gene therapy in arthritis and shows that local mTNFR-Is gene delivery modulates some pro-and anti-inflammatory gene cytokine expression within the joints.
Alpha-6 Integrin Subunit Plays a Major Role in Proangiogenic Properties of Endothelial Progenitor Cells
Alpha-6 integrin subunit expression is increased by proangiogenic growth factors such as Vascular Endothelial Growth Factor or Fibroblast Growth Factor. This increase of α6 integrin subunit expression is correlated with an increased vascular tube formation in vitro by endothelial cells and their progenitors. We thus decided to study the role of α6 in vasculogenesis induced by human endothelial progenitor cells called Endothelial Colony-Forming Cells (ECFCs) after hindlimb ischemia. We used small interfering RNA (siRNA) to inhibit α6 expression on the surface of ECFCs, and we tested the effect of this inhibition of α6 expression in vitro and in vivo. In vitro, we observed that siRNA directed against α6 (siRNA α6) inhibited ECFCs adhesion or pseudotube formation on Matrigel® and migration on Boyden chambers (respectively p < 0.0001, p < 0.001 and p < 0.0001 vs. scramble siRNA transfected ECFCs or untransfected ECFCs). For the in vivo study, human ECFCs were injected intravenously in a nude mouse model of unilateral hindlimb ischemia. After 14 days, reperfusion induced by ECFCs transfected with siRNA α6 was significantly lower than in the other groups (p < 0.01 vs. ECFCs transfected with scramble siRNA and p < 0.001 vs. untransfected ECFCs). In addition capillary density in gastrocnemius muscles was lower for the group injected with ECFCs transfected with siRNA α6 (p < 0.001 vs. scramble siRNA). Therefore α6 integrin subunit is involved in ECFCs adhesion, migration and vascular tube formation in vitro and also in vascular repair after ischemia in vivo. We thus demonstrated the major role of alpha6 in proangiogenic properties of ECFCs.
Identification of the Host Cell Nuclear Proteome Modifications Induced After Infection of Hepatocarcinoma Cells with Herpes Simplex Virus Type 1
We are developing new HSV-1 derived oncolytic vectors, designed to strictly target and eradicate human hepatocellular carcinomas (HCC), the most common liver cancer of adults (see abstract Pourchet et al.). Upstream of these studies, we carried out fundamental proteome studies to define the interactions between the incoming HSV-1 particles and the target cells, i.e. the human hepatocarcinoma Huh7 cells that are being used as host cells to test the new oncolytic vectors. Indeed, although the HSV-induced alterations of cellular protein expression have been extensively investigated in epithelial cells, those induced in hepatocarcinoma cells are still missing.
HSV-1 replication occurs in the host cell nucleus. As a consequence, HSV-1-infected cells undergo a variety of changes including dramatic modifications of the nuclear architecture and composition. We therefore undertook a comprehensive analysis of the modifications of nuclear proteomes induced in Huh7 cells after infection with a wild type and an attenuated strain of HSV-1. We compared the synthesis rates of host nuclear proteins before and after infection. We combined 35S Methionine in vivo labelling of neo-synthesized proteins, two-dimensional gel electrophoresis separation of nuclear proteins. The proteins whose synthesis rate was modified after infection were characterized, then identified using image analysis software and tandem mass spectrometry and database searching respectively. The identified proteins are involved in different cellular pathways (DNA/RNA metabolism, stress, structure); some being down regulated, and others up-regulated after infection. The later proteins are believed to play essential roles in viral replication.
Inserm U844, Unité ImmunoRhumatologie Thérapeutique, CHU Lapeyronie, Université Montpellier , Montpellier, France
PBEF (Pre-B cell colony-enhancing factor/visfatin) is a hormone released by adipose tissue described as a new marker of inflammation in rheumatoid arthritis (RA) with pro-inflammatory and matrix-degrading activities. We previously showed that in vitro PBEF lead to the expression of pro-inflammatory mediators (IL-6, TNF) and matrix degrading enzymes (MMPs) in synovial fibroblasts and/or monocytes. The purpose of this study was to examine the therapeutic effects of systemic delivery of anti-PBEF siRNA lipoplexes in mouse arthritis pre-clinical model. Collagen-induced arthritis (CIA) was induced in male DBA/1 mice, and 0.5 mg/Kg small iinterfering (si)RNA against mouse PBEF (siPBEF) or non targeting siRNA (siCT) sequences formulated with the cationic liposome RPR209120/DOPE were injected intravenously weekly during arthritis progression. Systemic siPBEF delivery significantly decreased clinical disease activity scores of CIA as evidenced by paw swelling measures. Importantly, RNAi-mediated PBEF silencing significantly decreased IL-6 secretion in both sera and spleen, without altering serum anti-collagen (bCII) antibodies levels, bCII-specific T cell proliferation, and Th1- or Th2-type cytokines (IFN-γ and IL-5). Finally, the frequency of CD4+CD25+FoxP3+ T regulatory cells was increased in the liver of siPBEF-injected mice. Histological scores of inflammation and cartilage damage, bone erosion, and mRNA levels of pro-inflammatory cytokines and MMPs in the joints will be investigated, as well as immunohistochemical staining of PBEF in the joints. These results provide novel evidence that systemic PBEF inhibition prevents inflammation and joint destruction in murine arthritis model and might be proposed as novel therapeutic target for RA.
Improved Lentiviral Vectors for Intrathymic Gene Transfer Application
One of Severe Combined Immunodeficiencies (SCID) variant is caused by an absence of ZAP-70, a protein tyrosine-kinase recruited to the T cell receptor (TCR) following its antigenic stimulation. The manifestation of this deficiency is the absence of functional T cells.
The proof of concept for a novel gene therapy strategy of SCID has been done based on intrathymic gene transfer with lentiviral vectors. The vector used introduced the CD4 promoter together with proximal CD4 enhancer upstream of ZAP-70 together with GFP downstream of an IRES (Adjali, O. et al. 2005).
In order to make a better and safer lentiviral vector, several modifications should be incorporated: (i) modify the regulatory sequences to obtain a better specificity and appropriate expression level, (ii) use insulators to reduce the oncogenic risk and (iii) introduce a suicide gene.
The lentiviral vector used in the previous studies (Adjali, O. et al. 2005) has been modified to integrate a 1.1 kb regulatory sequence from the CD4 gene to improve its T cells specificity. Indeed, in previous studies GFP expression could be detected in some B cells. In contrast, expression in the B cell compartment was never detected with the longer regulatory sequences in transgenic mice. The modified vectors have been tested in vitro in different cell lines and in vivo, in NOD/SCID γcnull mice, showing their functionality.
INSERM, U 844, INM, Hôpital St Eloi, Université Montpellier 1, UFR de Médecine, Montpellier, France
The objective is to engineer biodegradable vectors for sustained RNA interference in vivo. Tumor necrosis factor (TNF)-α being a key mediator of inflammation in most of the immune-mediated disorders, we developed poly (DL-lactide-co-glicolide) (PLGA) microspheres to deliver small interfering (si)RNAs against TNF-α, and investigated their potential in the treatment of arthritis. PLGA microspheres were prepared by a modified multiple emulsion-solvent evaporation method, and then characterized in terms of morphology by scanning electron microscopy and mean diameter by laser light scattering. The encapsulation efficiency and in vitro release profile were investigated by UV spectrophotometry. TNF silencing was assessed at both mRNA and protein levels in the mouse monocytic cell line J774 following LPS challenge, using RT-qPCR and ELISA respectively. PLGA microspheres with a mean diameter of about 30 μm and characterized by a siRNA encapsulation efficiency of about 70% (actual loading of 0.2 nmol siRNA/mg PLGA) were obtained. In vitro release studies showed only a slight burst effect, followed by a slow and sustained release of the siRNA for over 40 days. LPS-induced TNF expression on monocytes was decreased compared with empty microspheres, as evidenced by 50% and 70% reduction of TNF mRNA and protein levels respectively. Following intra-articular injection of collagen-induced arthritic mice, TNF expression was still 50% less in anti-TNF microsphere-treated joints, 3 weeks after injection. Thus, slow and prolonged anti-TNF siRNA release makes possible the use of microsphere in treatment of chronically inflamed tissues, such as joints in case of rheumatoid arthritis.
Study and Characterization of Different Forms of Clusterin Using Different Strategies Including Exon Skipping
The aim of this study was to determine the role of clusterin (clu) in NF-κB regulation in rheumatoid arthritis (RA). Clusterin is a multifunctional protein that plays numerous roles in mammalian cells. The clu transcription process is complex and generates several protein isoforms differentially localized in the cell. By mean of transcriptomic analysis, we previously demonstrated that lower expression of clu both in tissues and cultured fibroblast-like synoviocytes (FLS) of RA patients compared to osteoarthritic patients.
Recently, we and others demonstrated CLU implication in NF-κB pathway regulation by IkB-α stabilization. We showed that CLU interacts with phospho- IkB-α and decreases the translocation of p50/p65 to the nucleus. To specify the interaction sites of CLU with its partners and to study the CLU isoforms roles, we generated several molecular constructs coding for various CLU regions of interest and test their role on NF-κB pathway and CLU subcellular localization. We have also developed a new approach of “exon skipping” in order to induce preferential expression of the nuclear spliced form of the gene. This strategy will allow a good understanding of nuclear forme poorly characterized.
Transient Expression of Genes Delivered to Newborn Rat Liver Using Recombinant AAV2/8 Vectors
Epitope Display on Adenovirus Capsid as a New Strategy of Vaccination
Recombinant adenovectors (Ad) have been used as vaccine vectors for antigen delivery. However, their efficacy in humans is confronted to the high prevalence of anti-Ad antibodies that hamper gene transfer. To circumvent this limit, we developed a strategy of vaccination based on Ad bearing epitopes. As a proof of concept, the sequence of two immunodominant epitopes (MHC-I-restricted epitope SIINFEKL or MHC-II-restricted epitope TSQAVHAAHAEINEAGR) derived from ovalbumin (OVA) has been genetically inserted into either the hexon (AdHOVA-I and AdHOVA-II) or the fiber (AdFOVA-I and AdFOVA-II) protein. All viruses were produced at titers comparable to an Ad with a wild-type capsid (AdWT). Moreover, OVA epitopes on Ad capsid were detected by ELISA or western blotting. Ad bearing OVA epitopes were injected intraperitoneally into C57Bl/6 mice (108 pfu/mouse) to document their ability to generate anti-OVA immune responses. First, an anti-OVA antibody response was observed following AdHOVA-II or AdFOVA-II injection that increased after a second virus injection. Interestingly, anti-OVA antibody levels were higher in mice injected with AdFOVA-II than in mice injected with AdHOVA-II, suggesting a better epitope exposure/accessibility at the viral surface. In parallel, we observed that splenocytes from AdHOVA-I- but not from AdWT-injected mice were able to produce IFNγ upon restimulation with OVA-I peptide, thereby demonstrating that virus injections have elicited memory CD8+ T-cells.
Altogether, these data suggest that Ad bearing heterologous epitopes on their capsid trigger efficient humoral and cellular responses. Efforts are currently made to better understand how the site of epitope insertion (hexon or fiber) influences vaccination efficacy.
IFR3 Vectorology Platform—Lentiviral Vectors Production
Re-engineered viruses are a widely used tool for nucleic acid delivery, transgene expression, and gene therapy. Among the various available viral vectors, lentiviral vectors allow long term, stable in vitro and in vivo gene transfer. They can transduce both dividing and quiescent cells in a stable manner. Their tropism can be modified by changing their envelope glycoprotein. The “Institut Fédératif de Recherche n°3” (IFR3) based in Montpellier, has recently set up a vectorology platform which is providing a service of lentiviral vectors production to the scientific community (http://ifr3.igf.cnrs.fr/plateforme/index.php?page = vectorologie).
The service includes: (1) The purification and concentration of the viral particles produced after co-transfection of the gene transfer plasmid (supplied by the customer), the envelope and packaging plasmids into HEK-293T cells.
(2) The titration of the lentiviral particles. The p24 capsid protein concentration is measured by ELISA to assess the amount of total particles produced. When the transfer vector bears a reporter gene (eGFP, DsRed, etc.), we perform FACS analysis to determine the infection titer.
(3) Finally, a safety test is performed to check the replication-defectiveness of the viral particles.
The platform also provides a support to costumers to define the best experimental strategies to set up in their research programs (pseudotypes, use of specific promotors, choice of the best vector, etc.).
A research and development activity has also been set up in order to optimize the current vectors and investigate the use of other viral systems for gene transfer.
The vectorology platform is open to academic and private partners.
Inserm U781, Hôpital Necker -Enfants Malades, Bât M. Lamy, 149, rue de Sèvres, 75015 PARIS, e-mail: olivier.danos@inserm.fr
Most cases of Duchenne muscular dystrophy are caused by mutations in the dystrophin gene that disrupt the mRNA reading frame. In some cases, forced skipping of an exon can restore the reading frame, giving rise to a shorter but still functional protein. To induce exon skipping, antisense sequences directed against splice sites of pre-mRNA can be used. To enhance their stability and achieve long term correction, these sequences can be attached to small nuclear RNAs, such as U7 or U1 which can be delivered using viral vectors. Our recent work has demonstrated that AAV and lentiviral vectors efficiently deliver therapeutic antisense sequences for exon skipping in the skeletal muscle of mice, dogs, and in cells from Duchenne patients. This work has laid the foundation for clinical trials now in preparation but face the problem of large quantities of vector to be administered. Reducing this dose of rAAV is therefore a central issue that must be addressed by increasing the overall specific activity and bioavailability of each vector particle. This study focuses on optimizing the expression levels of therapeutical U7snRNAs. We describe modifications of the U7DTex23 expression cassette used for the skipping of exon 23 in the murine dystrophin pre-mRNA, in which a muscle specific transcriptional enhancer has been added, or new cassettes based on RNA polymerase III transcriptional elements from the U6 or 7SK genes. The new cassettes are packaged into AAV2/1 vectors and tested in dose response studies in cultured myoblasts or following direct injection into the skeletal muscle of mdx mice.
Inserm U614, University-Hospital , Rouen, France
Carcinoembryonic antigen (CEA) is a potential target for antigen-specific immunotherapy since it is frequently overexpressed in human carcinomas. Moreover, an epitope derived from CEA, designated CAP1 (YLSGANLNL), has been proposed as naturally processed and presented by tumors in the Human Leukocyte Antigen (HLA) A*0201 context. For future immunotherapy strategy development in colorectal cancer (CCR) patients, we attempted to dissect the HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) response against CEA. To do so, we took advantage of stable and potent artificial antigen presenting cells (AAPCs), which have already been shown to efficiently activate CTLs against tumor-derived epitopes after transduction with the antigenic peptides or full-length proteins. We found that AAPCs genetically modified to express CAP1, the agonist peptide CAP1-6D or the whole CEA protein were not able to activate CAP1-specific CTLs from HLA-A*0201+ healthy donors or patients with colorectal carcinoma, even after multiple stimulations. In addition, we showed that a CAP1-specific T cell clone, obtained after multiple stimulations of T cells of a HLA-A*0201+ healthy donor in vitro with autologous antigen presenting cells, recognized CEA− HLA-A*0201+ tumors transduced with a minigene encoding CAP1 but failed to react against HLA-A*0201+ tumor cells that naturally or ectopically expressed high levels of CEA. Finally, AAPCs expressing the whole CEA protein did not induce any specific CTL response against CEA+ HLA-A*0201+ tumor cells. Our data indicate that CAP1 is not efficiently processed and presented by CEA+ tumor cells and furthermore, that CEA antigen, regardless of the epitope, is not an appropriate target for T cell-based immunotherapy, in the HLA-A*0201 context.
Inserm U614, University-Hospital , Rouen, France
Colorectal cancers (CRCs) with microsatellite instability (MSI) due to replication errors (RERs) are characterized by a high density of tumor infiltrating lymphocytes (TILs) and a good prognosis, and TILs extracted from MSI tumors have recently been shown to be able to recognize HLA-restricted frameshift peptides associated to MSI, and to specifically kill MSI CRC cells.
With the goal of developing new immunotherapy strategies in MSI CRC patients, we attempted to characterize some of the potentially immunogenic antigens inducing the T cell response during the development of a MSI tumor. We determined in 61 MSI CRC, using a fluorescent multiplex PCR comparative analysis, the relative frequency of frameshift mutations within 19 target genes and we analyzed the correlation of these frameshift mutations with TIL density. The four most frequently mutated genes were ACVR2 (92%), TAF1B (84%), ASTE1/HT001 (80%) and TGFBR2 (77%). The vast majority (95%) of the tumors exhibited at least 3 frameshift mutations, and the number of frameshift mutations was associated with tumor progression. TIL density was associated with the overall number of frameshift mutations and the presence of frameshift mutations within 3 target genes: ASTE1/HT001, OGT and PTEN. The frequency of certain frameshift mutations, their presence in all tumor cells, and their association with TIL density, notably for ASTE1/HT001, OGT and PTEN, strongly argue for the clinical relevance of immunotherapy in MSI CRC.
Inserm U905, Rouen University-Hospital , Institute for Medical Research, Cancéropôle Nord-Ouest
Adoptive immunotherapy based on in vitro activation and expansion of tumor antigen-specific cytotoxic T lymphocytes (CTLs) is a very promising approach against cancer. Numerous techniques have been proposed to activate such CTLs, including the culture of peripheral T lymphocytes with allogeneic tumor cells (Labarrière et al., Cancer Immunol Immunother, 2008). Our team has developed a strategy based on the use of artificial antigen-presenting cells (AAPCs) co-cultured with peripheral T lymphocytes (Latouche and Sadelain, Nat biotechnol, 2000). These AAPCs have been transduced to express molecules involved in the immunological synapse, restricted to the HLA-A2.1 molecule, and essential to activate CTLs against MART-1, auto-antigen which is overexpressed in melanoma. We defined, using blood from A2.1 + healthy donors, the optimal conditions for proliferation and purification of MART-1-specific CTLs, using AAPCs and anti-MART-1 phycoerythrin (PE)-coupled pentamer staining combined with anti-PE magnetic bead sorting of specific cells. We will now apply this strategy to expand MART-1-specific CTLs from stage II, III or IV melanoma patients, and study quantity, function (cytotoxic assays against MART-1-pulsed or melanoma-derived cell lines), and phenotype (IFN-γ, TNF-α, IL-2 production and Vβ T Cell Receptor repertoire) of the obtained CTLs.
IRES-Based Vector Co-Expressing FGF2 and CYR61 Provides Synergistic and Safe Therapeutics of Lower Limb Ischemia
An In Vitro Beating Heart Model for Long-Term Assessment of Experimental Therapeutics
Within the framework of studies aiming at regenerative medicine in cardiology using embryonic stem cells, we have designed an in vitro model to analyse cells grafted into the myocardium. This model is based upon organotypic rat ventricular slices maintained in culture at air-medium interface on semiporous membranes. Survival and differentiation of human cardiomyocytes derived from embryonic stem cells could thus be assessed over months. As a major side result of this set-up, we observed that ventricular tissue slices not only exhibited normal histology, but also rhythmic contractions till the end of the experiments (up to 3 months). Similar results were obtained using ventricular slices obtained from two human foetuses at 8 and 9.5 weeks of age. Calcium transients were associated to the beating, and were modulated in a dose-dependent manner by epinephrine. Organotypic slice cultures on semiporous membranes appear as a relevant in vitro heart model for long term histological and physiological studies.
Biothérapies Hépatiques INSERM U948 , EA 4274, France
We report the anti-tumour response measured in an in situ tumour model of hepatocellular carcinoma (HCC) in mice, after direct immunization with a synthetic vector consisting of plasmid DNA encoding the tumour-associated antigen alpha-fetoprotein (AFP), associated to an amphiphilic block copolymer (704).
Therapeutic efficacy was evaluated in a chemically induced hepatocarcinogenesis model where 15 day-old mice receive an injection of Diethylnitrosamine (DEN). AFP-expressing tumour nodules are detected 5 months later. Groups of 10 DEN-treated mice received two intramuscular injections of synthetic vector (DNAmAFP/704) at 4 and 5 months and were sacrificed at 8 months for detailed analysis of the liver. The AFP-specific immune response was analyzed by IFNγ ELISpot on CD8 + T cells incubated with the mAFP immunodominant peptide NEFGIASTL identified in this study.
Immunization with DNAmAFP/704 resulted in a long-lasting AFP-specific CD8 + T cell activation detected 3 and 8 weeks post immunization (95.0 ± 67.6 and 60.0 ± 30.4 spots/million CD8 + T cells). In therapeutic experiments, liver from DNAmAFP/704-immunized DEN-mice at sacrifice displayed less and smaller tumour nodules compared to control DEN-mice (50.3 ± 38.7 vs. 144.0 ± 86.7 mm2 tumour surface, respectively, p = 0.006) corresponding to a 65% reduction of the tumour burden. Interestingly, AFP-expression level was reduced in the remaining isolated tumours from DNAmAFP/704-immunized mice compared to control mice (0.008 ± 0.006 vs. 0.021 ± 0.013, p = 0.03). Regulatory T cell depletion was also tested on this HCC model. A long-term depletion of Treg cells resulted in a 72.97 ± 7.35% reduction of the tumour burden. Finally, there was no synergistic effect when the two therapeutic approaches were combined (61.27 ± 9.68% reduction).
DNAmAFP/704 is a potent synthetic vector for HCC immunotherapy in a chemically-induced HCC model. Considering the high relevance of this model to the human disease, we advocate that DNA-AFP/704 may be tested in a phase I/II clinical trial for HCC immunotherapy.
Inserm U905, IFRMP 23 and Institute for Biomedical Research (IHURBM), University of Rouen , France
Recombinant adeno-associated viruses (rAAV) are efficient vectors for gene transfer to muscle but their efficacy is limited by anti-vector and anti-transgene immune responses. The development of novel approaches to counteract these responses would benefit from a better understanding of the mechanisms governing tolerance to muscle-expressed proteins. For this, we developed transgenic SM-Ova mice that selectively express ovalbumin (Ova) in skeletal muscle and are naturally tolerant to Ova (1) and evaluated the capacity rAAV-Ova to induce an anti-transgene immune reaction. Intra-muscular injection of 1011 rAAV-Ova particles induced a strong immune response in control B6 mice characterized by the expansion of specific anti-OVA CTLs (assayed by H2-Kb/OVA257–264 pentamer staining) that culminated in the spleen 14 days after gene transfer (10% ± 5 of CD8+). These CTLs were fully functional as assessed by their capacity to reject transplanted Ova-expressing EG-7 tumors cells. In striking contrast, SM-Ova mice treated in the same condition did not develop any detectable CTL response and were not able to reject EG7 cells. Further depletion of CD4+CD25+ Tregs using PC-61 antibody did not allow the development of an anti-Ova CTL response in SM-Ova mice after rAAV-Ova administration. Hence, rAAV vectors can not break T cell tolerance to a muscle-expressed auto-antigen. Additional experiments are ongoing to better characterize the tolerance mechanisms in this model and to try to induce tolerance in mice that do not express Ova as an auto-antigen. The present results may be relevant to gene therapy of hereditary myopathies where the defective gene is still translated into a non functional but yet tolerogenic protein fragment, or in the presence of revertant myofibers.
(1) Calbo S., Delagreverie H, Arnoult C, Authier FJ, Tron F and Boyer O. 2008. Functional tolerance of CD8+ T cells induced by muscle-specific antigen expression. J Immunol 181:408–417.
Feinberg Cardiovascular Research Institute, Northwestern University , Chicago, IL 60611, USA
Institut de Génétique Moléculaire de Montpellier , CNRS 5535, Montpellier, France
Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.
Genetic instability: rare diseases and cancers, CNRS FRE 3086, Faculty of Medicine , Nice, France
Xeroderma pigmentosum (XP) is a rare, recessively inherited genetic disorder characterized by an extreme sensitivity to sunlight and a severe predisposition to UV-induced skin cancers. Cells from XP patients are deficient in nucleotide excision repair, a repair mechanism which removes UV-induced DNA lesions. Among the seven classic XP complementation groups known to date (XP-A to XP-G), XP-C is the most common one in Europe and North Africa and, in contrast to other groups, XP-C patients do not have neurological problems. In the absence of any curative treatment, ex vivo cutaneous gene therapy is a promising approach for XP-C patients. In order to meet the specifications inherent to skin graft in vivo and particularly to avoid any immunogenic response, we have set up a strategy allowing the selection of genetically modified clonogenic keratinocytes using a small epidermal protein, CD24. Keratinocytes from XP-C patients were transduced with retrovirus driving the expression of both wild-type XPC and CD24 cDNAs, thereby allowing the safe selection of corrected XP-C keratinocytes. In these cells, re-expression of the XPC protein was accompanied by recovery of full DNA repair capacity and cell survival properties after UVB irradiation. Furthermore, both in vitro and in vivo regenerated skin from corrected XP-C keratinocytes recovered normal repair capacity of UVB-induced DNA lesions. Our results open the first rational perspective toward ex vivo cutaneous gene therapy of the XP-C disease.
Expression Shift in the Intercellular Signalling Network During Pluripotency Induction in Human Post Natal Fibroblasts
Lipoplexes Targeting the CD44 Hyaluronic Acid Receptor for Efficient Transfection of Breast Cancer Cell
Lipoplexes containing a hyaluronic acid-dioleoylphosphatidylethanolamine (HA-DOPE) conjugate were designed to target the CD44 receptor on breast cancer cells. Cationic liposomes composed of a mixture of [2-(2,3didodecyloxypropyl)hydroxyethyl] ammonium bromide (DE) and dioleoylphosphatidylethanolamine (DOPE) with or without HA-DOPE were prepared, characterized, and used to form a complex with plasmid DNApCMV-luc. Lipoplexes displayed a negative zeta potential and a mean diameter between 250–300 nm. Cytotoxicity and transfection efficiency of the lipoplexes were determined on the MDA-MB-231and MCF-7 breast cancer cell lines. Cytotoxicity was not modified by the presence of HA-DOPE. However HA-DOPE increased the level of transfection on CD44-expressing MDA-MB-231 cells compared to the MCF-7 line, which express very low levels of CD44. The transfection on the MDA-MB-231 cells was highly inhibited by anti-CD44 Hermes-1 antibody but not by the non-specific anti-ErbB2 antibody. In conclusion, cationic liposomes containing the HA-DOPE conjugate mediated good transfection on CD44 expressing cell lines in culture.
Genethon, 1, rue de l'Internationale, 91000 Evry, France
Titin is a giant protein expressed in both skeletal muscles and heart. Several pathogenic mutations were identified in its last coding exon. The most frequent mutation commonly referred to as FINmaj, results in the replacement of 4 amino acids and affects a subset of patients in Finland. The mutation causes a Tibial Muscular Dystrophy (TMD) when present on one allele and a Limb Girdle Muscular Dystrophy phenotype 2J (LGMD2J) when present on both alleles.
To obtain a model for studying the physiopathology of these diseases and evaluating therapeutic strategies, we introduced the FINmaj mutation in the murine genome by a knock-in strategy by homologous recombination. This model reproduces symptoms of both the human TMD and LGMD2J.
Considering the large size of the titin cDNA (about 100kb), classical gene transfer strategies are not feasible. To bypass this hurdle, we are testing spliceosome-mediated RNA trans-splicing (SMaRT) to replace mRNA titin last exon. HER911 cells expressing a titin minigene with the FINmaj mutation in the last exon were transfected with a plasmid coding for a 3′ pre-trans-splicing molecule encoding wild-type last exon. RT-PCR analyses showed that specific replacement of the mutated 3′ portion of the minigene TTN transcript has occurred through trans-splicing. We could see the reduction of the level of mutant mRNA and the apparition of the wild-type corrected gene. Experiments to delivered trans-splicing RNAs in the FINmaj mouse model using Adeno-Associated Viral vectors are currently investigated in our laboratory.
INSERM U972, Bicêtre Hospital , Kremlin-Bicêtre, France
Ex vivo gene therapy is becoming an alternative to liver transplantation for the treatment of liver metabolic diseases since lentiviral vectors have the ability to transduce efficiently human hepatocytes. However, hepatocyte engraftment efficiency is poor in rodent models and this strategy needs to be evaluated in large animal models such as non-human primates a) to define an approach which induces hepatocyte engraftment and proliferation b) to evaluate the long-term expression of the transgene in situ.
The aim of this work was to investigate whether reversible portal vein embolization combined with ex vivo lentivirus-mediated gene transfer is a safe and effective approach for successful hepatocyte engraftment in Macaca and whether GFP expression under the control of the human Apolipoprotein A-II promoter persisted at long-term.
Simian hepatocytes were isolated after left lobe resection and labelled with Hoechst dye or transduced with APOA-II-GFP lentiviruses then transplanted via the inferior mesenteric vein. The left and right anterior portal branches of animals were embolized with gelfoam. The volume of non-embolized liver volume increased and hepatocytes significantly proliferated (10.5 ± 0.4% on day 3 post-embolization). Liver repopulation after transplantation with Hoescht-labelled hepatocytes was 7.4 ± 1.2%. It was 2.1% ± 0.2% with transduced hepatocytes with a transduction efficacy of 35%, suggesting a three-fold increase in repopulation for these transplanted cells. Transgene expression persisted at 16 weeks post-transplantation. Conclusion: we have developed a new approach to significantly improve hepatocyte engraftment and to express a transgene at long-term in Macaca, which could be suitable for clinical applications.
The Pleiotrophin Inhibitor P111 Increases the Expression of the Pro-Apoptotic Protein CHOP and Radio-Sensitizes Human Glioblastoma Cells In Vivo
P111 (HARPΔ111–136) is a truncated protein lacking the last 26 amino acids of the pleiotrophin. Over-expression of this recombinant protein in the human glioblastoma cell line U87MG inhibits the proliferation of these cells in vitro, and tumor growth and angiogenesis in vivo. This study focused on identifying the underlying mechanisms for the observed antitumor effect using Ad-P111, an adenovirus expressing the truncated protein. We used the findings of microarray analysis to demonstrate that P111 triggers endoplasmic reticulum (ER) stress and induces the expression of the protein CHOP leading to apoptosis. SiRNA-mediated CHOP gene silencing abolished P111-induced apoptosis. Moreover, Ad-P111 increased the expression of the death receptor DR5 and sensitized U87MG cells in vitro to TRAIL and radiation-induced apoptosis with activation of caspases 3 and 8. In vivo, the combination of Ad-P111 and radiation therapy (bitherapy) resulted in a striking inhibition (92%) of the growth of established U87MG xenografts, resulting from the potent synergistic effect of bitherapy on U87MG tumor apoptosis. Based on these and previous data, P111 appears to be a promising new agent that could, in combination with standard treatments, improve the outcome of glioblastoma patients.
Properties of DNA Binding Domains to Specifically Locate Transgene in the rRNA Genes
Gene transfer technologies offer promising prospects for the treatment of several human diseases. The integration vectors, derived from transposons or retroviruses, are used to maintain and to sustain transgene expression. But, to date, integration vectors integrate randomly within the genome of cells. To be used in gene therapy, new molecular tools are required to target transgene integrations in a safe locus to confer innocuity on the genetically-modified cells (see communication Solenne Bire), and to prolong expression of the therapeutic cassette. Proofs of concept have shown that chromosomal gene clusters encoding the ribosomal RNA (rRNA) correspond to optimal integration loci.
Our work investigated the properties of two kinds of DNA binding domain (DBD) able to bind specifically within a 100-bp region of the rRNA genes that is highly conserved among eukaryotes. Two Myb-like DBDs, originating from the endonucleases encoded by R2 non-LTR retrotransposons (NterR2P), and three synthetic polydactyl zinc finger domains were designed and synthesized using database information. Our findings demonstrate that NterR2P DBDs are promising candidates since they (i) specifically recognize their DNA target with high affinity, (ii) work as monomers, and (iii) have a 20-bp binding site that is long enough to be specific in the human genome.
So, this DBD could be used with several types of vectors. The efficiency of such a targeting system will nevertheless have to be extensively investigated to confirm its specificity and innocuity for the purposes of gene therapy.
mRNA as a Source of Transposase to Secure Transgene Integration
In order to promote
Our goal is to calibrate mRNA transposase transfection and expression in a relative short period of time allowing only
These experiments are the prerequisite to produce secure ex vivo
Comparison of the Bone Forming Capacity of Different Cell-Based Tissue-Engineered Constructs in Sheep
An animal ectopic model has been developped in sheep to test in a well controlled environment the bone forming capacity of clinically relevant volumes of granular biomaterials loaded with stem cells (Bensidhoum et al., Termis 2008). In this model, the materials to be tested are placed in a pouch delineated by a subcutaneously PMMA induced membrane. The purpose of the present study was to determine and compare in this model the osteogenic potential of clinically relevant volumes of four different granular constructs engineered from materials which are currently available for bone tissue engineering purposes in the clinical setting.
Autologous-derived bone marrow stem cells (MSCs) were loaded onto each of four different particulate materials (coral Porites, coral Acropora, β-TCP and bank-bone) prior to. After 8 weeks, explanted pouches were analysed, with radiography and undecalcified histology for evidence of material resorption and bone formation
AP-HP, Groupe Hospitalier Pitié -Salpêtrière, Service de Biothérapies, Paris, 75013, France
Human natural regulatory T-cells (nTregs), defined as CD4 + CD25 + FOXP3 + CD127low cells, have a great potential in cell therapy for induction of tolerance against allogenic cells and/or transgenes and for the control of auto-immune diseases (AID). Recently, we reported a clinical grade process (Guillot M. et al., 2008) that gave rise to a cell product composed of 60–70% FOXP3+ nTregs and that has a clear immunosuppressive activity. However, before administering such cells to patients, it has to be considered that: (i) the potential contamination of cultured Tregs with activated T-cells that also expressed CD25, and could be enriched in patients with AID, cannot be excluded; and (ii) the induction of a prolonged uncontrolled immunosuppression might be deleterious. Hence, it might be valuable to improve the safety of nTreg-based therapeutic products by transducing them with a Thymidine Kinase (TK) suicide gene capable of conditionally killing undesired dividing cells in the presence of ganciclovir (GCV). We engineered a lentiviral vector co-expressing TK and the human CD90 reporter gene. nTregs were selected, activated overnight, infected with lentiviral supernatant and cultured for 3 weeks. While ≥ 60% of nTregs were transduced, cells were then enriched by immunomagnetic selection using anti-CD90 biotinylated beads giving rise to ≥ 95% CD90 + cells. Transduced CD90 + cells could suppress the proliferation of autologous effector T-cells and could be ablated by GCV. Thus, the use of “secured nTregs” should be taken into consideration for future cell therapy clinical trials based on nTregs.
Ultra-Pure Retroviral Production for In Vitro and In Vivo Applications
Vectalys is a gene delivery platform providing viral recombinant vectors for gene target identification and validation. With these tools, Vectalys provides an integrated and flexible service going from DNA to animals. Retroviral production and purification are one of our core activities to improve ex vivo and in vivo gene transfer. These applications require development, well-characterisation, standardization and up-scaling of production and purification methods. Crude supernatants contain contaminants that need to be removed to increase the potency and safety of the final product. Impurities usually come from serum, plasmid DNA or are released by intact or disrupted producer cells.
Vectalys has optimized a robust process for the production of high titer viral supernatant in serum free medium. Following the production step, two different processes of purification have been developed depending on the applications. First, ultrafiltration allows rapid and significant concentration/purification of large lentiviral vectors volumes with high vector recovery. This leads to 1E7 to 1E9 TU/ml purified samples and offers efficient transduction of primary and stem cells for in vitro or ex vivo strategies. Second, for in vivo experiments, the need in higher purity is required. So, Vectalys has included a chromatography step to get ultra-pure viral preparations. These stocks samples are injected into rat and mouse tissues in order to validate gene transfer efficiency, diffusion and toxicity.
Transplantation of Human Liver Adult Progenitors in NOD/SCID Mice: A New Therapy for Liver Failure
UMR INSERM 745, Faculté des Sciences Pharmaceutiques University Paris-Descartes , Paris
Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder of the CNS due to the deficiency in arylsulfatase A (ARSA) involved in the degradation of sulfatides that results in lethal demyelination and neuronal degeneration. The rapidity of the neurological deterioration in the infantile form of MLD requires to deliver rapidly the ARSA enzyme in the brain, possibly through the intracerebral injections of gene therapy vector encoding human ARSA.
Using an AAV vector of serotype 5 encoding ARSA, we demonstrated correction of MLD mouse phenotype. Before engaging towards potential clinical application, we evaluated the tolerance and efficiency of intracerebral delivery of AAV5-ARSA vector in the brain of Macacus fascicularis.
To optimize our clinical therapeutic strategy, we then evaluated a new serotype of AAV (Rh10) that was shown to result in better diffusion in mouse and rat brain after intracerebral injections. Symptomatic MLD mice were injected bilaterally in the striatum. We demonstrate a stronger expression throughout the all brain compared to AAV5 (7 to 8 fold higher) as well as a better correction of mouse phenotype with AAVRh10. Despite AAV transduction limited to neurons in vivo, we also prove correction of specific sulfatide accumulation in oligodendrocytes. Our data indicate that significant amount of ARSA enzyme was secreted by transduced neurons and efficiently recaptured by oligodendrocytes resulting in a complete biochemical correction 2 months after injection.
Altogether, these data demonstrate substantial improvement of our gene therapy vector, an encouraging step towards clinical application in MLD patients.
Searching for Genetic Variants Involved in the Progression of Cerebral Demyelination in X-Linked Adrenoleukodystrophy (ALD): A Way to Improve Hematopoietic Stem Cell Transplantation
ALD is a severe neurodegenerative disease, characterized by a progressive demyelination within the central and peripheral nervous system, adrenal insufficiency (Addison's disease) and accumulation of very-long-chain fatty acids (VLCFA) in plasma, fibroblasts and tissues. ALD is a clinically heterogeneous disorder ranging from asymptomatic persons to adrenomyeloneuropathy (AMN), and cerebral ALD leading to death. ALD results from mutations in ABCD1 gene encoding the adrenoleukodystrophy protein (ALDP). But there is no correlation between ABCD1 genotypes and ALD phenotypes, suggesting that modifier genes could influence the course of this disease. Allogeneic hematopoietic stem-cell transplantation (HSCT) is currently the only effective long-term treatment for cerebral ALD however only when performed at an early-stage of disease. Unfortunately, mechanisms acting on ALD evolution are currently unknown.
Taking together, these observations prompt us to search for modifier genes in order to better understand the pathogenesis and increase the efficacy of HSCT to treat cerebral ALD. Among them, ELOVL1, coding for an elongase involved in the elongation of VLCA, appears to be a promising candidate gene. We genotyped ELOVL1 polymorphisms in control, AMN, and cerebral ALD patients. We detected a polymorphism in ELOVL1 promoter associated with cerebral ALD (P = 0.017, OR = 2,092). Functional studies are undertaken to know if this polymorphism could influence ELOVL1 expression and accumulation of VLCA in tissues.
This polymorphism may be useful to better predict the evolution of ALD and thus, to improve the efficacy of HSCT to treat cerebral ALD.
Laboratory of Bioengineering and Biomechanics for Bone Articulation (UMR CNRS 7052), Faculty of Medicine University Paris Diderot , 10 Avenue de Verdun, 75010 Paris, France
Mechanical loading plays an important role in bone homeostasis. Bone mass loss to microgravity is well illustrated using the hindlimb unloading (HU) through tail-suspension model. The aim of this study was to assess growth and differentiation potential of bone-marrow derived mesenchymal stem cells (MSCs) from the femurs of skeletal unloaded rats. It is hypothesized that HU induced bone alterations are related to an altered proliferation and differentiation potential of the MSCs. A multi-scale approach including cellular, molecular and 3D-morphometric analysis was adopted. Four-months old female Wistar rats were randomized to 2 groups of 5 animals to be either tail-suspended for 9 days or to act as controls (CTL). Bone marrow samples isolated from the femora were cultured for four passages. Fibroblastic colony formation and growth potential was tracked. Osteogenic and adipogenic differentiation potential was assessed by means of phenotypic staining and real time quantitative PCR analysis for alkaline phosphatase, Runx2 and osteocalcin expression. Bone volume and structure changes in the proximal tibia in response to unloading were quantified by micro-computed tomography (micro-CT). The results revealed that 9 days HU negatively affected the number of CFU-F. Meanwhile, HU did not decrease the growth potential of the MSCs neither their osteogenic and adipogenic differentiation potential. No significant differences in tibial morphometric parameters between 9-days HU and CTL rats could be observed by micro-CT analysis. The results suggest that microgravity conditions primarily affect the quantity of CFU–F, but not their differentiation ability.
Modelisation of Alzheimer Disease by In Vivo Inhibition of Cholesterol-24-Hydroxylase
A hallmark of Alzheimer disease (AD) is an abnormal accumulation of amyloid-β (Aβ) peptides in specific brain regions. Aβ peptides are generated by sequential proteolytic processing of the amyloid precursor protein.
The production of amyloid fragments seems to be paralleled by cholesterol content in cultured cells and in transgenic mouse models of AD, yet mechanisms by which cholesterol modulates the amyloidogenic pathway are poorly understood.
The Cyp46A1 gene that encodes cholesterol-24-hydroxylase, controls the efflux of intracerebral cholesterol by converting it into 24S-hydroxycholesterol, that crosses the blood-brain barrier and is degraded in the liver.
We demonstrated that selective overexpression of CYP46A1 in neurons of APP23, mouse model of AD, reduces Aβ peptides, amyloid deposits in vivo and improves cognitive deficit.
To further evaluate if viral AAV vector delivery of CYP46A1 could find therapeutic application in AD, we conversely studied the effect of cholesterol 24-hydroxylase down-regulation on the pathological features of the APP23 mouse.
RNA interference strategy was used to down-regulate the expression of CYP46A1 in vivo. Three shRNA were selected among 20 for their efficacy to inhibit CYP46A1 expression in vitro. Among the three corresponding AAV5-shRNA produced, AAV5-sh271 was chosen for its best capacity to decrease CYP46A1 expression and brain 24S-hydroxycholesterol (−40%) in vivo after injection in the cortex and the hippocampus of C57B6 mice. Injections in APP23 mice was associated with an threefold increase of Aβ 40/42 peptides, and major hippocampal lesions as early as one month after injection, Further investigations are in progress to understand the mechanisms of neuronal loss.
These preliminary inhibition results confirm that CYP46A1 is a relevant potential therapeutic target in Alzheimer disease.
INSERM U935, University Paris 11, Villejuif, France
Recent data suggest that there is a major discrepancy between the expression of BCR-ABL in primitive hematopoietic stem cells as compared to their differentiated counterparts where the expression is much lower. This high expression level predisposes this compartment to a major genetic instability leading to a mutator phenotype. T315I is the most problematic of these mutations leading to a resistance to all three clinically available tyrosine kinase inhibitors (TKI). In order to study the effects of both mutated and non-mutated BCR-ABL in a primitive stem cell context, we have transduced retrovirally both BCR-ABL and BCR-ABL-T315I, into day 5 embryoid bodies (EB's) derived from D3 murine embryonic stem (ES) cells. The transduction efficiency was found 10 to 45% as evaluated by GFP expression at day 3 post-transduction. EB's were then dissociated and put in hematopoietic differentiation conditions using clonogenic assays and in liquid culture in the presence of Op9 stroma and hematopoietic growth factors. In clonogenic assays, BCR-ABL and T315I- transduced cells induced GFP + growth-factor-independent colonies. In liquid conditions, empty vector and BCR-ABL-transduced cells underwent 8-population doublings whereas BCR-ABL-T315I transduced cells underwent 16-population doublings in 4 weeks. Interestingly, phenotypic analysis demonstrated the appearance of CD45 + CD34 + cells in BCR-ABL-transduced cells as early as week 2–3 whereas this hematopoietic differentiation appeared to be delayed in BCR-ABL-T315I-transduced cells. Current experiments underway are testing the long-term repopulating ability of BCR-ABL and BCR-ABL-T315I-expressing cells in NOD/SCID reconstitution assays as well as the occurrence of ABL-kinase mutations in the presence and in the absence of TKI.
INSERM U972, Hôpital de Bicêtre , 80, rue du Général Leclerc 94276 Le Kremlin-Bicetre Cedex, France
Hepatocyte transplantation is an attractive alternative to liver transplantation for the treatment of metabolic diseases. However there is a lack of donor organs and isolated mature hepatocytes are difficult to manipulate in vitro. Human pluripotent cells, either embryonic stem cells (ES) or induced pluripotent stem cells (iPS), represent a potential source of hepatocytes, but the conditions to expand and differentiate them in conditions transposable to the clinics remain to be defined.
The aim of this work is to compare the derivation of human ES and iPS into hepatic progenitors by reproducing the major steps of embryonic liver development in vitro.
We generated 40 iPS cell lines from human foreskin fibroblasts, using Moloney-derived retroviral vectors containing the sequences coding for human Oct4, Sox2, Klf4, c-Myc. All lines expressed pluripotency markers (Oct4, Sox2, Nanog, TRA1-60, SSEA-3) as determined by immunocytochemistry.
We set up chemically defined conditions, devoid of serum, to derive human pluripotent cells into endodermal cells expressing specific markers (Goosecoid, Mixl1) in absence of feeder layer. We then generated hepatic progenitors within 10 days from these cells. Cell phenotype was analysed by QRT-PCR and immunocytochemistry which showed that both ES and iPS-derived cells expressed hepatic markers (Albumin, AFP, CK19, AAT, HNF4). Differentiation of these hepatic progenitors into more mature hepatocytes is currently being assayed.
We will generate pluripotent cells from fibroblasts isolated from patients with metabolic diseases and differentiate them into hepatic cells using our conditions. These cells will be useful to evaluate in mouse models ex vivo gene therapy applications.
Inserm U 972, Hôpital de Bicêtre , France
Until now, clinical trials, based on the permanent secretion of the therapeutic human Factor IX (hFIX), were not satisfying because of the low and transient levels of circulating hFIX obtained. Our approach is based on time and space regulated secretion of hFIX stored in platelet α granules in response to a hemorrhagic stress. The strategy consists in the engraftment of hematopoietic stem cells (HSCs) transduced with a lentivector encoding hFIX, fused to an α granule targeting peptide, driven by a megakaryocyte (MK)-specific promoter.
We had shown that: (i) the human glycoprotein Ib (hGPIb α) promoter restricted GFP expression to the human MKs differentiated from transduced HSCs, (ii) GFP fused to the Platelet Factor 4 (PF4) was targeted to human MK α granules.
We demonstrated recently (i) that hFIX fused to a PF4-derived granule-targeting peptide was addressed to the α granules of human and mouse MKs, as shown by electron immuno-microscopy analyses and (ii) using transplantation experiments that the hGPIb α promoter strictly restricted GFP expression to mouse mature MKs and to circulating platelets.
Our approach thus validated, we have performed transplantations of HSCs transduced with the therapeutic lentivector into irradiated hemophilic mice. After hematological reconstitution, we are currently assessing both secretion and activity of hFIX synthesized in vivo. The coagulant activity of hFIX is tested by thrombin-generation time measurements and by chromogenic assay in a platelet releasate assay. Correction of the hemophilia syndrome is studied by tail-clipping bleeding-time assays. Preliminary results will be presented.
Development and Characterization of a New Model of Wiskott Aldrich Syndrome Induced by RNAi in Humanized Mice
Wiskott-Aldrich syndrome (WAS) is rare genetic disease caused by lack of WAS protein (WASp) in hematopoietic cells, leading to multiple anomalies in cellular activation and migration. The study of WAS patient cells is hampered by the lack of available cells and is largely restricted to in vitro assays. To test in vivo the role of WASp in human lymphoid development, we developed a humanized mouse model of WAS using lentiviral-mediated RNA interference. Following transduction with the shRNA-W7dc (which reproducibly inhibits more than 80% of WASp) or shRNA-control, cord blood CD34 + cells were injected intrahepatically into irradiated newborn (gamma)c-/-Rag2-/- mice. After 10–14 weeks, 60% of mice display a sufficient human reconstitution with, as average, 13 ± 3% in bone marrow, 14 ± 3% in spleen and 90 ± 3% in thymus. WASp inhibition persisted in vivo and had no significant effect on human cell reconstitution. Cytometric analysis revealed the existence of multiple lineages such as T, B, NK and myeloid cells confirming that the lack of WASp does not block central hematopoiesis. However, the thymopoiesis of WASp-deficient progenitor cells was abnormal. Thymocytes expressing the highest levels of shRNA-W7dc have elevated levels of CD3/TCR while the CD4 + CD8+ thymocytes were often reduced. The CD34 + and CD4 + CD25 + T cells seemed to be more prevalent than in controls. To improve this model, transduced cells were intravenously injected in temporal vein. First results showed no improvement of the percentage of reconstruction. Altogether, we find that the RNAi model provides interesting information on the development of the immune system in the absence of WASp that could not be obtained in human patients.
Pluripotent Stem Cells: A Cell Model of Laminopathies: Large Scale Genetic and Epigenetic Study, the Search for New Therapeutic Targets
Laminopathies are a group of genetic disorders caused by mutations in LMNA gene which encodes nuclear type A and C lamins. Among hundreds of mutations so far indentified in lamin, our team works more specifically on two mutations (H222P and DelK32) resulting in several cardiac defects including autosomal dominant Emery-Dreifuss muscular distrophy (AD-EDMD). Pluripotent stem cells harbouring LMNA mutations represent a powerful model to study the genetic and epigenetic processes underlying the pathology in earlier embryogenesis. Both LMNA mutations in mouse embryonic stem cells cause a delay in cardiac cell differentiation. This observation could be explained by two hypotheses: the genetic and the structural one. To study the genetic hypothesis, expression of mesodermal and cardiac genes were monitored and these genes are less expressed in these two mutations than in wild type cells. Furthermore, cell contractility was impaired when compared to wild type cells: H222P mutated cells featured a more severe phenotype than DelK32. These results were confirmed in transgenic mouse models that carried theses mutations.
To further examine these abnormalities, we are monitoring by immunochemistry protein expression in mouse ES cell derived embryonic bodies (EBs) at day 5 (Lamins A/C and B, Mef2c, Nkx2.5, Tbx5 and Isl1) and day 12 (Beta-MHC and Alpha-Actinin).
Furthermore, development of a human model of laminopathies is on going using iPS from reprogrammed cells isolated from patients who carried H222P and DelK32 mutations. This model might allow us to identify therapeutics targets by a large scale genetic and epigenetic study.
Glucose Supply, but Not Long-Term Exposure to Continuous Hypoxia, Affect Mesenchymal Stem Cells (MSCs) Function
Although therapy using MSCs has enormous potential, they clinical exploitation is hampered by unability to deliver large numbers of viable and functional cells into injured tissues. Whereas accumulation of cell metabolic waste products, lack of nutrients, and oxygen deprivation are side-effects of ischemia, their specific contributions to cell viability and function are poorly understood.
To better understand the biological cascades leading to cell death, an in vitro model of ischemia, in which changes in the local physico-chemical environment were driven by cell metabolism, was established. In contrast to other models that exposed cells to intermittent hypoxia, our model maintained and investigated the effect of continuous hypoxia (<0.5 mm Hg) on MSC viability and function. The effects of increased local carbon dioxide content, limited availability of nutrients and accumulation of metabolic waste were assessed. MSCs exposed to continuous hypoxia for 12 days survived and were functional as evidenced by cell morphology and proliferation. Upon transfer to standard (21%O2, 10% FBS) culture conditions, the doubling time of MSCs pre-exposed to hypoxia was significantly (p < 0.01) lower than that of control cells (24.7 ± 1.9 and 29.4 ± 1.7 hours, respectively). Whereas extensive glucose stock depletion did not affect MSC survival under standard conditions, it led to massive cell death when the cells were exposed to continuous hypoxia for 12 days.
Optimization of Integration-Defective Lentiviral Vectors: Production and Transgene Expression Level
Lentiviral vectors (LV) are powerful tools for stable gene transfer. LV efficiently integrate into the host genome, with a potential risk of insertional mutagenesis. Such a risk may be bypassed with the use of integrative-defective lentiviral vectors (IDLV).
IDLV synthesis is based upon the natural formation during the viral cycle of circular episomal forms. These forms are actively transcribed. However, compared to native LV, IDLV production is less efficient and transgene expression level is lower. We aimed at improving both IDLV production and transgene expression level.
IDLV were produced by transient co-transfection of (1) vector plasmid, (2) packaging plasmid encoding D64V-defective integrase, and (3) VSV-G envelope-coding plasmid. Viral titers and transgene expression level were analysed by flow cytometry and qPCR.
With a LV standard production protocol, a 100-fold lower production rate was achieved, compared to native LV. By optimizing plasmids quantities and/or cellular density, we significantly improved IDLV production, with a viral titer only reduced by 5-fold as compared to that of native LV. To evaluate transgene expression lever, strong and weak promoters were tested. Promoter activity was assessed by measurement of GFP Mean Fluorescence Intensity. We showed that in a IDLV context all promoters tested had a very low level of activity, which was 200-fold lower than that with native LV.
In conclusion, we determined optimal conditions for efficient IDLV production, and showed that transgene expression could not be improved by increasing the promoter strength. Other strategies should be explored to improve transgene expression.
Progress in minicircle Manufacturing and Performance Testing
The dissemination of antibiotic resistance genes, as well as the uncontrolled expression of backbone sequences may have profound detrimental effects. Additionally, unmethylated CpG motifs have been shown to contribute to silencing of episomal transgene expression. Therefore, an important goal in vector development is to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle DNA.
PlasmidFactory's minicircle production technology facilitates the large scale production of highly pure minicircle DNA for applications in gene therapy and vaccination as well as e.g. virus production.
The production technology is based on two processes: (1) An inducible, sequence specific, close to 100% efficient in vivo recombination process and (2) a novel affinity-based chromatographic purification technology for the isolation of the minicircle DNA. The latter only consists of the gene of interest and a tiny residual sequence stretch resulting from the recombination. The chromatographic purification results in an exceptional purity proven by various QC tests which is extremely important since even small amounts of contaminants can produce dramatic experimental artefacts.
Within first efficacy studies, reporter genes for different types of analyses within various tissues, cells, animals and for testing the mode of administration (e.g. electro gene transfer, sonoporation, lipofection, magnetofection, etc.) were used. Also biodistribution studies using these constructs have been performed. Additionally, minicircle constructs for vaccines, virus production etc. are currently investigated.
The results demonstrate a significant increase of gene expression of minicircle DNA in comparison to a standard plasmid.
As a result of the work presented here, PlasmidFactory has launched the first commercially available minicircle DNA product now.
11-kDa Fragment of Midkine Acts on Neuroblastoma and Endothelial Cells and Inhibits Tumor Development
Pleiotrophin (PTN) and midkine (MDK) are members of a new family of neurotrophic factors that are considered as rate-limiting growth and angiogenic factors in the onset, invasion and metastatic process of neuronal tumors including neuroblastoma. We first evaluated the serum PTN and MDK levels in human neuroblastoma patients and their mRNA expression in 8 neuroblastoma cell lines. We showed that only MDK is highly expressed in the serums of patients compared to control serums (10.5 + 0.19 ng/ml versus 1.45 + 0.12 ng/ml, p < 0.0001) and that MDK mRNA was more highly expressed than PTN in almost all neuroblastoma cells lines showing that MDK is a critical player in neuroblastoma development which may hence represent an attractive therapeutic target. Thus, we showed that the knockdown of MDK and PTN expressions by siRNA led to a marked and significant decrease of neuroblastoma cell proliferation in vitro. We then investigated in a new strategy to evaluate the anti-tumor effect of a truncated MDK, lacking the C-terminal 81–121 portion of the molecule (MDKΔ81-121), which may act as a dominant negative effector for its mitogenic, angiogenic and tumorigenic activities by heterodimerizing with the wild type protein. In vitro studies showed that MDKΔ81-121 selectively inhibited MDK-dependent neuroblastoma cells and that in vivo, the electrotransfer of a plasmid encoding a secretable form of MDKΔ81-121 into tibialis cranialis muscles of nude mice was shown to dramatically inhibit tumor development and growth. This anti-tumor effect was correlated with the detection of the MDKΔ81-121 molecule in the plasma and the suppression of neovascularization within the tumors.
CHUV-Experimental Surgery Pavillon 4 Avenue de Beaumont 1011, Lausanne and Laboratory of stem cell dynamics Ecole polytechnique fédérale de Lausanne , Suisse
Recessive dystrophic epidermolysis bullosa (RDEB) is caused by loss of function mutations in COL7A1 encoding type VII collagen, the major component of anchoring fibrils, which are key structures for dermal-epidermal adherence. RDEB patients suffer from skin blistering and develop severe complications resulting in cancer. Lacking a specific treatment for RDEB, ex vivo gene transfer to epidermal stem cells shows therapeutic potential. Using a clonal strategy that enables us to fully characterise corrected stem cells before transplantation to patients we hope to satisfy the safety concerns.
Keratinocytes isolated from a skin biopsy of an RDEB patient were infected with self inactivating (SIN) retroviruses bearing the COL7A1 cDNA under a human promoter. Infected cells were cloned and expanded to identify corrected stem cells. We have isolated stem cells that stably secrete type VII collagen and engineered a functional epithelium that can generate anchoring fibrils when grafted onto SCID mice. Moreover, the safety aspects of the corrected stem cells were addressed (life span and tumorigenic potential, proviral insertion number and proviral integration sites).
We have demonstrated by proof of principle experiments the safety and the feasibility of a single cell approach for ex vivo gene therapy and we see permission for a first clinical trial.
Footnotes
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Presentors are underlined for all abstracts.