Spanish Society of Gene and Cell Therapy 2009 Oral Presentations

Or1 53 STEM CELLSImmunoresponses Against Exogenous Transgenes Limit the Engraftment of In Utero Transplanted Fetal Liver Stem Cells Transduced with Lentiviral Vectors

In utero gene therapy is an alternative to the postnatal treatment of diseases that can be diagnosed in early stages of development. We have previously demonstrated that in utero transplantation of transduced hematopoietic progenitors from adult syngeneic mice efficiently engraft in the long-term. The transplantation of fetal liver transduced cells constitutes a more realistic option because these cells could be obtained from the affected fetus, subjected to genetic treatment and then re-injected in utero. Herein, hematopoietic fetal liver progenitors from 14.5 days old fetuses were transduced (6h, MOI 10) with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP), in the presence of different combinations of cytokines. Up to 70% of transduction efficiency was obtained when cells were transduced in retronectin-coated dishes and with mSCF, TPO and Flt3L in the culture medium. The analysis of the in vivo repopulating ability of transduced cells after in utero transplantation showed that the chimerism was inversely related to the transduction efficacy. We tested whether in utero transplanted recipients could develop immunoresponses against the EGFP transgene. Non-engrafted recipients presented antibodies and cellular responses against EGFP, indicating an immunoreaction against the transduced cells. However, some animals showed EGFP expressing cells and did not show immunoresponse against the transgene, indicating immunotolerance. In summary, these results show for the first time that mouse fetuses can develop both humoral and cellular immunoresponses against exogenous transgenes, an observation that should be considered for future strategies of in utero gene therapy.

Or2 64 STEM CELLSGeneration of Inner Ear Sensory Cells from Bone Marrow-Derived Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) are able to self-renew and to differentiate into multiple cell types, and thus have received significant attention in regenerative medicine. Sensory hair cells and auditory neurons are the two central components of the inner ear that have to be protected or repaired in order to avoid hearing loss. To achieve this objective replacement by hMSCs is a novel and interesting treating option. We are using different approaches to promote differentiation of bone marrow-derived hMSCs into inner ear progenitor cells in vitro. For the differentiation of hMSCs cell cultures are established and treated with growth factors including IGF, FGF, EGF, BDNF and NT-3 that stimulate the formation of neuronal progenitors and their amplification. Neuronal progenitors are then transfected or transduced with Atoh1, a transcription factor essential for inner ear sensory cell formation. The Atoh1 gene has been cloned into different vectors, including Herpes and retroviral vectors. Viral infection or electroporation of these vectors resulted in efficient expression of Atoh1 in hMSCs. The generation of inner ear sensory cells is confirmed by monitoring specific hair cell and neuronal markers using quantitative RT-PCR and immunocytochemistry. These experiments may open novel treatment options for hearing loss based on autotransplants in patients.

Or3 70 STEM CELLSMechanisms Involved in the Immunosuppressive Properties of Adipose Tissue-Derived Mesenchymal Stem Cells

In previous studies we demonstrated that Ad-MSCs suppress activated T cells proliferation through a cell-to-cell contact and soluble factors, and that they could control graft-vs-host disease in a haploidentical hematopoietic transplantation mouse model (Yañez, Stem Cells 2006). In this study we focused on the mechanisms involved in these immunosuppressive effects. We found that MSCs inhibit the maturation of dendritic cells (DCs), evidenced by decreased CD83 and CD80 expression, and by a decreased secretion of TNF-α by m-DCs, and an increased production of IL-10 by p-DCs. The blockade of PGE2 present in the cultures by indomethacin (IDM), gave way to the maturation of p-DCs, but not of m-DCs. These results show that PGE2 accounts for the immunosuppressive effects of MSCs over p-DCs, but not over m-DCs. On the other hand, we observed that MSCs in co-culture with activated T lymphocytes inhibited their proliferation and their production of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6 and IL-12), and increased the immunosuppressive IL-10. In these co-cultures, high levels of PGE2 were detected. Its blockage with IDM preserved activated T lymphocyte proliferation, but the cytokine secretion profile was not restored. Additionally, an increase in the expression of the proliferation factors, transcription factors and cytokine mRNA implied in the pathway of TH1/TH2 differentiation of the T lymphocytes was detected. These results show that PGE2 plays a key role in the immunosuppressive effects of MSCs over activated T lymphocyte proliferation, but that the blockage of the mediating soluble factor PGE2 has no immediate effect on the restoration of the cytokine secretion profile of activated T lymphocytes.

Or4 82 STEM CELLSA Census of LINE-1 and Alu Elements Expressed in Human Embryonic Stem Cells

Almost half of the human genome has been generated by the activity of mobile DNA elements, and their activity continues to shape the genome. Long Interspersed Element-1 (LINE-1 or L1) is a family of active non-LTR retrotransposons that comprise 17% of the human genome. An active LINE-1 element code for the proteins required to mediate their mobility (known as retrotransposition), and these proteins are also used by Short Interspersed Elements (SINEs) like Alu to mediate their mobility in trans. The mobility of L1 and Alu has resulted in a diverse range of diseases and it has been estimated that 1/10 newborn may contain a de novo L1/Alu retrotransposition event. As a type of selfish DNA, L1 and Alu might ensure their evolutionary transmission to new generations through the accumulation of new insertions in germ cells or during early embryonic development. Human Embryonic Stem Cells (hESCs) represent an excellent model to study the accumulation of L1/Alu in early human development, and we recently described that L1 is expressed in these cells and that hESCs can accommodate a low level of L1 mobilization using a cell culture-based assay. In the present study, we adapted a previously characterized L1 antisense promoter to precisely map expressed L1s in undifferentiated hESCs and a straight cloning approach to determine the bulk of Alus expressed in hESCs. We found that several subfamilies of Alu are expressed in hESCs, and that the core from these elements is active in a cell culture-based assay. Surprisingly, we found that the antisense promoter is conserved trough LINE-1 evolution, and that almost half of the expressed L1s are not present in the Reference Human Genome (RHG). Furthermore, we also described several loci shared by all hESCs lines analyzed that are characterized by active L1 expression, which suggest that L1 expression in the developing embryo might be linked to defined and discrete places of the genome.

Or6 161 NEURODEGENERATIVE DISEASESSubstantia Nigra Dopaminergic Neurons Are Protected by Intraestriatal Carotid Body Grafts in a Chronic MPTP Mouse Model of Parkinson's Disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN). Previous work from our laboratory shows that intrastriatal transplantation of dopaminergic carotid body (CB) cells produce an amelioration of parkinsonism in animal models mainly due to a trophic effect, and can also induce significant beneficial clinical effects in PD patients. The mechanisms that underlie the trophic effects mediated by CB cells over the nigral neurons remain unknown. In order to study these mechanisms we have developed a new systemic chronic 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) mouse model that better resembles the features of PD, without the high mortality and variability inherent to other MPTP protocols. In this model of parkinsonism we have studied: i) stereological and densitometric analysis of the nigrostriatal pathway after TH- and NeuN immunohistochemistry, ii) striatal dopamine content determinations by HPLC, and iii) behavioral tests. Unilateral striatal CB transplants with a sham graft in the contralateral striatum were performed in this animal model. Besides these grafts we used fluorescent latex beads to trace in a retrograde manner the neurons of the SN that were in contact with the transplanted CB cells. Intrastriatal CB grafts clearly protected MPTP-induced degeneration of SN dopaminergic neurons. Confocal microscopy analysis showed that the protected SN neurons were those exposed to the grafted CB cells. This model can be now used both to better understand the nature of the CB trophic effect over the nigrostriatal pathway and to test other cell therapy or pharmacologic therapeutical approaches in PD.

Or7 175 NEURODEGENERATIVE DISEASESBDNF Regulation Under GFAP Promoter Provides Engineered Astrocytes as a New Approach for Long-Term Protection in Huntington Disease

Brain derived neurotrophic factor (BDNF) is the main candidate for neuroprotective therapeutic strategies for Huntington's disease. However, the administration system and the control over the dosage are still important problems to be solved. Here we generated transgenic mice overexpressing BDNF under the promoter of the glial fibrillary acidic protein (GFAP) (pGFAP-BDNF mice). These mice are viable and have a normal phenotype. However, intrastriatal injection of quinolinate increased the number of reactive astrocytes and enhanced the release of BDNF in pGFAP-BDNF mice over wild-type mice. Coincidentally, pGFAP-BDNF mice are more resistant to quinolinate than wild-type mice, suggesting a protective effect of astrocyte-derived BDNF. To verify this, we next cultured astrocytes from pGFAP-BDNF and wild-type mice for grafting purposes. Wild-type and pGFAP-BDNF derived astrocytes behave similarly in non-lesioned mice. However, pGFAP-BDNF derived astrocytes showed higher levels of BDNF and larger neuroprotective effects than the wild-type ones when quinolinate was injected 30 days after grafting. Interestingly, mice grafted with pGFAP-BDNF astrocytes showed important and sustained behavioral improvements over time after quinolinate injections as compared to mice grafted with wild-type astrocytes. These findings demonstrate that astrocytes engineered to release BDNF can constitute a therapeutic approach for Huntington's disease.

Or8 187 NEURODEGENERATIVE DISEASESLentiviral Delivery of LMX1A Enhances Dopaminergic Differentiation from Human Embryonic Stem Cells

Parkinson's disease (PD) is an incurable, chronically progressive neurodegenerative disease leading to invalidity and death. A promising potential treatment for halting the progression and restoring lost function in PD patients is cell replacement using specific and functional neurons derived from human pluripotent stem cells. A deeper understanding of mDA neuronal specialization and survival will facilitate the generation of an optimal cell source for cell replacement therapy for PD. Here, we used lentivirus-mediated transgene delivery to generate hES cell lines that overexpress, under the control of the neural specific enhancer of Nestin, the transcription factor LMX1a, which was reported to be a key player in the embryonic development of mDA neurons. Transduced hES cells displayed normal growth characteristics and remained pluripotent for more than 20 passages, as judged by their in vitro differentiation and teratoma-formation abilities. Time-course analyses of in vitro differentiating LMX1a-expressing hES cells showed increased Nurr1 and En1 mRNA expression compared to mock-transduced cells at early time points of neuronal differentiation. Furthermore, LMX1a expression resulted in a robust DAT and Girk2 expression, at late stages of the differentiation process, and increase in the total numbers of tyrosine hydroxylase (TH) -expressing cells in neural progenitor cultures, thus emphasizing the capacity of the LV transduced cells to generate high numbers of dopaminergic neurons with the full characteristics of mDA neurons. Our studies underscore the role of LMX1a in promoting mDA neuronal fate and provide a strategy for the directed and efficient differentiation of human pluripotent stem cells into dopaminergic neurons with potential value for cell therapy applications.

Or9 38 CANCERCharacterization of Human Glioblastoma-Derived Cancer Stem Cells and Establishment of Two Rat Models for Therapeutic Assays

The cancer stem cell (CSC) hypothesis suggests a hierarchical organization of cells within the tumor, in which only a subpopulation of stem-like cells is responsible of the rise and progression of the tumor. This population of CSCs exhibit extensive self-renewal and proliferative potential, being able to differentiate to cells with limited or no proliferative attributes. CSC populations have been identified in a variety of haematopoietic and solid tumors, and are selectively resistant to conventional therapies. This concept has critical therapeutic implications and may explain the relapses following treatment. In this work, we describe the establishment of two model systems of human glioblastoma from CSCs, and a preliminary therapeutic assay. The CSCs were derived from surgical resections and were capable of self-renewal and long-term proliferation as neurospheres that express the neural stem cell markers CD133, nestin, Sox2 and vimentin. The CSCs obtained possess multipotentiality, as demonstrated by their ability to give rise to a differentiate progeny (neurons, astrocytes and oligodendrocytes) with limited proliferative potential. Furthermore, orthotopic xenografts of both patient-derived CSCs in nude rats drive the formation of human glioblastomas that were a phenocopy of the original patient's tumor. We have developed a novel gene therapy approach based on the adenoviral transfer to tumor cells of a gene encoding a betaglucosidase, linamarase, which in the presence of the innocuous substrate linamarin and nontoxic level of glucose oxidase, produces cyanide and hydrogen peroxide. The combined system is effective in the eradication of all glioma cells, including CSCs.

Or10 65 CANCERDevelopment of a Graft-Versus-Chronic Myeloid Leukemia Mouse Model

Graft-Versus-Host Disease (GVHD) is a frequent complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), mediated by donor T cells reacting against host antigens. It cannot be separated from the beneficial Graft-Versus-Leukaemia effect (GVL), in which the donor T lymphocytes recognize tumor antigens as non-self and destroy residual leukaemic cells. In order to study factors that could affect GVL, we aimed to generate a leukemic mouse model, with a slow progress disease, induced by retrovirally transduced hematopoietic stem cells with the chronic myeloid leukemia (CML) BCR/ABL gene. First, we transduced syngeneic lin-hematopoietic progenitor cells with a retroviral vector carrying the BCR/ABL fusion cDNA, and the marker gene NGFR. The infusion of the transduced cells to lethally irradiated mice developed a CML, characterized by hemorrhagic lungs, hepatomegaly, splenomegaly, granulocytosis, in which NGFR⁺ cells were detected, as well as in bone marrow. Mice died on day +18-20 after infusion. The additional infusion on day 0 of donor allogeneic bone marrow cells developed a slower course of the disease, and the mice died of leukemia between days +28-72. In order to generate a GVL effect, donor allogeneic T lymphocytes were infused on day 0. This prevented the development of leukemia, detected clinically, and by the absence of NGFR⁺ cells in all the studied organs. The mice died of GVHD between days +13-36, similar to the GVHD control group. These results show that we have developed a GVL mouse model applicable for the study of factors that can affect this essential curative effect in allo-HSCT.

Or14 69 GENE THERAPY VECTORSClonal Analysis Demonstrates Widespread Transcriptional Termination Read-through in Keratinocytes Transduced with Self-Inactivating Lentiviral Vectors

Inappropriate gene expression caused by the insertion of retroviral vectors in the vicinity of cellular protooncogenes is a major cause of concern in gene therapy. Safe cutaneous gene therapy is envisioned through the use of individual keratinocyte stem clones in which insertional mutagenesis risk can be assessed. While transactivation of cellular genes by the retroviral long terminal repeat enhancer has been clearly proved in multiple experimental and clinical settings, the formation of chimeric viral-cellular transcripts originated by the inefficient transcriptional termination (read-through) of retroviral vectors remains to be studied in depth. This is particularly necessary for self-inactivating (SIN) lentiviral vectors, in which a deleted U3 region containing the retroviral enhancer, aimed at mitigating inappropriate expression of cellular genes by transactivation, results in increased read-through transcription demonstrated in transient transfection experiments. We have analyzed the formation of viral-cellular fusion transcripts in HaCaT keratinocytes transduced with K14 driven SIN lentiviral vectors. Our work proves the existence of significant levels of readthrough transcription, detected as high molecular weight RNAs in Northern blot analysis of lentiviral expression in individual cell clones. Using RACE-PCR techniques, we have cloned and characterized some of these fusion transcripts and verified that they correlate with genes located at the lentiviral integration sites that we have identified by LM-PCR in each clone. Our data show that lentivirus-mediated artificial gene expression results in high expression levels of viral-cellular chimeric transcripts in every clone that we have analyzed. The ability of these transcripts to cause abnormal expression of cellular proteins is under investigation. We are currently characterizing new lentiviral vectors that avoid these untoward effects.

Or15 108 GENE THERAPY VECTORSTreatment of Chronic Viral Hepatitis in Woodchucks by Prolonged Intrahepatic Expression of Interleukin-12

Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus (HBV) infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks with chronic woodchuck hepatitis virus (WHV) infection, a condition that closely resembles chronic hepatitis B. Woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral load below 1010 viral genomes/ml showed a marked and sustained reduction of viremia accompanied by a reduction in hepatic WHV-DNA, loss of e- and surface antigens and improved liver histology. In contrast, none of the woodchucks with higher viremia responded to therapy. In the responder animals, the antiviral effect was associated with induction of T cell immunity against viral antigens and reduction of hepatic expression of Foxp3. Studies performed to elucidate the rational behind the lack of antiviral activity of IL-12 in animals with high viremia showed that the presence of high amounts of WHV-DNA in serum is associated with the absence of IL-12 responsiveness in vitro. Thus, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in all cases with viral load below 10¹⁰ vg/ml. The elucidation of the precise mechanism responsible for the lack of response to IL-12 in animals with high viral load will be extremely important for the development of more efficient therapies against chronic hepatitis infection.

Or17 34 METABOLIC AND AUTOIMMUNE DISEASESIGF-I Expression from AAV Vectors Leads to Total Fibrosis Reversion in Cirrhotic Rat Livers

Liver transplantation is the only curative treatment for advanced liver cirrhosis. Therapies aimed at halting the progression of the disease are urgently needed. Previous studies have shown that the administration of insulin-like growth factor-I (IGF-I) from a SV40 vector (SVIGF-I) reduces liver cirrosis. However, SV40 vectors need further development before translation to the clinic. As an alternative, we have evaluated the effect in liver cirrhosis of AAV vectors delivering IGF-I (AAVIGF-I). First, we have showed that both healthy and cirrhotic livers are transduced with similar efficacy by AAV vectors. Then we have injected saline, AAVLuc, SVIGF-I or AAVIGF-I in cirrhotic rat livers. Treatment of AAVIGF-I in rats with established cirrhosis lead to a total reversion of liver fibrosis and restoration of liver function. We have analyzed the molecular mechanisms involved in this response. IGF-I expression is detected in the liver at 4 days post virus administration. At this time, expression of some hepatoprotective factors such as HGF and HNF4a is increased and levels of pro-fibrogenic factors such as TGFb are decreased in AAVIGF-I treated animals. Surprisingly, at only 4 days post-vector injection, we already detect a decrease in activated HSCs, which are the cells that secrete the collagen deposits that result in liver fibrosis. Interestingly, these changes were not observed with SVIGF-I. Two weeks after vector administration there is a tremendous activation of matrix metalloproteinases, which could be in charge of removing the collagen deposits. Two months after treatment with AAVIGF-I animals showed liver function tests identical to healthy animals and 2 months later liver fibrosis was reverted completely. This has never been observed with SVIGF-I treated animals. We believe that AAVIGF-I gene therapy should be tested in patients with advanced liver cirrhosis that do not have access to timely liver transplantation.

Or18 102 METABOLIC AND AUTOIMMUNE DISEASESAdipose-Derived Mesenchymal Stem Cells Ameliorate Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Introduction: MSCs are nonhematopoietic, multipotent cells present in various adult tissues such as bone marrow, fat and umbilical cord blood, which possess potent immunomodulatory properties making them a promising therapy for autoimmune disease. Multiple sclerosis is a chronic inflammatory disease of the CNS. Injections of bone marrow-derived MSC in animal models of MS, experimental autoimmune encephalomyelitis (EAE), have been shown to suppress inflammation and tissue destruction. However, no studies have evaluated the efficacy of mouse adipose tissue-derived (Ad) MSC in EAE. Aim: To study the effect of mouse AdMSC on immune responses in vitro and in a model of MOG-induced EAE in C57Bl/6 mice. Results: AdMSC suppressed splenocyte proliferation in vitro and suppression was reversed by adding the iNOS inhibitor L-NAME and the COX-1/2 inhibitor indomethacin. Dendritic cells (DC) cocultured with AdMSC in the presence of LPS failed to upregulate CD40/CD86, did not produce TNF-alpha and were less efficient in inducing T cell proliferation in an MLR. Adding indomethacin, but not L-NAME, to the DC:MSC co-culture partially reversed the block in DC maturation. Administration of AdMSCs into mice with EAE ameliorated disease, decreasing the number of IFN-gamma- and IL-17-producing cells in draining lymph nodes and spleen. Also MOG-specific recall responses were decreased in AdMSC-treated mice. Interestingly, DC obtained from the draining lymph nodes from AdMSC-treated mice expressed lower levels of CD40 compared to DCs from control mice. Conclusions: AdMSCs possess potent immunosuppressive capacities in vitro and in vivo, modulating the T cell response partly through their effect on DC function.

Or19 118 METABOLIC AND AUTOIMMUNE DISEASESDifferentiation of Human Embryonic Stem Cells (HESC) to β-Cell-Like Cells: The Role of UCP2 in Insulin Secretion

Embryonic stem cells have the capacity to differentiate into many cell types and therefore represent a potential source of cells for the treatment of several diseases including type-I diabetes. The relative lack of successful pancreatic differentiation of human embryonic stem cells (hESCs) suggests that directed differentiation of these cells into definitive endoderm and subsequent commitment towards a pancreatic fate are not readily achieved. The aim of this study was to optimize an efficient differentiation protocol that mimicked in vivo pancreatic organogenesis (definitive endoderm, pancreatic endoderm and endocrine precursor). Results: (1) Exposure of hESC to low serum concentration and activin A induce definitive endoderm differentiation as detected by the expression of markers (Sox17, FoxA2). (2) Addition of retinoic acid (RA) promoted pancreatic differentiation (expression of the early pancreatic progenitor markers Isl1 and Ngn3). (3) Suspension cultures with medium supplemented with bFGF, fibronectin and insulin-transferring selenium (ITS) induce maturation (expression of islet specific markers such as Pdx1, GLUT-2, glucagon and insulin). (4) Finally, we have studied the role of mitochondrial transporter uncoupling protein 2 (UCP2) on clusters maturation and insulin secretion. Up-regulation of the UCP2 results in failure of cells to increase ATP levels after glucose stimulation then decreasing insulin release. Here we demonstrated that the inhibition of UCP2 levels through small molecule induction of Sirt-1 resulted in increased insulin release. Our strategy allows the progressive differentiation of hESCs into pancreatic endoderm capable of generating mature beta cell-like cells and demonstrates that UCP2 is involved in the maturation process during the differentiation.

Or21 41 CARDIOVASCULAR DISEASES, ISCHEMIA AND INFLAMMATIONTransplantation of Adipose Derived-Stromal Vascular Fraction in a Model of Chronic Myocardial Infarction Induces a Long-Term Improvement of Cardiac Function and Proves Multilineage Differentiation Capacity

The aim of our study was to determine the long-term effect of the adipose derived-stromal vascular fraction (SVF) cell transplantation in a rat model of chronic myocardial infarction (MI). MI was induced in 87 rats by coronary artery ligation and 5 weeks later, rats were allocated to receive intramyocardial injection of 107 GFP-expressing SVF-cells or medium as control. Heart function and tissue metabolism, determined by echocardiography and 18FFDGmicroPET respectively, were performed together with histological studies 3 months after transplantation. Cell fate was analyzed along the experiment (1, 3 days, 1, 2 weeks, and 1, 3 months). SVF induced a long-lasting (3 months) improvement in cardiac function with an increase in the LVEF (26.46 ± 2.26% to 38.25 ± 4.01%, mean ± SD, p < 0.01) and in infarct tissue metabolism, quantified by 18F-FDG uptake (50.55 ± 4.41% to 60.64 ± 5.26%, mean ± SD, p < 0.05). Functional improvement was associated with a smaller infarct size, thicker infarct wall, lower scar collagen content and a higher revascularization degree. Importantly, injected cells engrafted and remained in the treated hearts for at least 3 months, contributing to the vasculature and to a lesser extent to the cardiac tissue. Furthermore, SVF-release of angiogenic (VEGF and HGF) and pro-inflammatory (MCP-1) cytokines, was demonstrated in vitro and in vivo, strongly suggesting their trophic effect. Altogether, these results show the potential of SVF to contribute to the regeneration of the ischemic tissue and to provide a long-term functional benefit in a rat model of chronic MI.

Or22 44 CARDIOVASCULAR DISEASES, ISCHEMIA AND INFLAMMATIONDevelopment of Vascular Endothelial Growth Factor (VEGF)-Loaded Poly(Lactide-co-glycolide) Microparticles to Stimulate Angiogenesis in a Rat Model of Myocardial Infarction

Cardiovascular diseases are the most common pathologies with the highest morbidity and mortality rates in the occidental countries. Early therapies to improve the regeneration of the damaged tissue, like the delivery of growth factors to promote neovascularization and cell survival, are being explored. Several clinical trials have been performed in patients with cardiac and limb ischemia, by injection of pro-angiogenic factors like VEGF or bFGF, but has been unsuccessful, due, partially at least, to suboptimal delivery strategies and protein instability. The aim of this work was to develop Poly-lactide-co-glycolide (PLGA) biodegradable and biocompatible microparticles loaded with the Vascular-Endothelial-Growth-Factor (VEGF-165) in order to deliver the cytokine with a more localized, stable and controlled release pattern. Angiogenesis stimulation was analyzed in a rat model of cardiac ischemia-reperfusion.

VEGF was encapsulated by solvent extraction/evaporation method using the Total-Recirculation-OneMachine-System (TROMS), producing microspheres in the size range of 2-30 μm with 20-30% encapsulation efficiency and cumulative release kinetics of 28% VEGF-release during the first 24 h. Biological activity of the released VEGF was confirmed by induction of the VEGF receptor (KDR) activation and proliferation stimulation of an endotelial cell line (HIAECs).

In vivo studies were carried out by intramyocardial injection of the microparticles, four days after injury. Mean particle size of 5 μm was more compatible with a myocardial in situ administration and histological evaluation showed the stable presence of Rhodamine-labeled-microparticles one and four-weeks after injection and stable VEGF release. Thus, the use of microparticles as a cytokine delivery system constitutes a promising strategy for ischemic cardiovascular application.

Or23 75 CARDIOVASCULAR DISEASES, ISCHEMIA AND INFLAMMATIONHuman Adult Stem Cells Derived from Adipose Tissue Protects Against Sepsis

The invasion of the peritoneal cavity by intestinal bacteria is a serious complication following abdominal surgery. It involves the exposure to microbial products that cause hyperactive and out-of-balance network of endogenous proinflammatory cytokines, which can lead to lethal septic shock. The objective of any antisepsis agent is to control this exacerbated systemic inflammatory response, without compromising the antimicrobial defense. Recently, it has been reported that stem cells present in the mesenchyme of several adult tissues (MSCs), have a potent immunomodulatory action. Our objective was to investigate the antiinflammatory and protective effect of human MSCs isolated from adipose tissue (ASCs) in two experimental models of sepsis with clinical relevance. Treatment with ASCs protected against mortality caused by high dose of endotoxin (LPS) and caecal ligation and puncture (CLP). ASC injection to septic animals decreased the levels of inflammatory mediators and increased IL10 in the major affected organs, and diminished the inflammatory infiltration into the peritoneal cavity, lung, liver and intestine. Interestingly, treatment with ASCs did not affect the natural response against infection. In contrast, septic mice ASCs-treated showed less bacterial load in the peritoneum and affected tissues. However, ASCs showed no direct bactericidal effect, suggesting that ASCs would promote bactericidal activity in other cells. These results indicate that ASCs rescue mice from endotoxemic death by downregulating the exacerbated inflammatory response. By their relative abundance and easy acquisition/expansion, ASCs are attractive candidates for the design of cellular therapies in the treatment of sepsis and other complications associated with systemic infections by intestinal bacteria.

Or24 90 CARDIOVASCULAR DISEASES, ISCHEMIA AND INFLAMMATIONCellular Therapy of Hemorrhagic Cerebral Ictus: Experimental Anatomical Study

Intracerebral hemorrhage (ICH) is a neurological disorder derived by the breakage of a blood vessel in the brain. The associated mortality of ICH is approximately 40% to 50%, and only 20% of patients are living independently at 6 months. Currently, there is no effective treatment to improve the functional deficits caused by this disease. Keeping in mind that cell therapy promises new strategies to treat some disorders in the Central Nervous System, in the present study, ICH was induced by stereotactic injection of 0.5 U of collagenase type IV in the striatum of adult Wistar rats, and 72 hours later, intralesional administration of 2 × 10⁶ allogeneic BMSC in saline (n:10), or saline only (n:10) was performed. In the following 30 days, functional outcome was evaluated in each animal by rotarod and the modified neurological severity score (mNSS) tests. Our present study suggests that the biological role of intralesional administration of allogeneic BMSC is not only to differentiate into neurons and glial cells into the injured brain but also increasing endogenous neurogenesis in the SVZ. These synergic functions can help to replace the cells lost after ICH and to improve the neurological deficits. Our present findings support that local administration of allogeneic BMSC could be a useful treatment to reduce the neurological deficits caused by ICH.

This proyect has been partially financed by Mapfre Foundation, FIS PI06650 and Fina-Biotech Proyect.

Or27 146 VECTOR DEVELOPMENT AND INFECTIOUS DISEASESMeasles Virus GP Pseudotyped LVS Allow Efficient Transduction of Quiescent Healthy and Cancer B-Cells

B-lymphocytes are attractive targets for gene therapy of diseases associated with B-cell dysfunction and also for immunotherapy by their potential to induce specific immune activation or tolerance. However, up to now lentiviral transduction of true quiescent B-cells has not yet been achieved. Recently, we generated high-titer HIV-based lentiviral vectors (LVs) pseudotyped with measles virus glycoproteins (H/F-LVs) that allowed the transduction of completely quiescent human T-cells after a single exposure. Here we demonstrate for the first time that H/F-LVs can efficiently transduce quiescent B-cells in the absence of any stimulus. We systematically achieved up to 50% of transduction of resting adult B-cells with H/F-LVs where VSVG-LVs remained completely refractory. Most importantly, the naïve phenotype of transduced B-cells was conserved and no upregulation of early or late activation markers was detected. No differential expression of the B7 molecule CD86 was shown, implying that B-T interactions should be maintained. Importantly, H/F-LVs represent the first tool permitting stable transduction of leukemic cancer cells, B-CLL cells, blocked in G0/G1 early phase of the cell cycle. In addition, when we co-transduced quiescent B-cells with H/F-LVs and VSVG-LVs we observed that H/F-LV does not trigger or facilitate VSVG-LV entry, pointing out a differential entry mechanism between the two vectors. Thus, H/F-LV transduction overcomes the limitations of current LVs by making B-cell-based gene therapy and immunotherapy applications feasible. These new LVs will facilitate antibody production and the study of gene functions in healthy and cancer immune cells.

Or29 111 RARE DISEASESProspects for Gene Therapy of Friedreich's Ataxia

Friedreich's ataxia (FA) is a very-early-onset neurodegenerative disease caused by recessive mutations resulting in a deficiency of frataxin. Gene therapy for FA should therefore raise frataxin expression and/or increase the survival of the affected neurons in the spinal ganglia, spinal cord, brainstem and cerebellum. As a “proof of principle”, we had previously shown that the injection of a herpesviral amplicon vector carrying frataxin cDNA rescues the motor coordination deficit which is triggered by a locally-restricted frataxin gene knock-out in mice (Lim et al. 2007; Mol Ther. 15:1072-8). However there remain important challenges which must be addressed to develop a realistic gene therapy approach for FA. One is the assurance of a physiological regulation and long-term persistence of frataxin gene expression. In this respect, we have used another herpesviral amplicon vector with a 135-kb bacterial artificial chromosome insertion containing the complete genomic locus of human frataxin. We have demonstrated that frataxin expression in vivo is more persistent when this genomic herpesviral vector is used. We have also developed neural cell models of the disease for the testing of neurotrophic factor genes with the potential to compensate for the neurodegenerative process triggered by frataxin gene silencing. Thus, we have found that brain-derived neurotrophic factor (BDNF) is capable of ameliorating cell death of frataxin-deficient neurons. Interestingly BDNF is a secreted protein that could act in both an autocrine and paracrine manner. These results pave the way for a combinatorial approach for the gene therapy of FA using both frataxin and neurotrophic factor genes.

Or30 121 RARE DISEASESCell Therapy for Enterocutaneous Fistula Associated with Crohn's Disease: A Clinical and Biological Comparison of Protocols With and Without Cell Expansion

Introduction: Enterocutaneous fistulas (ECF) associated with Crohn's disease are a therapeutic challenge due to recurrence and bad response to available treatments. Expanded adipose-derived stem cells (ASC) have shown their effectiveness in treating Crohn's patients with digestive fistulas. We aimed to study if unexpanded cells from the stromal vascular fraction (SVF) of lipoaspirate may also be effective. Material and methods: We compared two groups of Crohn's patients with refractory ECF and similar selection criteria; first one is part from a previous phase I clinical trial who received autologous expanded ASCs (n = 5) and the other one underwent SVF implantation (n = 4). The implantation procedure consisted in tract curettage, superficial injection of cellular product into the tract walls, and sealing of the fistulous tract with a mixture of fibrin glue and cells. ASCs are obtained by SVF expansion and cell selection by adhesion properties. Both cell products were characterized by immunofluorescence staining. Results: SVF cells are CD-34⁺, CD-90⁺, C-kit⁺ and Vimentin⁺⁺⁺; ASCs are CD-34⁻, cd-90⁺⁺⁺, C-kit⁺⁺⁺ and Vimentin⁺⁺⁺. In the ASCs group, three of the four evaluated fistulas healed (follow up: three years). In the other group one of the four cases cured (follow up: one year). No adverse events related to cell products were observed. Discussion and conclusions: Although a comparison of case series cannot be considered firm evidence, cell therapy can reach healing in refractory ECF. ASC protocol seems to be more effective (maybe due to better stem-cell quality, higher ASC concentration or for their immunomodulating action). This therapy is feasible and safe for humans.

Or31 193 RARE DISEASESAAV-Mediated Sulfamidase Expression in the Liver Prevents Development of Somatic Alterations in MPS IIIA Mice

Mucopolysaccharidosis IIIA (Sanfilippo syndrome type A) is a lysosomal storage disease caused by the lack of sulfamidase (Sgsh) activity, which is needed for the complete degradation of heparan sulfate (HS) glycosaminoglycan. This results in widespread HS accumulation and causes both neurodegeneration and somatic pathology, leading to death around puberty. There is no effective therapy available.

The aim of this study was to evaluate a new gene therapy approach for this disease based on the genetic manipulation of either the liver or the skeletal muscle of a MPSIIIA mouse model to secrete the lacking enzyme to the bloodstream. Circulating sulfamidase can be taken up by non-transduced cells via the mannose-6-phosphate receptor, where it is targeted to the lysosomes and can correct the HS accumulation. With this objective, we generated a serotype 8 adeno-associated viral vector that expressed the mouse sulfamidase cDNA (AAV2/8-Sgsh). MPSIIIA mice receiving an intravenous or an intramuscular administration of AAV2/8-Sgsh showed a high expression and activity of sulfamidase in liver or in muscle, respectively. These tissues were able to secrete the enzyme to the bloodstream, as the sulfamidase activity in the serum almost reached the levels of control mice, whereas it was undetectable in MPSIIIA untreated mice. Glycosaminoglycan (GAG) accumulation was decreased in all tissues, even reaching normalization in many of them, demonstrating that the enzyme was being taken up by non-transduced cells. Accordingly, the urinary excretion of GAGs was completely normalized. Furthermore, a significant reduction in the GAG accumulation was found in the brain of the treated animals.

Our results suggest that a therapy for the MPS IIIA based on the genetic modification of the liver or the muscle could benefit both the peripheral tissues and also the central nervous system. Therefore, our study suggests that this therapeutic approach may be of great interest, as it would improve the lifespan and the quality of life of MPSIIIA patients.

Or32 195 RARE DISEASESOptimized Lentiviral Transductions Allow High Transduction Efficacies of Murine Fetal Liver Progenitors but Induce Immunoresponses Against the Transgene Limiting In Utero Hematopoietic Engraftment

In utero gene therapy is an alternative to the postnatal treatment of diseases that can be diagnosed in early stages of development. We have previously demonstrated that in utero transplantation of transduced hematopoietic progenitors from adult syngenic mice efficiently engraft in the long-term. The transplantation of fetal liver transduced cells constitutes a more realistic option because these cells could be obtained from the affected fetus, subjected to genetic treatment and then re-injected in utero. Herein, hematopoietic fetal liver progenitors from 14.5 days old fetuses were transduced (6 h, MOI 10) with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP), in the presence of different combinations of cytokines. Up to 70% of transduction efficiency was obtained when cells were transduced in retronectin-coated dishes and with mSCF, TPO and Flt3L in the culture medium. The analysis of the in vivo repopulating ability of transduced cells after in utero transplantation showed that the quimerism was inversely related to the transduction efficacy. We tested whether in utero transplanted recipients could develop immunoresponses against the EGFP transgene. Non-engrafted recipients presented antibodies and cellular responses against EGFP, indicating an immunoreaction against the transduced cells. However, some animals showed EGFP expressing cells and did not show immunoresponse against the transgene, indicating immunotolerization. In summary, these results show for the first time that mouse fetuses can develop both humoral and cellular immunoresponses against exogenous transgenes, an observation that should be considered for future strategies of in utero gene therapy.

Or33 100 STEM CELLSLCN-2 Overexpressing Bone Marrow-Derived Macrophages Promote Renal Regeneration

Recent studies revealed a reparative role of macrophage therapy against different types of human pathologies. Specifically, re-injection of macrophages ex vivo genetically modified towards an antiinflammatory phenotype has been described to protect against nephrotoxic nephritis. Lipocalin-2 (Lcn-2) is a 25 kDa protein of the lipocalin superfamily that is synthesized from macrophages and that exerts a positive modulation of acute inflammation. In the kidney, it is known that Lcn-2 is produced at sites of injury and may modulate renal repair. In this study, we found that ex vivo genetically modified macrophages towards lipocalin-2 (Lcn-2) overexpression were capable of modulating injury and inflammation outcome in ischemic kidneys, promoting kidney repair and tissue regeneration.

Renal function markers BUN and creatinine were decreased upon adoptive transfer of these macrophages and the expression of pro-inflammatory mediators was attenuated. Results indicated that macrophagederived Lcn-2 promotes the proliferation of tubular epithelial cells. Immunostaining for the regeneration markers stathmin and PCNA showed markedly positive expression in the kidney sections with Lcn-2macrophage treatment. Real-Time RT-PCR of the proliferation markers Ki-67 and PCNA further confirmed these effects. In conclusion, we provide a novel role for macrophage-derived Lcn-2 on kidney regeneration and protection from experimental renal ischemia/reperfusion injury. Our results indicate a possible target for further therapeutic use in disease, since Lcn-2 not only modulates the macrophage phenotype, but also its prorepair properties. The provided results of this study have recently been patented and are expected to be soon translated into clinical approaches.

Or34 158 STEM CELLSGrowth in Low Oxygen Increases the Genetic Stability of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSC) have a significant potential for the treatment of many pathologies associated with chronic inflammation or aging. However, the large numbers of hMSCs needed for therapeutic doses generally imposes a long cell culture expansion period prior to their use in the clinic; this is typically performed in pro-oxidative (20% O₂) conditions and here mutations and chromosomal abnormalities can arise posing a significant bio-safety concern. Our work shows that culture of hMSC in low oxygen tensions promotes the growth, retards cellular senescence, and increases the genetic stability of these cells, thus increasing their potential for regenerative medicine. Our data also indicates that the growth of hMSC in standard conditions is associated with a significant increase in aneuploidy and defective mitosis concurrently with the transcriptional deregulation of a set of genes previously associated with ploidy control. These genes are proposed to be biomarkers of the genetic stability in human mesenchymal stem cultures.

Or36 182 STEM CELLSBcl-XL Affects the Cell Cycle of Human Neural Stem Cells

The promising idea of the use of human Neural Stem Cells (hNSCs) as a source for cell replacement therapies for neurodegenerative diseases needs a deep study of the basic biology of these cells to make sure of its success and safety. One of the aspects being intensively studied is the fine balance between proliferation and cell death in the process of the differentiation of hNSCs into the three main cell types of Central Nervous System: neurons, oligodendrocytes and astrocytes. In this direction, our laboratory has published that the antiapoptotic protein Bcl-XL is able to modulate the hNSCs differentiation; increasing the amount of neurons and decreasing the glia phenotype. We proved that this is not due to a cell death counteraction but to an increase in the uncommitted and committed progenitor proliferation. Following the study, we have investigated if Bcl-XL has effects on the cell cycle of the hNSCs. First, we report in this work an increase of the cell cycle length, a phenomenon linked with a higher frequency of asymmetric cell divisions in Bcl-XL overexpressing cells. Secondly, we observe a differential regulation of important regulators of the cell cycle; specifically, p53 and p21Cip1 are up-regulated in Bcl-XL overexpressing cells. On the other hand, p27Kip1, a cell cycle inhibitor regulating the cell cycle mainly in committed progenitors is down-regulated. We also observe an increased in cyclin D3. Overall, we show that Bcl-XL is able to affect the differentiation of hNSCs by the modulation of some cell cycle regulators.