Spanish Society of Gene and Cell Therapy 2009 Invited Speaker Presentations

Inv1 Safety and Efficacy of Gene Therapy for Severe Retinal Dystrophy

rAAV vector-mediated gene replacement therapy improves retinal function and visual behaviour in animal models of retinal degeneration caused by defects in the gene encoding RPE65. The purpose of the present study is to determine whether gene therapy for retinal dystrophy caused by RPE65 mutations is safe and effective in humans. In a phase I/II dose-escalation trial, we have administered by subretinal injection a rAAV-2/2 vector expressing human RPE65 cDNA under the control of a human RPE65 promoter in human subjects with early onset severe retinal dystrophy caused by mutations in RPE65. There have been no complications associated with the surgical delivery of vector in the subjects enrolled to date. We have detected no systemic dissemination of vector genome and no evidence of immune responses to rAAV vector capsid or RPE65 proteins. We have found no evidence of any significant adverse effect on retinal function. There has been no clinically significant change in visual acuity or in peripheral visual fields on Goldmann perimetry in any of the four patients. We have detected no change in retinal responses on electroretinography. One subject has demonstrated significant improvement in visual function on microperimetry and on dark-adapted perimetry. This subject has also showed improvement in a subjective test of visual mobility. The outcomes in the first subjects to date suggest that subretinal administration of rAAV vector is safe in humans. These findings provide support for further clinical studies of this experimental approach in other patients with mutant RPE65.

Inv2 Regenerative Cell Therapy in Parkinson's Disease

Parkinson's disease (PD), one of the most frequent neurodegenerative disorders, is due to the progressive loss of neurons both in the central and peripheral nervous system. Cell death is particularly prominent within the ventral dopaminergic mesencephalic neuronal population, the projections of which constitute part of the nigrostriatal (NS) pathway. Intrastriatal transplantation of dopamine-secreting cells has been assayed for over two decades in preclinical and clinical studies. However, in more recent studies the emphasis has shifted towards the protection of NS neurons by means of the striatal delivery of trophic factors, such as the glial cell line-derived neurotrophic factor (GDNF). The absolute trophic dependence of adult dopaminergic substantia nigra neurons on GDNF has recently been demonstrated in a conditional GDNF-null mouse model. The neuroprotective effects of GDNF, alone or in combination with trophic factors, is a quite appealing phenomenon since these factors are naturally produced by some cell types, i.e., carotid body (CB) glomus cells. Transplantation assays using glomus cells yielded good results in parkinsonian animal models, although the clinical efficacy in PD patients was less obvious due, among other factors, to the scarcity of the CB tissue transplanted. We have recently discovered the presence in the adult CB of neural crest-derived progenitors, which are induced to proliferate and to differentiate to new glomus cells upon exposure to hypoxia. Therefore, in vitro expansion of CB tissue is a feasible methodology that could have potential applicability in regenerative cell therapy in PD.

Inv3 Reprogramming Somatic Cells for Modeling and Treating Human Disease

The generation of induced pluripotent stem (iPS) cells by ectopic expression of a defined set of factors has enabled the derivation of patient-specific pluripotent cells and provided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold great therapeutic potential, although several shortcomings should be addressed before iPS cell technology can be implemented clinically. Here, I will review recent results by our laboratory and others on the usefulness of iPS cells to model human disease, the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications, and novel strategies aimed at the generation of clinically-safe iPS cells.

Inv4 Transcription Factor Induced Immune Cell Switching

Data will be presented describing the direct reprogramming of committed hematopoietic cells with transcription factors. It is shown that a single factor, C/EBPalpha, can efficiently switch committed B and T cell precursors to acquire a macrophage phenotype. For both lymphoid lineages, a 1 to 3 day exposure to the inducer leads to transgene independence, resulting in more than 60% of the cells committing to a macrophage fate. In addition, activation of the myeloid program required activation of PU.1 while extinction of the B and T cell programs were found to be PU.1 independent. However, the effects of C/EBPalpha on the two lineages differed, in that it reprogrammed fully mature B lymphocytes while cells within the T cell lineage already became partially resistant at the double positive stage. These observations suggest that differentiation plasticity varies widely within the hematopoietic system and that the B lineage is particularly prone to transdifferentiation into macrophages.

Inv5 Inducing Immune Tolerance with Mesenchymal Stem Cells

Maintenance of immune tolerance is critical to keep the homeostasis of the immune system and to avoid autoimmune reactions. The mechanisms and cells involved in the induction of tolerance are still unravelled. Mesenchymal stem cells (MSCs) are mesoderm-derived, fibroblast-like somatic cells that reside in the stroma of solid organs and function as precursors of non-hematopoietic connective tissues. Beside their capacity to differentiate into mesenchymal and non-mesenchymal cell lineages, and their potential clinical application for the repair of damaged tissues, many recent studies have shown that MSCs from different sources regulate the immune response, including in vitro inhibition of T-cell proliferation, B-cell function, and dendritic cell maturation. Here, I will focus on the most recent data supporting the therapeutic potential of adipose-derived MSCs (ASCs) on autoimmunity. In different experimental models of multiple sclerosis, rheumatoid arthritis and Crohn’ disease, we have shown that the systemic infusion of human and mouse ASCs significantly ameliorated the clinical signs of these diseases, by educating the immune system to be more tolerant. After in vivo administration, ASCs migrate to injured/inflamed tissues, where they can inhibit the release of inflammatory cytokines, deactivate Th1-type reponses, induce the generation of antigen-specific regulatory T cells and promote the survival of damaged cells. Therefore, ASCs emerge as key regulators of immune tolerance in physiological conditions and as attractive candidates for a cell-based therapy for autoimmune and inflammatory diseases.

Inv6 Directed Neuronal Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESC) provide a unique model to study early events in human development. In addition, the hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury including spinal cord injury. Development of efficient and robust differentiation protocols which have the capacity to generate region-specific and mature active neurons under in vitro conditions without embryoid body formation, co-culture with stromal cells and without presence of cells of mesodermal or endodermal lineages is required long-term strategy to identify factors which drive targeted differentiation of hESC. For instance, exposing the hESC and their progenitor cells to retinoic acid suppress differentiation to rostral forebrain dopamine neural lineage and promote that of spinal neural tissue including motor neurons. Application of other different inductive signals involved in early patterning of the cerebellar region followed by application of different factors directs hESC differentiation into cerebellar neurons. We were able to derive cells which show T-shaped polarity phenotype expressing similar markers as developed human cerebellum. In vivo implantation of differentiated hESC transfected with MATH1-GFP construct into neonatal mice resulted in cell migration across the molecular, the Purkinje cell and settlement in the internal molecular layers which demonstrate that the universal mechanisms involved in development of cerebellum can be efficiently recapitulated in vitro. This enables design of new strategies for intense cell replacement therapy, to study early human development and pathogenesis of neurodegenerative diseases.

Inv7 Differentiation of Human Embryonic Stem Cells Towards Blood: Scientific Opportunities

Human embryonic stem cells (hESC) have been shown to differentiate into the hematopoietic cell fate. From a developmental standpoint, hESC-derived hematopoietic CD45⁺ cells emerge around day 10 of embryoid body (EB) development from a subset of embryonic endothelium expressing PECAM-1, Flk-1, and VE-Cadherin, but lacking CD45 (CD45^negPFV). These CD45^negPFV precursors arise about day 5-7 EBs and are exclusively responsible for hematopoietic potential of differentiated hESCs. hESC-derived hematopoietic cells show similar clonogenic capacity and primitive phenotype to somatic sources of hematopoietic progenitors but possess very limited in vivo repopulating capacity in immunodeficient mice.

Human ESC-derived hematopoietic cells and hESC-derived hemangioblasts will be hailed as a unique tool for a range of biomedical applications, such as cell replacement therapy, developmental biology, drug discovery and disease modelling. We therefore have compared multiple hESC lines and iPS to assess their differential propensity for blood differentiation. As expected, some hESC lines are very prone to hematopoietic differentiation whereas others do not. Unfortunately, the CD45⁺ or CD45⁺CD34⁺ hematopoietic cells generated from hESCs lack in vivo function when transplanted in NOD/SCID mouse models. In an attempt to overcome this issue, we aimed at studying the implication of key hematopoietic transcription factors in blood differentiation from hESCs. We speculate that key hematopoietic transcription factors will need to be either over-expressed or knocked-down in order to enhance or skew the hematopoietic multilineage differentiation potential an timing of blood development from differentiating EBs.

In an attempt to use hESCs for modelling infant leukemia, we have generated MLL-AF4-expressing hESCs in order to assess the developmental impact of this fusion oncogene known to arise in utero in prenatal human stem cells. Some of this preliminary data will be discussed. Human ESCs were also used as a model to test the effects of etoposide on human early embryonic development. Etoposide induced MLL rearrangements in hESCs and their hematopoietic derivatives. After long-term culture, the proportion of hESCs harboring MLL rearrangements diminished and neither cell cycle variations nor genomic abnormalities were observed in the etoposide-treated hESCs, suggesting that MLL rearrangements are insufficient to confer hESCs with a selective proliferation/survival advantage. However, continuous exposure to etoposide induced MLL breaks and primed hESCs to acquire other major karyotypic abnormalities. These data show that chronic exposure of developmentally early stem cells to etoposide induces MLL rearrangements and make hESCs more prone to acquire other chromosomal abnormalities than post-natal CD34⁺ cells, linking embryonic genotoxic exposure to genomic instability.

Inv8 What Makes a Cell a Cancer Stem Cell?

A cancer dogma states that inactivation of oncogene(s) can cause cancer remission, indicating that oncogenes are the Achilles' heel of cancers. This current “hands on” model of cancer is in agreement with the fact that in human cancers, all cancerous cells, with independence of their cellular heterogeneity, carry the same oncogenic genetic lesions and has kept oncogenes firmly in focus as therapeutic targets. This rule is now broken in a study of the effect of BCR-ABL oncogene, linked to chronic myeloid leukemia (CML) disease in humans, in cancer development by limiting oncogene expression to the stem cell compartment. This study shows that by restricting the oncogene expression to Sca1⁺ cells CML arises indicating that silencing of BCR-ABL is not critical for generation of differentiated tumor cells and showing a “hands off” role for BCR-ABL in regulating cancer formation. Here we provide an update on the use of this system for modeling human cancer and its potential application for therapeutic targeting of the cancer stem cells (CSCs) and the hands-off function of oncogenes.

Inv9 Vascular Niche Factor PEDF Modulates Notch-Dependent Stemness in Adult Neurogenic Niches

We have addressed how vascular niches regulate the balance between self-renewal and commitment in stem cell populations. Pigment epithelium-derived factor (PEDF) is released by endogenous endothelial cells and promotes self-renewing divisions of the neural stem cells (NSCs) of the adult subependymal zone (SEZ). Here we show that soluble PEDF regulates the balance between self-renewal and commitment in NSCs by enhancing the Notch-dependent coordinated transcription of genes required for both proliferation and undifferentiation. In vivo, Notch activation is restricted to GLAST⁺/Sox2⁺ radial glia-like subependymal cells, as observed in transgenic Notch activity-reporter mice, and reporter activity correlates with neurosphere and multilineage differentiation potential. Moreover, the two incipient daughters of a dividing NSC can either exhibit equivalent or unequal levels of Notch activation and cells which inherit higher levels of Notch activity express higher levels of EGFR and Hes1 and are more self-renewing. In this scenario, PEDF intensifies Notch-dependent transcriptional activity in cells with low levels of Notch activation, thereby subverting the output of an asymmetrical stem cell division to the production of two stem cells. Our biochemical data indicate that PEDF upregulates Notch-mediated expression of Hes1 and EGFR genes by inducing the dismissal of co-repressor N-CoR from its binding to Notch-transcriptional effector CBF1 and that PEDF-induced N-CoR redistribution from the nucleus to the cytoplasm requires a non-canonical activation of the NFκB pathway. Thus, PEDF alters the balance between Notch-dependent symmetric and asymmetric self-renewing divisions, providing a basis for the regulation of stem cell maintenance in vascular niches.

Inv10 Carotid Body Neurogenic Niche and Parkinson

The carotid body (CB), the main peripheral chemoreceptor in mammals, is a neural crest-derived organ whose major physiological role is to detect oxygen tension in the arterial blood. The CB parenchyma is organized in clusters of highly dopaminergic sensory (glomus) cells innervated by numerous afferent nerve fibers, which in response to acute hypoxemia activate the brainstem respiratory centers to evoke hyperventilation. Our group has shown that transplantation of CB glomus cells into the striatum of Parkinsonian animals ameliorates the symptoms of this neurological disease. However, extrapolation of this procedure to Parkinson disease (PD) patients would need previous expansion of the glomus tissue due to the small size of the organ in humans. In situations of chronic hypoxia (as experienced by high altitude residents or by patients with chronic obstructive lung disease), the CB parenchyma grows in size, thus allowing adaptation of the organisms to a maintained low oxygen tension. We have recently shown that this classic adaptive response of the CB to chronic hypoxia depends on the activation of a population of neural progenitors able to proliferate and differentiate into new neuronal cells (Pardal et al., 2007) that could be used in cell therapy for PD. Deep characterization of the molecular and cellular events taking place in the CB neurogenic niche upon progenitor activation is necessary to improve our utilization of CB stem cells for glomus tissue expansion and transplantation against PD.

Pardal R., Ortega-Sáenz P., Durán R., and López-Barneo J. (2007) Cell 131, 364–377.

Inv11 Gene Virotherapy and Adenovirus

Virotherapy uses oncolytic viruses that grow selectively in tumor cells to treat cancer. Unlike chemotherapy drugs, viruses can respond to the absence of key molecular targets such as tumor suppressors and they amplify in tumors. Adenovirus is at the forefront of current virotherapy strategies. Clinical experience shows safety but limited efficacy. To gain efficacy adenovirus is genetically modified with mutations and transgenes. Random mutagenesis has revealed that substantial improvement can be achieved at the level of virus release from the infected cell and has led to new strategies to modulate such a release pharmacologically. Transgenes can be used to tackle key barriers for intratumoral spread of the virus but requires appropriate backbones to avoid detrimental effects on replication. We will present examples using fusogenic and matrix-degrading transgenes. Finally, virotherapy depends on the immunity against the virus and against the tumor. The development of immunocompetent models to assess adenovirus-mediated virotherapy is allowing to test for virocentric and immunocentric virotherapies.

Inv12 New Approaches for In Situ Expression of Recombinant Antibodies

In vivo production makes the antibodies less immunogenic and better tolerated and offers additional benefits by achieving sustained and effective concentration of therapeutic antibodies directly at points of target intervention. We have demonstrated the feasibility of in vivo production and systemic delivery of antibodies by different human cells. Human T lymphocytes, transduced ex vivo to secrete a bispecific (αCEA/αCD3) diabody on tumor site, inhibited the growth of CEA-positive tumors. However, activated T lymphocytes possess a short life span, and this implies a limitation to their application in a gene therapy strategy.

Cell-based tissue engineering is an evolving interdisciplinary area that offers new opportunities for clinical applications. We demonstrated that human mesenchymal stem cells genetically modified to express a bispecific (αCEA/αCD3) diabody and seeded in a synthetic extracellular matrix scaffold supported the release of functional antibody into the bloodstream at detectable levels for at least eight weeks after implantation. Furthermore, when therapeutic scaffolds were implanted in a location distant from the primary tumor, an effective anti-tumor response and tumor regression are induced. The biomedical potential of antibody factories will be discussed.

Inv13 Molecular Basis of Xeroderma Pigmentosum Group C DNA Recognition by Engineered Meganucleases

Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases, such as homing endonucleases, also known as meganucleases, constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules, Amel3-Amel4 and Ini3-Ini4, cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.

Inv14 Design of Multifunctional, Modular Proteins as Nonviral Vehicles for Gene Therapy

Most of the viral activities that are required for gene therapy purposes (including DNA condensation, self-assembling, specific cell attachment, internalization, endosomal escape, intracellular trafficking and nuclear transport) can be combined in single chain polypeptides by the appropriate selection of functional peptides or protein domains from different origins. Different combinations of functional modules, the relative position of critical segments, and the whole organization of the modular protein dramatically influence the final size of the protein-only assembles and DNA-protein complexes, as well as the percentage of transfected cells and the transgene expression levels. The semi-rational selection and proper organization of functional peptides, combined with strategies for cost-effective protein production and purification permits to foresee self-assembling modular proteins as promising, safer alternatives to viral vehicles and provide intriguing proof-of-concepts about the construction and functional tailoring of ‘artificial viruses'.

Inv15 Metabolic and Autoimmune Diseases Gene Therapy

No abstract available.

Inv16 Expanded Adipose-Derived Stem Cells for the Treatment of Complex Perianal Fistula

Management of complex perianal fistulas remains a challenge as a consequence of the serious limitations of currently available treatments. Expanded adipose-derived stem cells (ASCs) obtained from lipoaspirates appear to be a novel tool for the regeneration and/or repair of damaged tissues. Their use exploits two coordinated biological effects, namely, immunoregulation on one hand, and the proliferation and differentiation of cells on the other. Importantly, in a clinical setting, liposuctioned fat is available in large quantities and can be harvested with minimal adverse effects on the patient, unlike other sources of adult stem cells. In addition, adult stem cell therapy is not a subject to major ethical concerns generated by the use of embryonic stem cells. In the clinical program, expanded ASCs achieved promising results in a Phase I proof-of-concept study but firm conclusions could not be drawn in view of the small number of patients. A subsequent Phase II trial, including both Crohn's and non-Crohn's patients, was therefore conducted. The results have shown that healing rates with ASCs in combination with fibrin glue are significantly higher than those with fibrin glue alone. We stress that, as expected from the results of preclinical studies, the safety profile of the product in patients treated in the clinical trials conducted so far has been extremely good. The international multicenter Phase III trials are intended to provide further proof of the efficacy of ASC therapy. The marketing authorization of the product for application in perianal fistulas of cryptoglandular origin is estimated for the end of 2010. Once available, we believe the product will fulfill a clear unmet medical need and will contribute for quality of life improvement in patients with this distressing condition.

Inv17 Cardiac Regenerative Cell Therapy with Stem Cells

During the last 10 years we have witnessed the development of a new field in research termed Stem Cell Therapy. Classically, it was considered that cells had a limited division and differentiation ability; however, this dogma was challenged when new exciting results about cell multi/pluripotency were presented to the scientific community. Thus, cells from one adult tissue source were able to originate cells of a very different type. The possibility to transplant these cells in damaged organs with the aim to substitute the sick or death tissue, triggered many studies to understand the plasticity of the stem cells and their potential in pathological situations. Nowadays, much more is understood about stem cells, although of course, many questions, especially about their mechanism of action still need to be answered. Their benefit after transplantation has been shown experimentally and even clinically in some cases; however, the degree of stem cell contribution through their own differentiation into the transplanted tissue, has turned out to be generally low, and increased evidences indicate that a trophic effect must play an important role in such benefit. A better understanding of the paracrine mechanisms involved could be of great relevance in order develop new therapies focused on stimulating endogenous cells. On the other hand, more sophisticated methods for cell transplantation combined with bio-engineering techniques, have been started in cardiac disease models.

In this review we will try to critically provide an overview of the stem cell studies performed until now and to discuss some of the questions raised about the origin and mechanisms that are involved in their putative reparative effect in the cardiovascular diseases.

Inv18 Translational Research: From Basic Research to Clinical Trials

No abstract available.

Inv19 Translational Research: From Basic Research to Clinical Trials

No abstract available.

Inv20 The Organizational Model to Facilitate NonCommercial Clinical Translation in Advanced Therapies Developed by the Andalusian Initiative for Advanced Therapies

The Andalusian Initiative for Advanced Therapies (AIAT) created by the Andalusian Government coordinates and reinforces the research programmes on “Cell Therapy and Regenerative Medicine”, “Clinical Genetic and Genomic Medicine” and “Nanomedicine” supported by a network of research centres, biobanks and GMP facilities. Also, the Initiative finances research projects, develops recruitment and training programs (some of them aimed at technicians working in GMP facilities), promotes clinical research sponsoring clinical trials (CT) and encourages the public-private collaboration to develop new therapies and technologies.

In order to help the development of CT, the Initiative is promoting the creation of GMP facilities for cell and gene therapy as well as for tissue engineering. 6 GMP facilities are already constructed and 3 others are undergoing construction. The AIAT gives support designing technical installations, assessing on quality systems for laboratories, elaborating technical dossiers, counseling for regulatory agencies audits, hiring and training personnel. The AIAT helps in the elaboration of Investigational Medicinal Product Dossiers, counselling in product characterization and assessing in quality controls. Finally, the AIAT promotes the collaboration between basic and clinical researchers and gives support designing CT, hiring insurance policies, performing agreements with the centers and clinicians, handling of clinical research ethics committees and regulatory agencies authorization paperwork, coordinating the pharmacovigilance and hiring CROs for monitoring, supervising and performing the results analysis. As a result of this work, during 2009 we have recruited patients for 6 CT in cardiology, neurology and peripheral vascular diseases. In another 3 CT we are pending the regulatory agencies approval.

Inv21 Genome Editing in Human Stem Cells Using Zinc Finger Nucleases

Precise modification of human stem cells holds tremendous potential both in basic research and in the clinical application of stem cell therapies. However, methods which combine the necessary efficiency and specificity to accomplish such genome editing have been lacking. A solution to this problem emerges from the application of two highly conserved biological processes: DNA recognition by zinc finger proteins and the pathways of DNA double-strand break (DSB) repair. Zinc finger proteins engineered to bind a DNA sequence of interest can be fused to a nuclease domain to yield a zinc finger nuclease (ZFN). These ZFNs are capable of recognizing an investigator-specified target sequence in the stem cell genome and induce a DSB with high efficiency and precision. Subsequent repair of this targeted DSB can result in gene disruption. Alternatively, if suitably designed extrachromosomal “donor” DNA is provided, a process of homology-directed repair can be employed resulting in the site-specific addition of novel genetic information to cell. Importantly, simple transient expression of the ZFNs is sufficient to provoke permanent and heritable genetic changes. ZFN-mediated genome editing can be performed in primary CD4 T cells, hematopoetic stem cells (HSCs), as well as human embryonic stem (ES) cells, the latter resulting in multipotent stem cells that possess the targeted gene modification but otherwise divide and differentiate normally. Clinical translation of the ZFN approach to cell-based therapy is underway with the initiation of the first clinical trial of an engineered ZFN in patients with HIV/AIDS.

Inv22 Retroviral Vectors and Insertional Mutagenesis

Retroviral vector-mediated gene transfer into hematopoietic stem cells (HSC) may be used to treat a great variety of genetic and acquired disorders. However, adverse reactions related to insertional mutagenesis have created substantial uncertainty. We have developed murine models to address the impact of target cell type, cell culture conditions and vector technology on the induction of insertional mutants. To this end, we compare results obtained using a cell-culture based assay with insights from studies of vector-modified HSC performed in serial transplantation with long-term follow-up (up to 20 months). In the worst case, we found that a single insertion of a vector with a strong viral enhancer-promoter next to a proto-oncogene such as Evi1 or Prdm16 may be sufficient to induce leukemia. Further experiments identified a hierarchy of factors governing the emergence of insertional mutants. The progeny of multipotent cells with a priori HSC potential were considerably more sensitive to insertional mutations than multipotent progenitor cells, more mature myeloid cells and T cells. The second most important factor in the induction of insertional mutants is the presence of a strong enhancer-promoter in the vector backbone. The third factor in this hierarchy is the insertion pattern of retroviral vectors, with the gammaretroviral insertion pattern showing a greater risk to induce insertional mutants than the lentiviral pattern. However, lentiviral vectors harboring strong internal enhancer-promoter sequences with high activity in HSC were still able to trigger insertional transformation. These studies provide the basis for rational approaches to improve the therapeutic index of integrating vectors.

Inv23 Lentiviral Vectors as Vaccines

The aim of my group is to engineer viral vectors to be used as effective vaccines. Lentiviral vectors based on human immunodeficiency virus type 1 (HIV-1) transduce dendritic cells in draining lymph nodes after subcutaneous injection. Using vectors encoding ovalbumin, or the tumour antigen NY-ESO-1 we have shown that lentiviral vector injection initiates potent antigen-specific CD4⁺ and CD8⁺ T cell responses. Protective and therapeutic anti-tumor immunity can be induced. To improve safety and efficacy, we have developed non-integrating lentiviral vectors, and also vectors with uptake and antigen expression targeted to antigen presenting cells. We have also expressed constitutive activators of NFkappaB or mitogen-activated protein kinase pathways. Triggering of NFkappaB or p38 led to activated DC, and substantially enhanced the anti-tumor immune response. Activation of ERK increased TGF-β expression, suppressed the immune response and stimulated expansion of regulatory T cells. These results provide a toolkit to regulate immune responses to immunization; vaccine responses to foreign or tumor antigens can be enhanced and harmful responses to self-antigens or introduced transgenes can be reduced.

Inv25 Tissue Engineering as an Alternative to Improve Transplant's Outcome

No abstract available.

Inv26 Regenerative Medicine for Primary Immunodeficiencies

No abstract available.

Inv27 Skin Regeneration

Regenerative medicine involving cell therapy is an emerging field that seeks to combine the knowledge and expertise of diverse disciplines towards the aim of restoring impaired organ functions in the body. Its goal is not just to replace what is malfunctioning, but to provide the elements required for in vivo repair, to devise replacements that seamlessly interact with the living body, and to stimulate the body's intrinsic capacities for regeneration. A great deal of expectation has been put on the capabilities of both embryonic and adult stem cells towards these aims and certainly, revolutionary advances in the field are foreseen.

Skin is the outermost tissue of the body and the largest organ in terms of both weight and surface area. The main function of skin is to act as a barrier to the surrounding environmental dangers. It protects the body from friction and impact wounds with its flexibility and toughness. It also prevents water loss and regulates body temperature by blood flow and evaporation of sweat. The skin is formed by anatomically, functionally and developmentally distinct tissues: the epidermis and the dermis. The epidermis is the outermost component of the skin formed mostly by keratinocyes. The epidermis is morphologically divided into different layers or strata. Keratinocytes produced in the basal layer, where cell proliferation is confined, move upward to the outer surface in a process named as epidermal differentiation. During this turn-over, keratinocytes change their structures and physiological functions. One cycle of this turn-over process takes about 28 days. The dermis is the living layer that acts as a substrate and a support network for the epidermis. The essential dermal cell type is the fibroblast, which is responsible for the production and maintenance of the structural elements of skin. Finally, a complex basement membrane (BM) composed by specialized proteins serves as an epidermal and dermal anchoring structure. Mutations in the genes coding for the BM are responsible for rare inherited cutaneous diseases. Regeneration of epidermis throughout life is achieved by a specialized, discrete population of basal keratinocytes know as epidermal stem cells.

Bioengineered skin substitutes have emerged over the past 20 years as the most carefully studied and proven of the advanced wound management technologies. While the initial impetus for their development was to replace autograft and allograft in acute skin losses applications, they have found wider application in the treatment of chronic and even genetic cutaneous disorders. Although an ideal skin substitute has not yet been achieved, a robust product has been recently developed by our team that provides many of the desired clinical characteristics. Such a product was devised by carefully looking at the wound healing process. Thus, fibrin was chosen as a matrix suitable to host dermal cells in a bioengineered skin equivalent. Fibrin is the primary and temporary wound healing matrix allowing blood clotting and migration of both, epithelial and mesenchymal cellular elements that, in turn, will repair the damaged tissue. Autologous epidermal stem cells as part of a fibrin-based skin equivalent have been used successfully for permanent skin regeneration in different situations: extensive burns, necrotizing fascitis and graft-versus-host disease. Promising results are currently being obtained also in the management of Epidermolysis Bullosa through allogenic epidermal stem cell transplantation.

Inv28 Improved Immunotherapies for Liver Cancer

Advanced hepatocellular carcinoma (HCC) not elegible for transplantation or surgical resection lacks curative therapy. Current treatment is based on chemoembolization, internal radiation using yttrium-90 microspheres or administration of sorafenib. However all these approaches have only a modest impact on survival. Immunotherapy is an attractive approach to fight liver cancer as it has been shown that tumor infiltrating lymphocytes are able to kill cancerous cells. Antitumor vaccination can be performed by procedures targeting specific tumor-associated self-antigens (such as alpha-fetoprotein) or by stimulating the response against undefined tumor antigens using tumor lysates. These can be used to pulse autologous dendritic cells that can be reinjected into the patient after activation ex vivo.

On the other hand, antitumor immunity can be elicited using gene therapy vectors encoding immunostimulatory cytokines to transduce the tumor or the peritumoral tissue. Interleukin-12 (IL-12) has been employed in many studies due to its ability to activate cytotoxic T cells and NK cells. In addition IL-12 displays potent antiangiogenic effects that can contribute to inhibit tumor progression. Some clinical trials have been performed in patients with liver cancer applying intratumor injection of first generation adenoviral vectors encoding IL-12. These studies have shown that the procedure is feasible and well tolerated and that tumor nodules are very well transduced with type of vectors. However, the expression of the transgene lasts for only few days being the efficacy of the therapy limited by the brevity of transgene expression and by the fact that repeated administrations of the vector are not followed by repeated tumor transduction due to the generation of neutralizing antibodies. These studies showed that gene therapy is a promising strategy to fight cancer but they also revealed that there is a need to improve vector design to allow long term expression of the transgene. With this type of vectors it is necessary to control the intensity of transgene expression using inducible promoters. This can be achieved by utilizing third generation adenoviral vectors also named gutless adenovirus (gAd) or high capacity adenovirus. These vectors are devoid of all adenoviral genes and possess high cloning capacity allowing the use of inducible promoters.

In experimental animals gAd encoding IL-12 have been shown to inhibit tumor growth, to elicit potent antitumor responses and to prolong animal survival. We have observed that although IL-12 activates effector T cells, this cytokine also increases the presence of regulatory T cells (Treg) in the tumor tissue. Treg block effector T cells acting in part through TGFbeta. Interestingly, the use of TGFbeta inhibitors has been shown to increase the antitumor effect of IL-12-based gene therapy. Myeloid suppressor cells are another variety of cells that contribute to blunt antitumor immunity. We found that a low dose of cyclophosphamide reduces the abundance of these cells in the tumor environment resulting in increased efficacy or IL-12 gene therapy.

Tumor cells have acquired mechanisms to become invisible to the immune system by downregulating the expression of HLA and immunostimulatory molecules. Recently we found that during the initiation of the immune response, dendritic cells (the most relevant immunostimulatory antigen-presenting cells) release not only type I interferon (IFN) but also oncostatin (OSM) in response to TLR stimulation. Interestingly we observed a strong synergy of these two cytokines in the activation of immunostimulatory functions of hepatoma cells. Thus, upon incubation of tumor cells with IFN type I plus OSM we detected a marked induction of ubiquitination processes, immunoproteasome activity, and upregulation of the expression of TAP1, TAP2, HLA molecules, ICAM-1 and IL-15 receptor together with increased IL-7 production. The treatment with IFN type I plus OSM conferred tumor cells the ability to present antigenic peptides more efficiently to T cell effectors, to increase the presentation of IL-15 to CD8⁺ T cells favoring the activation and expansion of memory cytotoxic T cells. According to these findings the combined treatment IFN type I plus OSM, either using recombinant proteins or gene therapy vectors, will promote specific antitumor responses and will make tumor cells more sensitive to the immune attack. This therapy can be combined with IL-12 gene therapy to achieve potent antitumor immunity. Further trials using these improved strategies are clearly needed.

Inv29 Gene Therapy of Experimental Viral Hepatitis

Hepatitis B and C are among the most prevalent viral diseases in the world. They lead to chronic liver disease in a high percentage of infected individuals, putting them at an increased risk for liver-related morbidity and mortality from complications of cirrhosis and hepatocellular carcinoma. Despite the success of universal hepatitis B vaccination in many countries, this disease remains a major public health problem, resulting in more than 500,000 deaths per year (1). In the case of hepatitis C the situation is aggravated due to lack of a prophylactic vaccine. It is estimated that HCV infects more than 170 million people worldwide and 20 to 30% of them will eventually face life-threatening symptoms (2). Existing therapies against chronic viral hepatitis are far from satisfactory due to low response rates, undesirable side effects and selection of resistant viral strains. Therefore, new therapeutic approaches are urgently needed. Gene therapy represents a promising alternative for the prevention and treatment of hepatitis B (HBV) and C (HCV) viral infections. Three basic strategies can be considered: (1) Immunological approaches: immunogene therapy and genetic vaccines; (2) Genetic antivirals; (3) Suicide gene therapy.

Immunogene therapy consists on the expression of cytokines with antiviral and immunomodulatory activities. Intrahepatic expression has the advantage of attaining a high liver-to-blood concentration ratio of the cytokine, this would increase the efficacy of the therapy while minimizing the toxic side effects that are associated with systemic treatments. Genetic vaccines are based on the expression of viral gene products alone or together with immunostimulatory cytokines in order to stimulate the host's immune response against infected cells. This strategy, seen primarily as preventive also holds a great therapeutic potential (3). Genetic antivirals were designed to block the viral life cycle at different points. In vitro a large number of different molecules have been shown to have a strong antiviral activity; however, in vivo only few studies using genetic antivirals have shown efficacy, most of them based on the use of antisense oligonucleotides and short interfering RNAs (siRNAs). Finally, suicide gene therapy represents an attractive novel strategy the goal of which is the specific elimination of infected cells by virus-mediated expression or activation of toxic molecules.

We found two main limitations for the clinical application of gene therapy protocols in chronically infected patients, the absence of data on clinically relevant animal models and the efficacy of the current gene delivery systems existing for liver transduction. Nowadays, the best animal model for the study of new therapies in the context of a chronic liver infection is the woodchuck infected with Woodchuck hepatitis virus (WHV). Using this relevant model we have analyzed the effect of the hepatic specific expression of cytokines with proven antiviral activity, using adeno-associated virus (AAV) and high capacity adenoviral vectors. Our data indicates that gene transfer of antiviral cytokines holds out great promise for new generations of antihepatitis therapies.

Inv30 Efficiency of Integration-Deficient Lentiviral Vectors (IDLVs) in the Spinal Cord

Integration-deficient lentiviral vectors can be produced through the use of integrase mutations. Such mutants generate normal amounts of viral DNA, which is then converted into circular episomes mostly by cellular DNA repair pathways. The circular molecules are metabolically stable but lack replication signals, being progressively diluted out in dividing cell populations. IDLVs support a number of processes, including gene expression and several forms of recombination (site-specific recombination and transposition, as well as homologous recombination-mediated gene targeting). Efficient gene expression from IDLVs results in transient transduction of dividing cells and stable gene expression in quiescent cells in vitro and in vivo. We have demonstrated stable gene expression in eye, brain and muscle, and have recently undertaken an extensive analysis of spinal cord transduction in vitro and in vivo with IDLVs. Direct injection of eGFP-expressing vectors in the ventral horn of the rat spinal cord led to efficient labelling of motor neurons, with lower transduction of astrocytes. In vitro motor neurons and dorsal root ganglia (DRG) neurons were also transduced efficiently. Astrocytes were remarkably susceptible to transduction at low multiplicities of infection. Purified cord microglia was the only cell type tested whose transduction efficiency was clearly higher with integrating than non-integrating vectors. Efficient RNAi mediated by IDLVs was demonstrated in DRG neurons. Expression of a therapeutic transgene (survival motor neuron, SMN, defective in spinal muscular atrophy) in cultured neurons was efficient regardless of lentivector integration proficiency, paving the way for preclinical tests in spinal disease.

Inv31 Prospects 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 to ameliorate 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.

Inv32 Gene Therapy of Pyruvate Kinase Deficiency

Human pyruvate kinase deficiency (PKD) is an autosomal recessive disorder produced by mutations in the PKLR gene. The gene deficiency produces a defect in the production of ATP in erythrocytes, which ultimately causes chronic nonspherocytic haemolytic anaemia. Clinical symptoms vary considerably from mild to severe anaemia. Severe cases require periodical blood transfusion, splenectomy and in some patients bone marrow transplantation. These characteristics make this disease a good candidate to be treated by gene therapy. To test this hypothesis, gammaretroviral vectors expressing the human RPK were developed. Wild type and pklr deficient mouse hematopoietic cells were infected with these vectors and transplanted into normal and deficient recipients. Expression of the transgene was observed for long periods of time in primary and secondary recipients. A complete recovery of the disease phenotype was observed in the deficient animals transplanted with corrected cells, when more than 25% of genetically corrected cells were transplanted. Additionally, corrected cells were injected in utero into 14.5 days old deficient fetuses. A partial correction was observed late after birth. To improve the gene therapy tools, optimized lentiviral vectors with the expression of the human PKLR gene driven by the erythroid specific promoter sequences of the same gene, have been developed. A moderate and stable expression of the transgene has been demonstrated in human hematopoietic cells. The efficacy of these optimized vectors is now under study in the murine model of PKD. In summary, gene therapy approaches could represent an efficient therapeutic treatment in severe clinical cases of human erythrocyte PKD.

Inv33 Multipotent Cardiac Resident Progenitor Cells: Basic and Applied Studies

In the last six years the concept of the mammalian heart as a non-postmitotic organ is being consolidated. Several cellular populations, isolated from the neonatal or adult heart, have been described to possess self-renewal capacity, multipotency and the ability to promote functional recovery of infarcted areas upon local injection. Globally, they have been denominated resident cardiac stem cells (CSC), although neither their ontogenic origin or the putative relationships between the several populations described are totally understood.

Our group is addressing the study of some of these CSC populations in a multidisciplinary approach, together with more general questions associated with the practical development of Cell Therapy strategies. First, we are trying to define genetic programmes (both involving cDNAs and miRNAs) that could be relevant for stemness and/or multipotency. Second, we are analyzing the impact of ex vivo culture conditions on genomic stability and biosafety. Thirdly, we are also trying to get a deeper knowledge of the basic aspects that control the regulation of the balance between self-renewal and differentiation in vivo, under physiological and pathological conditions. These parameters are being also evaluated in the porcine animal model. In all these studies we are comparing CSC and mesenchymal stem cell (MSC) populations (from mouse, pig and human), under different conditions promoting an undifferentiated state or cell differentiation. Finally, we are beginning to study some functions that could have implications on the physiological aging of the CSC, either directly or through the progressive functional decline of the associated niche structures.

Inv34 Human Mesenchymal Stem Cells Versus Umbilical Cord Blood Cells for the Treatment of Myocardial Infarction

While emerging cell therapies for the treatment of ischemic heart diseases, there is no consensus about the best cell type to be used in clinical trials. In this work we have compared head to head the ability of human CD34 hematopoietic stem cells (HSC) and bone marrow mesenchymal stem cells (MSC) to treat myocardial infarction. Human bone marrow MSC or isolated CD34 HSC from umbilical cord blood (UCB) were injected in infarcted nude rats. Cardiac function was analyzed by echocardiography. Ventricular remodelling was evaluated by tissue histology and electron microscopy and neo-formed vessels were quantified by immunohistochemistry. The results showed that both cell types induced an improvement in cardiac function. However, transplantation of MSC prevented ventricular remodelling more efficiently. Thus, they could be more appropriated to treat severe chronic myocardial infarction.

Inv35 New Perspectives in Cell-Gene Therapy

No abstract available.