GATA4 induces liver fibrosis regression by deactivating hepatic stellate cells.

In response to liver injury, hepatic stellate cells activate and acquire proliferative and contractile features. The regression of liver fibrosis appears to involve the clearance of activated hepatic stellate cells, either by apoptosis or by reversion toward a quiescent-like state, a process called deactivation. Thus, deactivation of active hepatic stellate cells has emerged as a novel and promising therapeutic approach for liver fibrosis. However, our knowledge of the master regulators involved in the deactivation and/or activation of fibrotic hepatic stellate cells is still limited. The transcription factor GATA4 has been previously shown to play an important role in embryonic hepatic stellate cell quiescence. In this work, we show that lack of GATA4 in adult mice caused hepatic stellate cell activation and, consequently, liver fibrosis. During regression of liver fibrosis, Gata4 was reexpressed in deactivated hepatic stellate cells. Overexpression of Gata4 in hepatic stellate cells promoted liver fibrosis regression in CCl4-treated mice. GATA4 induced changes in the expression of fibrogenic and antifibrogenic genes, promoting hepatic stellate cell deactivation. Finally, we show that GATA4 directly repressed EPAS1 transcription in hepatic stellate cells and that stabilization of the HIF2α protein in hepatic stellate cells leads to liver fibrosis.

Viral gene delivery vectors: the next generation medicines for immune-related diseases.

Viruses have evolved to efficiently express their genes in host cells, which makes them ideally suited as gene delivery vectors for gene and immunotherapies. Replication competent (RC) viral vectors encoding foreign or self-proteins induce strong T-cell responses that can be used for the development of effective cancer treatments. Replication-defective (RD) viral vectors encoding self-proteins are non-immunogenic when introduced in a host naïve for the cognate virus. RD viral vectors can be used to develop gene replacement therapies for genetic disorders and tolerization therapies for autoimmune diseases and allergies. Degenerative/inflammatory diseases are associated with chronic inflammation and immune responses that damage the tissues involved. These diseases therefore strongly resemble autoimmune diseases. This review deals with the use of RC and RD viral vectors for unraveling the pathogenesis of immune-related diseases and their application to the development of the next generation prophylactics and therapeutics for todays' major diseases.

How Simian Virus 40 Hijacks the Intracellular Protein Trafficking Pathway to Its Own Benefit and Ours.

Viruses efficiently transfer and express their genes in host cells and evolve to evade the host's defense responses. These properties render them highly attractive for use as gene delivery vectors in vaccines, gene, and immunotherapies. Among the viruses used as gene delivery vectors, the macaque polyomavirus Simian Virus 40 (SV40) is unique in its capacity to evade intracellular antiviral defense responses upon cell entry. We here describe the unique way by which SV40 particles deliver their genomes in the nucleus of permissive cells and how they prevent presentation of viral antigens to the host's immune system. The non-immunogenicity in its natural host is not only of benefit to the virus but also to us in developing effective SV40 vector-based treatments for today's major human diseases.

LRH-1 agonism favours an immune-islet dialogue which protects against diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus.

Generation of a Vero-Based Packaging Cell Line to Produce SV40 Gene Delivery Vectors for Use in Clinical Gene Therapy Studies.

Replication-defective (RD) recombinant simian virus 40 (SV40)-based gene delivery vectors hold a great potential for clinical applications because of their presumed non-immunogenicity and capacity to induce immune tolerance to the transgene products in humans. However, the clinical use of SV40 vectors has been hampered by the lack of a packaging cell line that produces replication-competent (RC) free SV40 particles in the vector production process. To solve this problem, we have adapted the current SV40 vector genome used for the production of vector particles and generated a novel Vero-based packaging cell line named SuperVero that exclusively expresses the SV40 large T antigen. SuperVero cells produce similar numbers of SV40 vector particles compared to the currently used packaging cell lines, albeit in the absence of contaminating RC SV40 particles. Our unique SV40 vector platform named SVac paves the way to clinically test a whole new generation of SV40-based therapeutics for a broad range of important diseases.

Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

Absence of WASp Enhances Hematopoietic and Megakaryocytic Differentiation in a Human Embryonic Stem Cell Model.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.

The 'Omics' of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease that primarily affects motor neurons and is accompanied by sustained unregulated immune responses, but without clear indications of the ultimate causative mechanisms. The identification of a diverse array of ALS phenotypes, a series of recently discovered mutations, and the links between ALS and frontotemporal degeneration have significantly increased our knowledge of the disease. In this review we discuss the main features involved in ALS pathophysiology in the context of recent advances in 'omics' approaches, including genomics, proteomics, and others. We emphasize the pressing need to combine clinical imaging with various different parameters taken from omics fields to facilitate early, accurate diagnosis and rational drug design in the treatment of ALS.

SCL/TAL1-mediated transcriptional network enhances megakaryocytic specification of human embryonic stem cells.

Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.

Mesenchymal stromal cells express GARP/LRRC32 on their surface: effects on their biology and immunomodulatory capacity.

Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-ß1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency-associated peptide (LAP)/TGF-ß1 to the cell surface of activated Foxp3(+) regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF-ß1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-ß1 and reduced their proliferative capacity in a TGF-ß1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T-cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker.

Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome.

Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS), which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK) development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN) pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells) resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFß and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS patients and as a cellular model to study new therapeutic strategies.

Mesenchymal stem cells expressing vasoactive intestinal peptide ameliorate symptoms in a model of chronic multiple sclerosis.

Multiple sclerosis (MS) is a severe debilitating disorder characterized by progressive demyelination and axonal damage of the central nervous system (CNS). Current therapies for MS inhibit the immune response and demonstrate reasonable benefits if applied during the early phase of relapsing­remitting MS (RRMS) while there are no treatments for patients that progress neither to the chronic phase nor for the primary progressive form of the disease. In this manuscript, we have studied the therapeutic efficacy of a cell and gene therapy strategy for the treatment of a mouse model of chronic MS [myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE)]. We used allogenic mesenchymal stem cells (MSCs) asa therapeutic tool and also as vehicle to deliver fully processed 3.3-kDa vasoactive intestinal peptide (VIP) to the peripheral immune organs and to the inflamed CNS. Intraperitoneal administrations of MSCs expressing VIP stopped progression and reduced symptoms when administered at peak of disease. The improvement in clinical score correlated with diminished peripheral T-cell responses against MOG as well as lower inflammation,lower demyelination, and higher neuronal integrity in the CNS. Interestingly, neither lentiviral vectors expressing VIP nor unmodified MSCs were therapeutic when administer at the peak of disease. The increased therapeutic effect of MSCs expressing VIP over unmodified MSCs requires the immunoregulatory and neuroprotective roles of both VIP and MSCs and the ability of the MSCs to migrate to peripheral lymph organs and the inflamed CNS.

Prostaglandin E2-induced IL-23p19 subunit is regulated by cAMP-responsive element-binding protein and C/AATT enhancer-binding protein ß in bone marrow-derived dendritic cells.

We reported previously that prostaglandin E2 (PGE2) up-regulates IL-23 in vitro in bone marrow-derived dendritic cells and in vivo in models of collagen-induced arthritis and inflammatory bowel disease, leading to preferential Th17 development and activity. There is very little information on the molecular mechanisms involved in the PGE2-induced up-regulation of Il23a gene expression. In this study we investigated the signaling pathways and transcription factors involved in the stimulatory effect of PGE2. Although PGE2 does not induce IL-23p19 expression by itself, it synergizes with both extra- and intracellular Toll-like receptor ligands and with inflammatory cytokines such as TNFα. We established that the effect of PGE2 in conjunction with either LPS or TNFα is mediated through the EP4 receptor and the cAMP-dependent activation of both protein kinase A (PKA) and exchange protein activated by cAMP (EPAC). Using the EP4 agonist PGE(1)OH in conjunction with TNFα, we found that PKA-induced phosphorylation of cAMP-response element-binding protein ((P)CREB) and EPAC-induced phosphorylation of C/AATT enhancer-binding protein ß ((P)C/EBPß) mediate the stimulatory effect of PGE2 on IL-23p19 expression. This is the first report of CREB and C/EBPß involvement in Il23a promoter activation. Mutation within the putative CREB and C/EBP sites combined with in vivo DNA binding (ChIP) assays identified the distal CREB site (-1125) and the two proximal C/EBP sites (-274 and -232) as essential for PKA-activated CREB and EPAC-activated C/EBPß-induced IL-23p19 expression.

Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors.

Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.

Development of an all-in-one lentiviral vector system based on the original TetR for the easy generation of Tet-ON cell lines.

Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and gene therapy approaches. Combining LVs with drug-inducible expression systems should allow tight control of transgene expression with minimal side effect on relevant target cells. A new doxycycline-regulated system based on the original TetR repressor was developed in 1998 as an alternative to the TetR-VP16 chimeras (tTA and rtTA) to avoid secondary effects due to the expression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscript we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have also showed that this dual vector system can generate regulatable bulk cell lines (expressing high levels of TetR) that are able to modulate transgene expression either by varying doxycycline concentration and/or by varying the amount of CTetOE vector genomes per cell. Based on these results we have developed a new all-in-one lentiviral vector (CEST) driving the expression of TetR through the SFFV promoter and the expression of eGFP through the doxycycline-responsive CMV-TetO operon. This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) cells. Bulk doxycycline-responsive cell lines express high levels of the transgene with low amount of doxycycline and are phenotypically indistinct from its parental cells.

Cecal ligation puncture procedure.

Human sepsis is characterized by a set of systemic reactions in response to intensive and massive infection that failed to be locally contained by the host. Currently, sepsis ranks among the top ten causes of mortality in the USA intensive care units. During sepsis there are two established haemodynamic phases that may overlap. The initial phase (hyperdynamic) is defined as a massive production of proinflammatory cytokines and reactive oxygen species by macrophages and neutrophils that affects vascular permeability (leading to hypotension), cardiac function and induces metabolic changes culminating in tissue necrosis and organ failure. Consequently, the most common cause of mortality is acute kidney injury. The second phase (hypodynamic) is an anti-inflammatory process involving altered monocyte antigen presentation, decreased lymphocyte proliferation and function and increased apoptosis. This state known as immunosuppression or immune depression sharply increases the risk of nocosomial infections and ultimately, death. The mechanisms of these pathophysiological processes are not well characterized. Because both phases of sepsis may cause irreversible and irreparable damage, it is essential to determine the immunological and physiological status of the patient. This is the main reason why many therapeutic drugs have failed. The same drug given at different stages of sepsis may be therapeutic or otherwise harmful or have no effect. To understand sepsis at various levels it is crucial to have a suitable and comprehensive animal model that reproduces the clinical course of the disease. It is important to characterize the pathophysiological mechanisms occurring during sepsis and control the model conditions for testing potential therapeutic agents. To study the etiology of human sepsis researchers have developed different animal models. The most widely used clinical model is cecal ligation and puncture (CLP). The CLP model consists of the perforation of the cecum allowing the release of fecal material into the peritoneal cavity to generate an exacerbated immune response induced by polymicrobial infection. This model fulfills the human condition that is clinically relevant. As in humans, mice that undergo CLP with fluid resuscitation show the first (early) hyperdynamic phase that in time progresses to the second (late) hypodynamic phase. In addition, the cytokine profile is similar to that seen in human sepsis where there is increased lymphocyte apoptosis (reviewed in). Due to the multiple and overlapping mechanisms involved in sepsis, researchers need a suitable sepsis model of controlled severity in order to obtain consistent and reproducible results.

Docosahexaenoic acid prevents dendritic cell maturation and in vitro and in vivo expression of the IL-12 cytokine family.

BACKGROUND: Acute and chronic inflammation play essential roles in inflammatory/autoimmune conditions. Protective anti-inflammatory effects of the n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were reported in animal models of colitis, sepsis, and stroke. Since dendritic cells (DC) represent the essential cellular link between innate and adaptive immunity and have a prominent role in tolerance for self-antigens, we sought to investigate the impact of DHA on DC maturation and proinflammatory cytokine production. METHODS: Murine bone marrow-derived DC were treated with DHA and stimulated with various toll-like receptor (TLR) ligands. Flow cytometry was used to determine the levels of surface maturation markers and endocytic activity. Cytokine expression and secretion were measured by real-time RT-PCR and ELISA assays. PPARgamma and NFkappaB activity in nuclear extracts were determined by binding to specific oligonucleotide sequences using ELISA-based assays. In vivo effects of DHA were assessed in splenic DC from LPS-inoculated mice maintained on a DHA-enriched diet. RESULTS: DHA maintained the immature phenotype in bone marrow-derived DC by preventing the upregulation of MHCII and costimulatory molecules (CD40, CD80 and CD86) and maintaining high levels of endocytic activity. DHA inhibited the production of pro-inflammatory cytokines, including the IL-12 cytokine family (IL-12p70, IL-23, and IL-27), from DC stimulated with TLR2, 3, 4, and 9 ligands. DHA inhibition of IL-12 expression was mediated through activation of PPARgamma and inhibition of NFkappaBp65 nuclear translocation. DHA exerted a similar inhibitory effect on IL-12 and IL-23 expression in vivo in LPS-inoculated mice maintained on a DHA-enriched diet. CONCLUSIONS: Exposure of bone marrow-derived DC to DHA resulted in the maintenance of an immature phenotype and drastic reduction in proinflammatory cytokine release. DHA inhibited the expression and secretion of the IL-12 cytokine family members (IL-12p70, IL-23 and IL-27), which play essential roles in the differentiation of the proinflammatory Th1/Th17 effector cells. The effect of DHA on IL-12 expression was mediated through activation of PPARgamma and inhibition of NFkappaB. Inhibition of IL-12 and IL-23 expression was also evident in splenic DC from mice fed a DHA-enriched diet, suggesting that dietary DHA acts as an anti-inflammatory agent in vivo.

Dendritic cells transduced with lentiviral vectors expressing VIP differentiate into VIP-secreting tolerogenic-like DCs.

Dendritic cells (DCs) initiate immune responses as well as tolerance. We showed previously that the neuropeptide vasoactive intestinal peptide (VIP) suppresses innate immune responses, modulates adaptive responses by generating regulatory T cells (Treg) through the induction of tolerogenic DCs (tDCs), and has therapeutic effects in models of autoimmune/inflammatory disorders. Systemic VIP administration is limited by its short biological half-life and by its pleiotropic effects on the cardiovascular system and gastrointestinal tract. Therefore, we used lentiviral vectors to genetically engineer VIP-expressing bone marrow-derived DC (BMDC) and characterized the transduced LentiVIP-DC in terms of phenotype and therapeutic effects in models of experimental autoimmune encephalomyelitis (EAE) and cecal ligation and puncture (CLP) sepsis. LentiVIP-DCs secrete VIP, and resemble tDCs through lack of co-stimulatory molecule upregulation, lack of proinflammatory cytokine secretion, increased interleukin (IL)-10 production, and poor stimulation of allogeneic T cells. A single inoculation of LentiVIP-DC in EAE or CLP mice had therapeutic effects, which correlated with reduced expression of proinflammatory cytokines and increased IL-10 production in spinal cord and peritoneal fluid, respectively. In contrast to systemic VIP administration that requires repeated, high-dose inoculations, local delivery of VIP by LentiVIP-DC may represent a promising therapeutic tool for the treatment of autoimmune diseases and inflammatory disorders.

Was cDNA sequences modulate transgene expression of was promoter-driven lentiviral vectors.

Abstract The development of vectors that express a therapeutic transgene efficiently and specifically in hematopoietic cells (HCs) is an important goal for gene therapy of hematological disorders. We have previously shown that a 500-bp fragment from the proximal Was gene promoter in a lentiviral vector (LV) was sufficient to achieve more than 100-fold higher levels of Wiskott-Aldrich syndrome protein in HCs than in nonhematopoietic cells (non-HCs). We show now that this differential was reduced up to 10 times when the enhanced green fluorescent protein gene (eGFP) was expressed instead of Was in the same LV backbone. Insertion of Was cDNA sequences downstream of eGFP in these LVs had a negative effect on transgene expression. This effect varied in different cell types but, overall, Was cDNA sequences increased the hematopoietic specificity of Was promoter-driven LV. We have characterized the minimal fragment required to increase hematopoietic specificity and have demonstrated that the mechanism involves Was promoter regulation and RNA processing. In addition, we have shown that Was cDNA sequences interfere with the enhancer activity of the woodchuck posttranscriptional regulatory element. These results represent the first data showing the role of Was intragenic sequences in gene regulation.

Safer vectors for gene therapy of primary immunodeficiencies.

Primary immunodeficiencies (PID) are caused by mutations in genes that impair the development or activity of the immune system. Although bone marrow transplants achieve long time restoration in up to 90% of treated patients, morbidity and mortality are still high for some PID and adequate donors are not always available. Gene Therapy (GT) was envisioned as an alternative treatment for PID by inserting the correct gene into the patient's haematopoietic stem cells (HSCs). Up to date, GT for PID has succeeded in 40 of 44 patients treated in four clinical trials. However, five children enrolled in the SCID-X1 clinical trial developed leukaemia-like disease produced by aberrant expression of oncogenes. This phenomenon resulted fatal in one patient and represented a severe setback for gene therapy. Since then, vector development has been a priority in the GT field, by refining existing Murine laeukemia virus (MLV)-based vectors or by developing new ones. This review summarize existing methodologies for PID GT highlighting the importance of animal models in the PID GT success and focusing on new gene transfer vectors to achieve safe, efficient and stable gene modification.