Abstract
Lectures (chronological order)
1. Gene therapy for primary immunodeficiencies
Gene therapy has become an attractive alternative therapeutic strategy to allogeneic transplant for primary immunodeficiencies (PIDs) due to known genetic defects. Over 60 patients affected by severe combined immunodeficiencies (SCID) due to IL2RG deficiency (SCID-X1) or adenosine deaminase (ADA)-SCID have received gene therapy with hematopoietic stem cells (HSC) in the past 15 years using gammaretroviral vectors, achieving immune reconstitution and clinical benefit in the majority of them. Moreover, results of clinical trials for Wiskott-Aldrich syndrome (WAS) and Chronic Granulomatous disease (CGD) showed the proof of principle of gene therapy for these diseases. However, the occurrence of insertional oncogenesis in the SCID-X1 trials and subsequently in CGD and WAS, highlighted limitations of the technology, leading to the development of novel experimental approaches. Current approaches are based on vectors that can achieve more robust correction with less risk of insertional mutagenesis, such as self inactivating lentiviral viral vectors.
Results from our gene therapy trial for WAS, based on infusion of lentiviral vector-transduced HSC, show robust engraftment, polyclonal reconstitution, restored WASp expression, and immunological improvement with clinical benefit. Moreover, we did not observe in vivo selection of integrations near cancer genes or aberrant clonal expansions.
These data demonstrate the improved safety and efficacy attained by lentiviral vector-based gene therapy providing a new treatment option for WAS and conceivably, other genetic disorders.
2. The T-body approach: From the mouse cage to the patient's bed
The “T-body” approach is based on engineered T cells expressing chimeric antibody receptor (CAR) that endows them with antibody-type specificity to any pre-defined target antigen. We have designed the modular CAR construct so that the modified, redirected T cells will undergo activation and perform their effector or regulatory function upon encountering their target.
Accordingly, the CAR's composition includes extracellular recognition domain made of a single chain variable fragment (scFv) of an antibody linked to intracellular domains of various stimulatory and co-stimulatory molecules that dictate either the effector (Teff) or regulatory (Treg) function of T cells. For antitumor reactivity we constructed CARs made of scFv specific to human Her2/neu, CEA and CD24 (a cancer stem cell marker of certain adenocarcinomas including pancreatic and colorectal ones).
To optimize the antitumor effect in therapeutic settings, we have treated either immunodeficient mice bearing human cancer xenografts or transgenic mice that spontaneously develop tumors expressing human TAA. T-bodies effectively rejected primary as well as disseminated tumors. Multiple systemic administrations of T-bodies were required for complete elimination of xenografts and murine tumors. Of note, single intratumoral injection was sufficient to eliminate breast, prostate cancer bone metastases as well as colorectal cancers. Moreover, under certain conditions, we have established a time-window which allows allogeneic T-bodies to serve as universal donors for cancer immunotherapy. Altogether, these results were recently dramatically reproduced in human cancer patients.
In another project, we have generated Treg T-bodies and demonstrated their ability to suppress autoimmune inflammatory responses. In experimental models of acute and chronic colitis we established the conditions to modify Treg cells with specific CARs to rescue mice from death to TNBS induced colitis, and alleviate the chronic symptoms of colitis induced in CEA transgenic mice. Importantly, we have found that the antigen towards which the Tregs are redirected-to, should be an organ-specific rather than pathogen-specific.
3. “First AID” for T cell gene therapy: TCR affinity maturation by somatic hypermutation
Adoptive cell transfer (ACT) of tumor specific T lymphocytes, after host immunodepletion, was shown to mediate objective cancer regression of metastatic melanoma. Effective T cell activation depends, among other factors, on the functional avidity of the peptide-MHC complex (pMHC) to the T cell receptor (TCR), i.e., on the affinity and the number of pMHC-TCR contacts. Since the T cell repertoire is controlled by negative and positive selection in the thymus, naturally occurring TCRs have mostly low affinities, in the range of 1–100 μM. Moreover, unlike antibodies whose affinities improve over time by somatic hypermutation (SHM), TCR does not undergo SHM.
In our research, we developed a system that can increase the affinity of a TCR to its ligand by subjecting TCR genes to somatic hypermutation, directed by the mutator enzyme Activation Induced cytidine Deaminase (AID). Affinity maturation reactions are performed ex-vivo in easy transfectable cells and affinity maturated TCRs are selected by tetramer staining followed by FACS sorting. The affinity maturation system is designed to be modular so the maturation can be done in several cycles in order to optimize TCR affinity. The affinity maturated TCRs are used to create anti-tumor reactive T cells by means of gene transfer into naïve lymphocytes or anti-viral CTL. These transformed T cells are then functionally tested in-vitro and in-vivo with murine melanoma models. Using this system we are trying to define the parameters that govern the changes in affinity as well as the biological consequences of these changes. Such systems can potentially be used to augment T cell responses to low immunogenic Tumor Associated Antigen (TAA) peptides, for the treatment of cancer.
4. Journey from gene identification to first approved gene therapy – Story of LPL
Lipoprotein lipase (LPL) is a pivotal enzyme responsible for the clearance of triglycerides (TG) from blood. LPL deficiency presents in childhood with profound hypertriglyceridemia, failure to thrive, chronic abdominal pain, and potentially lethal pancreatitis. Worldwide, LPL Deficiency is a rare disease; however, due to a population founder effect, the highest incidence of LPL deficiency occurs in eastern Quebec, Canada (1 in 3,000). Previously, there have been no effective drug therapies for LPL deficiency. Purified LPL enzyme replacement was ineffective because of the short half-life of the enzyme. An extreme dietary restriction of fat was the only means to try and treat disease symptoms; however, TG levels often remained dangerously elevated, causing pain, and potentially lethal pancreatitis. Soon after identification of the defective gene causing LPL Deficiency, we began research towards the development of a treatment for LPL Deficiency using gene therapy. An effective means of treating animal models of LPL Deficiency was eventually developed using a modified adeno-associated viral (AAV)-vector. This gene delivery system utilizes a viral protein shell that efficiently carries the functional therapeutic LPL gene into patient cells, whereupon the gene functions to continually produce high levels of the therapeutic LPL enzyme to overcome the patient's enzyme deficiency. After successful pre-clinical studies and three interventional clinical trials conducted in the Netherlands and in Canada, on November 2, 2012, LPL gene therapy Glybera® (alipogene tiparvovec) received regulatory approval from the European Commission for the treatment of patients with LPL deficiency. This approval makes Glybera the first gene therapy approved by regulatory authorities in the Western world.
5. Selective eradication of cancer cells by adenovirus-based delivery of cytotoxic agents: An alternative method for targeting pancreatic cancer
6. Human T-cells engineered to express a PD1/28 costimulatory converter display enhanced anti-tumor activity
The adoptive transfer of T-cells genetically-modified to express cancer-specific receptors can mediate impressive tumor regression in terminally-ill patients. However, T-cell function and persistence over time could be hampered by the activation of inhibitory co-stimulatory pathways, such as PD1/PDL1, leading to T-cell exhaustion and providing tumor cells with an escape mechanism from immunosurveillance. In addition, the lack of positive co-stimulation at the tumor site can further dampen T-cell response. Thus, as T-cell genetic engineering has become clinically-relevant, we aimed at enhancing T-cell anti-tumor activity by genetically diverting T-cell negative costimulatory signals into positive ones using chimeric molecules we termed “co-stimulatory converters” which are composed of the PD1 extracellular domain fused to the signaling domains of positive costimulatory molecules such as CD28 and 4-1BB.
After characterizing the optimal PD1 chimera, we designed and optimized a tripartite retroviral vector that enables the simultaneous expression of this chimeric molecule in conjunction with a cancer-specific TCR. Human T-cells, transduced to express a PD1/28 chimeric molecule exhibited enhanced cytokine secretion and upregulation of activation markers upon co-culture with tumor cells. These engineered cells also proliferated better compared to control cells. Finally, we tested the function of these cells in an in ovo cytotoxic assay using a chorioallantoic membrane (CAM)-based model of human melanoma tumors and showed that PD1/28-engineered human T-cells demonstrated superior anti-tumor function.
Overall, we propose that engineering T-cells with a co-stimulatory converter can enhance their function which bears important implications for the improvement of T-cell immunotherapy.
7. Newcastle Disease Virus (NDV) – a novel oncolytic agent for cancer treatment
Cancer is a leading cause of morbidity and mortality in Western countries. Despite a huge research effort and funding, there is no cure for most advanced stage cancers. Oncolytic viruses may serve as a novel approach for treating cancer where conventional therapies have failed.
Oncolytic viruses preferentially infect and kill cancer cells while sparing normal ones. When modified, these viruses can be used as vectors enabling gene expression of anticancer proteins to be delivered to the tumour site.
The use of a unique ex-vivo tissue model, developed in our lab, as well as an animal model of colorectal carcinomas, enabled us to explore the tropism and mechanism of Newcastle Disease Virus (NDV) as an oncolytic virus.
NDV is a negative ssRNA paramyxovirus, naturally infects poultry, is not pathogenic to humans and has a limited replication capacity in mammalian cells. The oncolytic effect of NDV was previously demonstrated both in-vitro and in-vivo as well as in clinical trials. NDV-HUJ is an attenuated NDV strain, with a defective replication capacity (single cycle). Studies in our lab showed that the virus causes apoptosis in lung cancer cells as well as in chemo-resistant primary melanoma cells.
In-vivo results indicate that treatment with NDV-HUJ in an orthotropic colon cancer model caused major tumour regression and increased survival. Using the ex-vivo system, a different infection and replication pattern was demonstrated both in mice and in human tumor tissues as compared to normal ones. This data may lead to new strategy treatment modalities for advanced colorectal cancer.
8. A novel RNAi-based treatment for pancreatic cancer (PC): pre-clinical and clinical results
The major challenge for oligonucleotides therapeutics is delivery. Local prolonged delivery potentially overcomes this hurdle. Pancreatic cancer (PC) is an aggressive disease. Genetic alterations in the KRAS signaling pathway are involved in 90% of PC cases and the majority of KRAS mutations are gain-of-function mutations at codon 12 (KRASG12D). The tumor is addicted to KRASmt. The goal of this study was to investigate the impact of kras oncogene silencing on PC. We developed a controlled regional drug delivery system by design of a miniature biodegradable polymeric matrix that encompasses antiKRASG12D siRNA drug, named siG12D LODER® (sL). This LODER releases the drug regionally within a pancreatic tumor, during three months. Treatment of pancreatic cells with sL resulted in a significant inhibition of KRAS mRNA and protein levels. This was associated with a decrease in cell proliferation, reversing EMT and inducting cell death. In vivo, the growth of human PC cell lines was retarded, in subcutaneous and orthotopic models. The survival of mice implanted with sL was significantly improved.
Our data reveals: 1) that sL efficiently overcomes current siRNA delivery obstacles related to systemic approaches. The LODER pharmaco-kinetics enables dose reduction by orders of magnitudes and eliminates toxicity; 2) the strategy of local kras targeting by sL can be effectively used. We have initiated a phase I study with sL implanted into patients with locally advanced PC. Twelve patients were included in this study. No major safety issues were found. We observed significant and promising results of tumor marker decrease and tumor shrinkage.
9. Delivery of placental stromal cells by IM injections for mitigation of lethal radiation effects and enhancement of recovery from bone marrow failure
A very limited arsenal of treatments is available for the life-threatening exposure to high dose of radiation. Such exposure is mainly associated with the direct killing of cells, but may also be highly influenced by systemic factors, including immune modulation and cytokine expression. Mesenchymal multipotent stromal cells from different sources have been previously tested for their indirect by-stander effect in the reconstitution of failing organs in different clinical conditions. Placental stromal cells are not “stem cells” by definition, but it has been shown that they are involved in the regulation of the immune response to enhance tissue repair and may provide a basis for cell-based therapy.
Commercially GMP produced placental cell preparations “PLX cells”, were supplied by Pluristem Therapeutics to be tested for mitigation of radiation effects. Our preliminary experiments with a C3H mouse model showed that IV injection of PLX cells 24 hrs after total body high dose irradiation very significantly increased the survival rate of the irradiated mice. Nevertheless, a short term stress of the mice after the IV injection was often recorded and the cells were trapped for up to 2–3 days in the recipient's lungs until they faded away. In further studies, we identified special batches of PLX, based on a mixture of fetal and maternal stromal cells, which yielded much better protection to high dose radiation. These cells were termed PLX-RAD and they could be delivered safely with no apparent complications by repeated intramuscular injections. The survival of mice irradiated by 7.7 Gy and treated with PLX-RAD on days 1 and 5 after irradiation increased from ∼27% to ∼98% (p < 0.001). Delaying the initiation of the treatment by 48 hrs after irradiation yielded a similar effect. This was also reflected by the fast recovery of the bone marrow and the enhanced regeneration of the peripheral blood cell profile. Preliminary data on the human cytokine secretion in the treated irradiated mice contributed to the explanation for their radiation protection effects. Our promising results set a basis for a new easily available “off the shelf” therapy for nuclear disasters victims following radiation accidents. The vast effect of the cells could be attributed to the induction of enhanced bone marrow stem cell proliferation.
These findings were supported by a number of successful clinical mercy treatments of patients with pancytopenia at Hadassah Hospital, showing that this approach can be applied not only for the mitigation of radiation effects, but also for a variety of other clinical conditions associated with severe bone marrow failure.
10. Dangerous liaisons: gene transfer vectors and the human genome
Retroviral integration is a non-random process, whereby pre-integration complexes of different viruses recognize components or features of the host cell chromatin in a specific fashion. By using deep sequencing technology, we mapped >90,000 MLV, SIN-MLV and SIN-HIV integration sites in the genome of human CD34+ hematopoietic stem/progenitor, peripheral blood T-lymphocytes and skin keratinocytes, and defined genome-wide integration maps in all cell types. MLV integrations cluster around regulatory elements (promoters, enhancers, and evolutionarily conserved non-coding regions) of genes involved in hematopoietic functions, and to chromatin regions bearing epigenetic marks of active or poised transcription. On the contrary, HIV integrations are clustered in regions marked by histone modifications associated to the body of transcribed genes and are under-represented in regulatory regions.
Although >90% of the genes targeted by HIV integration are transcriptionally active, expressed genes are not equally targeted. We define a set of <300 genes that are targeted by HIV at significantly higher frequency than matched random controls after normalization for gene length, and a smaller set of genes that are targeted at significantly lower frequency. Functional clustering analysis shows that highly targeted genes are involved in chromatin remodeling and transcription, and are enriched in housekeeping functions. This analysis identifies a set of “high-risk” genes in hematopoietic cells, the function of which is more likely to be influenced by lentiviral vector integration in clinical gene therapy. We show that gene targeting by lentiviral vectors is influenced by topological constrains in the nucleus, and favors chromatin regions located in the vicinity of the nuclear pore.
Integration of lentiviral vectors into transcribed genes has the potential to de-regulate their expression at post-transcriptional level, by interfering with splicing and polyadenylation of primary transcripts. We tested this hypothesis in human T cells, myeloid cells and keratinocytes transduced with a “splice trap” vector or with vectors carrying expression cassettes for a reporter gene or a human β-globin gene under the control of a reduced-size locus-control region (LCR). Cells were randomly cloned and integration sites determined in individual clones. Aberrantly spliced, chimeric transcripts were identified for >50% of the targeted genes in all cell types. Aberrant splicing was caused by the usage of both constitutive and cryptic splice sites located in the viral intron and the U5 portion of the 5' long terminal repeat (LTR), and in the β-globin gene and LCR. A semi-quantitative assay revealed that in most clones, abnormal transcripts were accumulated at low level compared to constitutively spliced transcripts, in some cases as a consequence of nonsense-mediated mRNA degradation. A limited set of cryptic splice sites was responsible for most of the aberrant splicing events, providing a strategy for recoding lentiviral vector backbones and transgenes and reducing their potential post-transcriptional genotoxicity.
11. Liver to pancreas transdifferentiation occurs in predisposed cells
Despite pharmacological treatments for type-1 diabetes, an adequate control of blood glucose levels is often difficult. One way to obtain functional insulin-producing cells (IPCs) for transplantation is to derive islet-like-cells by transdifferentiation. However, transcription factors (TFs)-induced transdifferentiation is restricted to a limited number of adult cells in culture.
Based on our in vivo studies performed in mice, we have predicted that a certain population of cells in human livers preferentially reprogram along the fate of insulin production. We isolated these cells and irreversibly tagged them by eGFP and the rest by DsRed. The eGFP+ cells comprise about 15% of the human liver cells in culture, but could be easily propagated separately reaching numbers feasible for transplantation.
The cells were transdifferentiated by adenoviral treatment of pancreatic TFs and soluble factors. Both groups displayed similar infection efficiency and expressed identical levels of ectopic factors. Following transdifferentiation β-cell-like characteristics were compared in the separated groups. The eGFP+ cells displayed improved glucose sensitive insulin secretion reaching 12.2% of primary islet culture. The insulin and pancreatic TFs gene expression was substantially higher than in the DsRed2+ cells.
To identify factors which affect the distinct transdifferentiation efficiencies, we performed gene expression profiling by microarray analyses. Approximately 800 probes were differentially expressed. None of these genes were markers of “stemness” or progenitor cells. Several signaling pathways were suggested to be involved and are under investigation.
To summarize, we have isolated and propagated a population of cells that have better transdifferentiation capacities. The interplay between the ectopically expressed TFs and the intracellular milieu may complement each other and revealing this may be implemented in transdifferentiation processes in other tissues.
12. Reversing blindness to sight: efficient and long-term gene delivery to the retina using the femtosecond laser
13. Nanomedicines: directing the immune response?
RNA interference (RNAi)-based approaches have greatly contributed to a better understanding of gene expression and function in vitro. The capability to apply these strategies in vivo in order to validate the role of specific genes in normal or pathological conditions, and to induce therapeutic gene silencing, has opened new avenues for utilizing RNAi as a novel therapeutic modality. However, the translation of RNAi from an effective genomic tool into a novel therapeutic modality has been hindered by the difficulty to deliver RNAi molecules into their target tissues by systemic administration, especially to hematopoietic cells.
In this presentation, I will describe some of the challenges and opportunities in modulating leukocyte response using RNAi and discuss off-targets effects and adverse effects such as immuno-toxicity.
14. Phase I gene therapy trial in Israeli patients with Leber Congenital Amaurosis caused by a founder RPE65 mutation: safety and efficacy update with up to three years of follow-up
15. Derivation of oncolytic mutant HSV-1 vectors with enhanced replication in malignant cells
Herpes Simplex Viruses 1 and 2 (HSV-1 and HSV-2) are the causative agents of recurrent facial and genital infections. The host reactions to the infections include suicidal functions and immune response to eliminate viral replication. To overcome the host defense, the virus induces a global shutoff of host protein synthesis. We have previously identified the virion host shutoff (vhs) function and shown it to be an mRNAse which destabilizes/degrades host and viral mRNAs. The vhs protein of the incoming virion initiates host mRNA degradation immediately upon viral entry into the cells.
In the present study, we describe the association of vhs-mRNase with cellular proteins essential for its activity. Towards use of the virus as an oncolytic vector, we have derived vhs mutants that do not shutoff the host and induce suicide and cell death in tumor cells in mice harboring human glioblastoma tumors. Nude mice were injected with human glioblastoma multiforme cells resulting in large tumors. Whereas mice untreated with the vectors died within 2–3-weeks post tumor induction, in some of the mice treated with the vhs mutant vector, there was significant tumor shrinkage and increased survival and cure for several months. Based on 13 mice in each group, efficacy was comparable to a parallel treatment with HSV-1 vector (G47) currently proposed for use in human trials in the USA for glioblastoma multiforme. We aimed to produce an oncolytic vector that will replicate in tumor cells, but not in non-dividing human cells. We produced doubly mutated vectors, which retained high virus replication in tumor cells while having reduced viral replication in primary normal cells. The vector retains the highly oncolytic properties with virus replication in malignant cells. Whereas the wild type HSV replicated equally well in malignant and normal primary cells, the new mutant virus retained replication capacity in malignant U87 glioblastoma cell line, but had reduced replication in primary human astrocytes. This is of potential relevance to the oncolytic vector efficacy and safety.
16. High-throughput screening identifies small molecules that selectively eliminate human pluripotent stem cells and prevent teratoma formation
Human pluripotent stem cells (hPSCs) hold great promise for cell therapy treatments, as they can differentiate into all the cell types of the human body. However, the clinical use of hPSCs is hindered by the tumorigenic risk from residual undifferentiated cells. In this study, we performed a high-throughput screen of over 52,000 small molecules, and identified 15 pluripotent cell-specific inhibitors (PluriSIns), 9 of which share a common structural moiety. The effect of the PluriSIns is extremely selective, as they eliminate hPSCs rapidly and robustly while completely sparing a large array of progenitor and differentiated cells of all germ layers and developmental stages. Cellular and molecular analyses demonstrated that the most selective compound, PluriSIn#1, induces ER stress, protein synthesis attenuation, and apoptosis in hPSCs. Further characterization identified this molecule as an inhibitor of stearoyl-coA desaturase (SCD1), the key enzyme in oleate biosynthesis, revealing a previously unknown unique role for lipid metabolism in hPSCs. Remarkably, exogenous supplementation of oleate completely rescued the PluriSIn#1-induced cell death, demonstrating that oleate depletion is the direct cause of hPSC death following exposure to PluriSIn#1. Of note, structurally-similar PluriSIns were found to exert their cytotoxic effect on hPSCs through the same mechanism of action. PluriSIn#1 was also cytotoxic to mouse pluripotent stem cells and to mouse blastocysts, indicating that the dependence on oleate is inherent to the pluripotent state, and is evolutionarily-conserved. Finally, application of PluriSIn#1 prevented teratoma formation from tumorigenic undifferentiated hPSCs.
This novel method to eliminate undifferentiated cells from culture should thus enable the generation of pure differentiated cultures and increase the safety of hPSC-based therapies.
• This work has been recently accepted for publication in Cell Stem Cell.
17. Targeting neurogenesis in the ventral hippocampus with lentiviral Wnt3a gene transfer improves behavioral symptoms in an Alzheimer's disease model
Alzheimer's disease (AD), the most common form of dementia and cognitive decline, is a devastating illness, with one in eight people over the age of 65 having the disease. Non-cognitive symptoms, termed the Behavioral and psychological symptoms of dementia (BPSD), are frequently observed and affect 80% of patients. These include depression, disinhibition, delusions, hallucinations, agitation, anxiety and aggression. AD has been associated with impaired neurogenesis in the hippocampal dentate gyrus, which is linked to cognitive impairment along with several behavioral and psychiatric disorders.
Previous studies have shown that the Wnt/beta-Catenin Signaling pathway promotes baseline neurogenesis in the adult hippocampus. To investigate the effect of neurogenesis on behavioral function in a relevant disease model, the Wnt3a gene was delivered bilaterally to the dentate gyrus in the ventral hippocampus of a 3xtg-AD mouse model via lentivirus (LV-Wnt3a). Animals injected with LV-Wnt3a at a presymptomatic stage showed significantly improved neurogenesis and behavioral symptoms. Wnt3a treatment increased behavioral inhibition and risk assessment in the 3xtgAD model
Thus, our data indicate that targeting adult neurogenesis in the ventral hippocampus has potential as an alternative therapy of the behavioral changes in AD and possibly in other neurodegenerative disorders.
Posters (alphabetical order)
18. T Cell Tolerance as Function of Tumor Progression
Determining how tumor immunity is regulated requires understanding the extent to which the anti-tumor immune response “functions” in vivo without therapeutic intervention. To better understand this question, we used traditional ex vivo analysis of tumor specific T cells. By transferring admixed splenocytes of melanoma-specific CD8+ T cells (pmel-1 and OT-I.GFP), we found that B16 tumor growth was sufficient to induce naïve pmel-1 and OT-I.GFP CD8+ T cell proliferation and acquisition of effector phenotype. However, pmel-1 T cells, were not capable of killing target cells, showed decreased and low capacity to secrete IFNγ and TNF-α and were unable to mediate tumor regression. Within the tumor, pmel-1 T cells were proliferating extensively while their frequencies were decreasing rapidly. In contrast, OT-I.GFP T cells were able to mediate tumor regression and were capable of killing target cells in the tumor and in the periphery. While OT-I.GFP cells in the DLN were proliferating in low rates, cells isolated from the tumor showed extensive proliferation capacity and long term interaction with B16 tumor cells; this suggests that presentation of cognate peptide to pmel-1 T cells occurs during tumor growth already in the DLN which eventually leads to their elimination.
These data indicates that lack of anti-tumor efficacy is not solely due to general immunosuppressive mechanisms in the tumor microenvironment, but rather to active and specific peripheral tolerance mechanisms induced in the DLN. Recently, we have implicated the lymph node stroma in mediating CD8 T cell peripheral tolerance. We demonstrate that LN-resident lymphatic endothelial cells express peripheral tissue antigens and directly present an epitope derived from one of these, the melanocyte-specific protein PMEL17/gp100, to pmel-1 CD8 T cells, leading to their deletion.
These results establish lymphatic endothelial cells, and potentially other LN-resident cells, as systemic mediators of peripheral immune tolerance, preventing a protective immune response.
19. Amelioration of ulcerative colitis and colitis-associated colorectal cancer by adoptive transfer of antigen-redirected regulatory T cells
In recent years, the T-body approach that employs chimeric antigen receptors (CARs) to redirect T cells to tumor sites has been recognized as a promising therapeutic modality for both haematopoietic and solid tumors. While this approach has been mainly studied in the context of anti-cancer therapy using effector T cells, far less attention was given to the employment of regulatory T cells (Tregs) in other pathological scenarios, namely those characterized by unrestrained immune activation, including graft rejection and autoimmune and auto-inflammatory diseases. This can be achieved by endowing Tregs with a CAR that specifically recognizes an antigen that is expressed predominantly at sites of aberrant immune activity.
Since the importance of Tregs for the maintenance of a healthy intestinal mucosa and the prevention of inflammatory bowel disease has been widely demonstrated, we chose to explore whether Treg T-bodies can be engineered to suppress ulcerative colitis (UC). Based on the elevated carcinoembryonic antigen (CEA) levels in the epithelia of UC patients, we employed a model system of CEA transgenic mice and demonstrated the colitis-ameliorating potential of the CEA-specific Treg T-bodies.
Two disease models were employed: the azoxymethane-dextran sodium sulfate (AOM-DSS) model for colitis and colorectal cancer, as well as a T-cell-induced colitis model. Colitis severity and tumor progression were monitored by endoscopic examination in live animals. In this manner, we showed that the adoptive transfer of CEA-specific Treg T-bodies can lead to a reduction in colitis severity, this in comparison to mice treated with control Tregs. Furthermore, in the AOM-DSS model, CEA-specific Treg T-bodies significantly decreased the subsequent colorectal tumor burden. Hence, this study demonstrates how the T-body approach can be applied to combat inflammatory bowel disease. Furthermore, we believe that this approach may also hold promise in other autoimmune and inflammatory disorders.
20. A new encapsulation approach for the entrapment of insulin-producing stem and liver cells
Microencapsulation of living cells in a biocompatible and semi-permeable polymeric membrane was proven to be an effective method for continuous drugs delivery and for immunoprotection of the cells. The addition of extra cellular matrix (ECM) to the encapsulation system can regulate the activities of adherent cells, including proliferation and differentiation. The cellular secretion level of insulin can be also achieved by activating the desired cell signaling via integrin-ligand-bonds and subsequently stimulating the gene expression level. We therefore propose to encapsulate human liver cells (Hum-Hep) or human mesenchymal stem cells (hMSC) after transduction with pancreatic and duodenal homebox gene-1 (PDX-1), which induced trans-differentiation into functional insulin-producing cells, as a possible diabetic therapy application.
ECM is obtained by the decellularization of porcine pancreatic tissue, using a combination of physical, chemical and enzymatic cleaning, to produce a non-immunogenic yet bioactive material. After decellularization, the ECM is lyophilized and liquefied, using proteolytic enzymes. Hum-Hep cells and hMSC were transduced using recombinant adenovirus containing PDX-1 gene. Transduced cells were encapsulated in alginate-PLL mixed with ECM. The effect of encapsulation parameters on cell viability and insulin secretion was followed using AlamarBlue and C-peptide secretion, respectively.
Histological analysis of the ECM reveals complete cell removal while maintaining the ECM fibers architecture. The morphology of the encapsulated cells showed that they prefer to organize around large collagen depositions originating from the liquefied ECM. Encapsulated cells were viable for more than 120 days. C-peptide measured 5 days after initial exposure to the viral transduction, revealed that the encapsulated hMSC had significantly higher secretion levels than non-encapsulated hMSC.
Our findings demonstrate that the microenvironment within the microcapsule is permissive for cell survival and insulin secretion. Therefore, encapsulated insulin producing cells with native ECM can be considered as a platform to replace deficient pancreatic beta–cells while circumventing the shortage in tissue availability and the need for anti-rejection treatment.
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21. Enhanced survival and neurite network formation of cord blood neuronal-progenitors in three-dimensional (3D) collagen constructs and hematopoietic CD45+ cells induced protection in mice with traumatic brain injury*
Traumatic brain injury (TBI) is a major cause of death and disability. Cell therapy by human umbilical cord blood (CB), which contains hematopoietic and non-hematopoietic progenitors, has shown promising results in brain trauma animal studies.
In the first aspect of the present study, we demonstrated that hematopoietic CD45+ progenitors reduced the neuronal deficits which typically occur in a traumatic brain injury mouse model. The therapeutic neuroprotective effects were observed upon cell transplantation either by intracerebral (icv) or intravenous (iv) delivery route 1 or 7 days post-trauma insult. Cells, labeled with near-infrared dye, were detected at the site of injury 1.5 hours after iv injection. Anti CD45+ antibodies blocked the neuroprotective effect of these cells. These findings demonstrate the neuroprotective potential of CB-derived CD45+ cell fraction in animal model of TBI and may serve as the basis for clinical therapeutic application.
In the second aspect of the research, we investigated the survival and neuronal properties of CB non-hematopoietic, neuronal progenitors in 3D collagen constructs. In contrast to two-dimensional culture conditions, the cells survived in 3D for an extended period of time of more than two months. Under 3D conditions, CB neuronal progenitors (HUCBNP) underwent spontaneous neuronal differentiation, which was further enhanced by treatment with neuronal conditioned medium (CM) and nerve growth factor (NGF). Neurite outgrowth, quantified by assessing the fractal dimension (Df) of the complex neuronal networks, was significantly enhanced under 3D conditions in the presence of CM/NGF, concomitant with a reduced expression of the early neuronal marker nestin (1.9-fold), and increased levels of mature neuronal markers such as MAP-2 (3.6-fold), β-tubulin (1.5-fold), and neuronal specific enolase (6.6-fold) and the appearance of the synaptic marker synaptophysin. To assess the feasibility for clinical usage, HUCBNP were also isolated from frozen CB samples and cultured under 3D conditions. The data indicate the essential complete preservation of neurotrophic (survival) and neurotropic (neurite outgrowth) properties. In conclusion, 3D culture conditions are proposed as an essential step for both maintenance of CB neuronal progenitors in vitro and for investigating specific features of neuronal differentiation towards future use in regenerative therapy.
22. Redirection of T-cells for adoptive cell treatment of cancer in a spontaneously progressing mammary tumor model
Adaptive transfer of T lymphocytes redirected with chimeric antibody-type (or antigen) receptor (CAR) has been recently demonstrated as a dramatic curative effect in pilot trials of patients with blood-borne leukemias. The wide application of adoptive cell treatment for the therapy of bulky solid tumors and their metastases remains a major challenge. Most of the experimental models studying the antitumor effect of CAR-expressing effector human T cells (we nicknamed “T bodies”) applied immunodeficient mice transplanted with tumor xenografts which do not faithfully simulate tumor progression and its suppressive microenvironment.
In our study, we analyzed the T body activity in a transgenic mouse model (huHER2NG, Genentech) that develops spontaneous mammary tumors overexpressing the erbB2 gene. In this “all-in-a-mouse” model, we followed the antitumor effect of several Her2/neu-specific T-bodies as well as the persistence and accumulation of the T-bodies as well as the safety issues associated with their administration. We focused on various therapeutic settings – prophylactic, prior to tumor development or therapeutically, when tumor(s) became apparent, by either systemic administration or direct intratumoral inoculum.
Our results show that upon the systemic administration Her2/neu-specific T bodies, they reach and accumulate at the tumor site, recognize the tumor and eliminate it. Within a few weeks after T body administration, the rejected primary tumor usually relapses both in the treated mammary gland and remote glands. Repeated administration of T-bodies is required to eliminate and keep in check spontaneously developing mammary cancer. Since in reality spontaneous tumors can arise repeatedly, viewing cancer as a chronic disease, multiple administrations of T-bodies can serve to control relapsing disease.
23. The epigenetic of liver to beta-like cell transdifferentiation
Recent studies have demonstrated the efficiency of β-cell replacement therapy for type 1 diabetes mellitus. It is accepted that this therapy will become widely available only when new sources of islets or pancreatic β cells are found. Our laboratory has suggested the liver as an alternate source for surrogate insulin producing cells and demonstrated that transient and ectopic expression of the pancreatic and duodenal homeobox gene-1 (PDX-1) in-vivo resulted in the generation of functional and consistent insulin producing cells in the liver. We next applied this method in vitro by transient expression of PDX-1 in primary liver cell cultures. During transdifferentiation, the liver cells lost the hepatic gene expression repertoire and gained a pancreatic gene expression repertoire.
Epigenetic modifications are considered to be a key mechanism in cellular development and lineage commitment. Vast studies discussing the role of epigenetic modifications, such as histone modification and DNA methylation, were performed focusing on embryonic development and cell fate commitment.
Our research goal is to characterize the epigenetic modifications accompanying the liver to beta-like cell transdifferentiation, in the aspect of inducing and disabling the pancreatic and hepatic repertoire, respectively. By using chromatin immunoprecipitation (ChIP), we demonstrated changes in the histone modifications during the transdifferentiation process. We used liver primary culture cells, as well as isolated liver cells prone to the transdifferentiation process.
Our results can shed light on the chromatin state during transdifferentiation and distinguish between the normal liver cells and cells prone to beta-like transdifferentiation in the epigenetic manner. A better understanding of the chromatin structure during transition of mature cells into a different lineage can contribute to our knowledge regarding silencing and induction of genes in a mechanism not yet fully understood.
24. Kynurenine regulates the function of bone marrow-derived MSCs through Aryl hydrocarbon receptor signaling
Bone marrow-derived mesenchymal stem cells (MSCs) exert an immunosuppressive effect, and therefore are tested as a therapy for graft versus host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). However, clinical trials using MSC therapy for GVHD patients has shown inconclusive results.
MSCs induce an immunosuppressant effect on T cell proliferation, by the activation of key enzymes such as nitric oxide producing enzyme-inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2) producing enzyme-cyclooxygenase-2 (COX2) and Indoleamine 2,3-dioxygenase (IDO), an enzyme that catalyzes the catabolism of Tryptophan to Kynurenine metabolites. Aryl hydrocarbon Receptor (AhR) is a transcription factor, known as a regulator of drug metabolizing enzymes. Activation of AhR by Kynurenine negatively regulates dendritic and T cell immunogenicity.
In the present study, we aimed to examine the influence of Kynurenine, alone or in combination with cytokines, on the regulatory phenotype of MSCs. We show, for the first time, that MSC AhR activation by Kynurenine leads to decreased IL6 secretion. Pre-treatment of MSCs with Kynurenine, IFNγ and TGFβ allows for better inhibition of T cell proliferation than non treated MSCs in vitro, through combined mechanisms including IDO, iNOS and COX2 activation. Moreover, these cells show an elevated expression of the PDL1 regulatory molecule. Administration of pre-treated MSCs to GVHD mouse model significantly decreases GVHD score and improves mouse survival.
Taken together, these results suggest that pre-treatment of MSCs with Kynurenine, IFNγ and TGFβ enables an ultimate regulatory phenotype. The results of this research may lead to an improved treatment of HSCT patients, enabling a wider use of allogeneic HSCT.
25. Searching for efficient therapeutic avenues to treat Adult Polyglucosan Body Disease (APBD) and Lafora Disease (LD) using a High-Throughput Screening platform
26. Head and neck cancer cells exhibit resistance to zebularine effect via the p21/CHK1 pathway
Head and neck cancer (HNC) consists mainly of epithelial squamous cells and regardless of the latest progress, current treatment options remain substandard. Due to difficulties in treating drug resistant tumors, new therapeutic drugs are administered alongside conventional chemotherapeutics treatments, in order to promote development of novel therapeutic strategies.
Herein, we investigated the effect and the course of action of zebularine, a DNA methylation inhibitor, in two human head and neck cancer cells: SCC-25 cells from tongue carcinoma and Fadu cells from the pharynx carcinoma, normal fibroblasts were used as control. XTT and BrDu analyses were used for establishing the inhibitory effect of zebularine. Cell cycle, cell differentiation and induction of apoptosis, were conducted by FACS, TUNEL and Western blot assays, respectively.
Our results revealed that zebularine inhibited cell growth of SCC-25 cells in a time dependent manner. Conversely, Fadu cells showed resistance to zebularine treatment, given that they displayed minor reduction levels in cell proliferation and exhibited no effect on cell cycle and apoptosis following treatment. Gene expression profiling conducted on zebularine-treated Fadu cells revealed up-regulation of p21 and CHK1, however these alterations were not present in SCC-25 cells. We further investigated the role of p21 and CHK1 in the process of protecting cells from apoptosis and revealed that CHK1 gene expression contributed to the resistance of Fadu cells to zebularine effect. Furthermore, cell cycle analysis demonstrated that knockdown of CHK1 sensitized Fadu cells to the effect of zebularine.
These results demonstrate the function of the p21/CHK1 pathway in the process of protecting Fadu cells from the apoptotic effect of zebularine. Furthermore, in this study we presented an innovative strategy of combining zebularine with either over-expression of p21 or down-regulation of CHK1, which resulted in sensitized Fadu cells to zebularine.
27. The role of MicroRNAs in liver to pancreas transdifferentiation
The diabetes epidemic affects approximately 6–8% of the world population, and the number of newly diagnosed patients increases annually. The only available treatment modality for T1DM and late-T2DM is continuous insulin administration (injection, pumps or patches). Pancreas transplantation or islet cell implantation are by far better than insulin administration in achieving continuous control of blood glucose levels and preventing diabetes-related complications. However, these methods are restricted by a severe shortage in tissue supply from cadaver donors, and require extensive, life-long suppression of the immune system. Therefore, it is generally accepted that cell replacement therapy for diabetic patients will be widely used only when new sources of insulin-producing cells (IPCs) are found. Transdifferentiation of liver to pancreas may be one of these replacement therapies for diabetic patients. We have reported that ectopic expression of pancreatic transcription factors in liver induced insulin production both in vivo and in vitro.
MicroRNAs (miRNAs) are short non-coding RNAs (20–25 nucleotides) that regulate gene expression. miRNAs have been shown to participate in almost every cellular process investigated so far.
Specific miRNAs involved in β-cells' development and function and also with the upkeep of insulin transcription have been identified. However, while the expression of hundreds of genes was affected during the transdifferentiation process, no alterations in pancreatic specific miRNA levels occurred.
The aim of the present project is to analyze the role of manipulating the pancreatic specific miRNAs levels in the activation of the pancreatic lineage in liver cells.
Preliminary data showed that ectopic expression of miR-375 as part of the transdifferentiation protocol promoted the activation of pancreatic gene expression and increased the efficiency of the process. This data suggests that using miRNA in the transdifferentiation protocol of hepatic to pancreatic cells may be the missing link for generating effective insulin-producing cells (IPCs).
28. Enhancing the anti-tumor activity of T cells by improving the expression of the TCR chains using different leader sequences
Immunotherapy treatments based on the engineering of T lymphocytes to express tumor-specific TCR genes is an attractive strategy to generate anti-tumor response. This approach has been proven effective in mediating tumor regression in terminally-ill melanoma patients in several clinical trials. However, one of the main problems in this strategy pertains to the low expression of exogenous TCR complexes. The latter is composed of two chains that assemble in the endoplasmic reticulum (ER) and are then exported to the cell surface. It has been demonstrated that the leader sequence (also known as “signal peptide”), which is a protein stretch of 15–30 aa at the N-terminus of most proteins holds an important role in targeting and leading proteins through the ER membrane and may therefore influence protein surface expression.
Thus, in the present work, we hypothesized that it is possible to improve TCR expression by replacing the original TCR leader sequences with selected signal peptides. To test this, we generated a dozen of TCR chains with different signal peptide attached to the variable region of a MART-1 specific TCR (F4) chains. This TCR was selected as previous studies demonstrated that F4 expression and function were sub-optimal (Morgan et al, 2006). Following in silico prediction and construction of these leader-modified TCR chains, we engineered T lymphocytes to express these different chimeras. We assessed the expression of the different chimeras by flow cytometry and observed differential levels of expression of the modified chains, with some expressed at a higher level than the wild-type F4-TCR chain. This translated into an improved function as measured by cytokine secretion of IFN-gamma in co-cultures of TCR-expressing cells with melanoma lines.
In conclusion, the present findings indicate that it is possible to enhance TCR surface expression and function using leader peptide optimization. This may have direct consequences for the treatment of cancer patients using TCR gene-transfer approaches.
29. Designing DNA containing cell-derived vesicles targeted for cancer gene delivery
We aim to develop a novel targeted non-viral gene delivery system for cancer therapy, which is based on unique vesicles produced from the cell membrane of mesenchymal stem cells (MSC). MSC are known for their homing capability towards cancer cells and its microenvironment. The cell derived vesicles (CDV) will benefit the surface molecules of the MSC and thus may preserve their targeting mechanism. Into this “Trojan horse” CDV system, we will incorporate therapeutic encoding DNA, which will be expressed at the tumor site.
CDV were prepared, PEGylated (to produce stealth CDV) and characterized for morphology, size distribution, zeta potential and surface protein. CDV-PC3 (prostate cancer cells) interaction was evaluated by flow cytometry and confocal microscopy. DNA was complexed with different cationic agents (to overcome electrostatic repulsion between the DNA and the cell membrane) and incorporated into the CDV. DNA Incorporation efficacy was evaluated as well as the physical characteristics of the DNA incorporated CDV. CDV-pGFP expression was evaluated post incubation with PC3 cells, using flow cytometry.
CDV preparation resulted in round vesicles with an average size of 180nm and a zeta potential of about −16mV (−12mV for PEGylated CDV). Flow cytometry analysis demonstrated preservation of most of the MSC surface markers. CDV showed specific and time-dependent accumulation inside the cytoplasm and nucleus following incubation with PC3 cells. pGFP was successfully incorporated to the CDV system, and preliminary experiments demonstrated expression of GFP in PC3 after 6 hr incubation.
30. Study of p53 mutated proteins as differential tumor antigens
Tumor suppressor p53 is reported to be an attractive immunotherapy target because it is mutated in approximately half of human cancers, resulting in its inactivation and often an accumulation of the protein in the tumor cells. Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumor-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer therapeutic. It has been shown that PBLs transduced with a murine TCR isolated from T cell clones specific for the p53 (264–272) epitope, derived from HLA-A2.1 transgenic mice, were capable of lysing p53-expressing tumor cell lines, while p53-negative lines and normal cells were ignored. However, it was recently shown that there was no significant correlation between p53 expression in tumors and recognition by the anti-p53 TCR transduced T cells.
Thus, the purpose of this study is to understand the influence of p53 mutations on its presentation by the MHC system. To that end, we have generated several constructs encoding different p53 mutant proteins and have expressed them in HLA-A2+/p53- cells. Expression of the different mutants was assessed by intracellular flow cytometry. Upon co-culture with p53-specific T cells, we measured specific recognition of the p53 expressing cells by means of cytokine secretion as IFNγ and TNFα and the expression of the activation antigen CD69.
Our data show that there is an inverse correlation between intracellular expression and recognition by T-cells, and thus we hypothesize that p53 mutant stability can greatly influence its recognition by the immune system. This pattern was redundant in several cell lines tested, and was not associated with any influence on class I MHC levels of expression, suggesting it could represent an intrinsic property of p53 mutant proteins. Thus, these results may have significant therapeutic implications for p53-based immunotherapy of cancer.
31. Targeting of a broad spectrum of tumors by T–Lymphocytes engineered to express NCR-based chimeric receptors
Natural killer (NK) cells are an important component of the anti-tumor response. Tumor recognition by NK cells was found to be partly mediated by recently-discovered molecules termed natural cytotoxic receptors (NCRs). Adoptive cell transfer of tumor-reactive T-lymphocytes has been demonstrated to mediate the regression of large solid tumors in melanoma patients. It was shown that it was clinically feasible to modify genetically human T-lymphocytes with anti-tumor receptors, thus leading to cancer regression in melanoma patients. Nevertheless, the use of such treatments for other malignancies such as breast, colon, cervical, liver or lung cancer has barely been explored due to the lack of receptors that would mediate efficient recognition of those tumors.
Herein, we propose to combine the NK anti-tumor recognition potential with the genetic modification of T-cells, providing them with an additional mean to recognize tumors in a non-MHC restricted way. To test this, we have generated several constructs utilizing the extra-cellular domain of NCR-3 (Nkp30) or NCR-1 (Nkp46) fused to different signaling moieties. The different receptors were expressed in T-lymphocytes and their relative expression was analyzed by flow-cytometry. Following co-culture with tumors of different histologies, our results suggest that primary human T-lymphocytes expressing NCR-derived molecules are able to recognize specifically several tumor lines such as lungs, cervical carcinoma, leukemia, hepatocarcinoma and melanoma as measured by IFN-gamma, IL2 and TNF-alpha secretion tests. In addition the NCR-chimeric receptors mediated upregulation of the surface markers CD25, CD69, 4-1BB and CD107a, as well as significant antitumor activity against multiple tumor lines in cytotoxicity assays both in vitro and in ovo, using a chorioallantoic-based model of xenograft tumors.
Overall, this research has meaningful implications for the immunotherapeutic treatment of cancer using gene-modified T-cells.
32. Validation of the ampliseq™ cancer panel using the Ion PGM™ reveals mutations with possible clinical implications in KIT, JAK3 and PTEN genes
The genomic landscape of cancer is characterized by dozens of mutations. Currently, massive parallel sequencing (MPS) allows sequencing of hundreds of genetic hotspots in tens of genes that may provide prognostic and predictive information.
We examined 20 samples of melanoma, lung and colon cancer with previously identified BRAF, EGFR or KRAS mutations. Formalin fixed paraffin embedded (FFPE) tissue was examined and cancer tissue identified by a pathologist. DNA extracted and the ion ampliseq™ cancer panel applied. Four samples were loaded onto a 316 chip 100 Mb and run on Ion PGM.
Ninety five percent of the samples were amplified successfully. All 316 chips were run successfully. The average base call was 229 Mb per chip; on average, each base was read 3,503 times. All the preknown mutations and deletions were detected providing support for the system's robustness. In addition, mutations in APC, ATM, JAK3, KDR, KIT, MET, PTEN, STK11, TP53 genes were found.
We conclude that analysis of FFPE samples using the ampliseq™ cancer panel with the Ion PGM™ is doable and can identify known and new mutations in clinical samples. The use of this tool to treat oncological patients is feasible.