Abstract
Human Gene Therapy Division of Medical Genetics, Akdeniz University Faculty of Medicine , Turkey
Session: Cancer, immune-based approaches
Ahmedin Jemal CA Cancer J Clin
Ted Shih Clin Ther
Session: Cancer, immune-based approaches
The adenoviral vector Ad5/35 was evaluated for gene delivery into T cells to be used in adoptive T cell therapy for CD19 + B cell lymphomas. Also normal B cells express CD19 but not the hematopoietic stem cell. We propose that CD19-retargeted T cells may lyse all CD19 + cells. By the use of a transient expression of the retargeting module, the infused T cells may eventually lose their transgene expression. Hence, post cancer therapy the stem cells may recreate a new healthy B cell population in the patient. The serotype 5 adenoviral vector has been widely used in clinical trials for efficient gene delivery to a variety of cells. However, gene transfer into hematopoietic cells is impeded by the scarcity of its primary receptor target, the Coxsackie adenovirus receptor (CAR). Therefore, the Ad5 vector has been retargeted through alterations in the knob domain extending the tropism to serotype 35, resulting in a vector with significantly improved gene transfer to CAR deficient cells. This vector was used to transduce isolated OKT-3/IL2 stimulated T cells. Vector transfer was well tolerated (85% survival) and flow cytometry revealed a stabile transduction efficiency of approximately 30% of cells. A withstanding gene expression was detected during one week of culture despite the proliferative nature of these cells. After further culture the T cells lost their expression and showed poor survival. Taken together, the Ad5/35 vector can be used for efficient and transient gene transfer to proliferating T cells to create genetically engineered T cells for adoptive transfer to malignancies such as B cell lymphoma.
CD28 costimulation of redirected effector T cells sustains its repression by regulatory T cells
Session: Cancer, immune-based approaches
Tumor tissues are frequently infiltrated by regulatory T (Treg) cells which render a productive T cell anti-tumor response ineffective. As a consequence, tumors grow unrestricted despite the presence of tumor-specific, cytolytic T cells. Effector T cells redirected by a tumor-specific chimeric antigen receptor (CAR; immunoreceptor) are effectively repressed by Treg cells. Repression is more effective when effector T cells encounter antigen in the context of CAR mediated CD28 costimulation than without. Mutation of the CD28 signaling domain which prevents lck recruitment abolished IL-2 secretion and Treg cell repression. Data establish that CD3zeta-CD28 CAR mediated T cell activation feeds its repression by sustaining Treg cell survival. Consequently, IL-2 suppression during the redirected T cell response provides a strategy to render the cytolytic anti-tumor attack less sensitive to Treg cell repression.
Georg-Speyer-Haus, Institute for Biomedical Research , Germany
Session: Cancer, immune-based approaches
Medulloblastoma (MB) is a highly malignant ErbB2-positive childhood tumor. Current treatment options for advanced disease are limited and novel therapeutic alternatives are needed. ErbB2-specific NK-92 natural killer cells are an attractive therapeutic alternative since NK-92 cells are known to have a powerful anti-tumor effect, which is however mainly restricted to haematological malignancies. Retargeted NK-92 cells could provide a curative therapy for solid tumors expressing ErbB2. With up to 80% of MBs expressing ErbB2, this therapeutic alternative may be applied to the majority of patients and could also be extended to more prevalent ErbB2-positive cancers. We have used lentiviral vectors to genetically modify NK-92 cells. Culture conditions and transduction protocols were optimized to assure the establishment of a GMP-applicable protocol. Also, a careful selection of promoter together with the optimization of transgene expression is undergoing. Ultimately a vector is required which provides, in a single copy fashion, high, stable and long term expression of the transgene. Results from these studies will be crucial for the selection of functionally active retargeted NK-92 cell clones for production of a master cell bank and will later contribute to the collection of data required to enter the regulatory proceedings for clinical application.
Department of Hematology, Medical School Hanover , Germany
Session: Cancer, immune-based approaches
Clinical ex vivo production of dendritic cells (DC) is costly, time-consuming and difficult for large-scale clinical trials. We developed a novel technology for production of DC consisting of one day lentiviral vector (LV) transduction of growth factors and full-length antigens into monocytes that are able to self-differentiate. These DCs are called “SMART-DCs” (Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors) and have been extensively tested in mice for safety, protective and therapeutic potency against melanoma. Novel multicistronic self-inactivating lentiviral vector modalities containing interspacing 2A elements, a non-encoding Wpre, and expressing human GM-CSF, IL-4 and tumor antigens are therefore in development for immunotherapy clinical trials. MART-1 is a differentiation antigen which is found in > 90% of melanomas and is commonly recognized by T cells of the patient in an HLA-restricted manner. Here, we demonstrate that lentiviral co-delivery of human MART-1 (>80%) into fresh adherent monocytes obtained from blood draws allowed their self-differentiation into SMART-DCs with normal immunophenotypic profile (CD209, CD86, HLA-DR). HLA-A0201 positive SMART-DC/MART-1 were recognized by T cell clones specific for MART-1, assessed by IFNγ-ELISPOT-Assay. This shows that this novel method for DC generation drastically simplifies cellular production and T cell priming, allowing clinical development and testing with small samples (up to 50 ml) of peripheral blood. Further characterization of the potency and mechanism(s) of SMART-DC/MART-1 to stimulate and expand autologous T cells obtained from melanoma patients are underway with tricistronic vectors for process development.
Immunomodulation and Immunotherapy, Institut de Genetique Moleculaire de Montpellier , France
Session: Cancer, immune-based approaches
T cell activity can be modulated by direct gene transfer. This attractive therapeutic approach has multiple clinical applications but requires an in vivo expansion and persistence of the transduced lymphocytes. It is thus concerning that this strategy is often compromised by the transduction protocol; TCR stimulation, used to facilitate gene transfer, results in extensive ex vivo proliferation and acquisition of effector function, potentially limiting subsequent in vivo function. To date, T cells have always been transduced at 20% oxygen, even though physiological O2 concentrations in lymphoid organs range from 2–5%. As such, we tested whether culture of T cells under physiological oxygen conditions (2%) would be beneficial. Ex vivo culture of T cells in 2% O2 conditions did not reduce viability, but significantly decreased cell division. However, high level lentiviral- and MuLV-gene transfer in TCR-activated human T cells was achieved at both 2% and 20% O2 (60–80%). Notably, the relative distribution of effector memory and central memory T cells was significantly modulated by the O2 concentration in which T cells were activated; the vast majority of TCR-stimulated cells at 20% O2 acquired an effector phenotype whereas at 2% O2, approximately 30–40% were central memory cells that were efficiently transduced by MuLV-mediated gene transfer. Finally, T lymphocytes stimulated in 2% O2 demonstrated a higher metabolic activity, as measured by expression of the glucose transporter GLUT1 and glucose uptake. In conclusion, ex vivo TCR stimulation under physiological O2 conditions results in a preferential differentiation of naive T cells to a central memory phenotype and promotes efficient gene transfer under conditions of minimal proliferation.
M. Tettamanti Research Center, Pediatric Unit University Milano-Bicocca , Italy
Session: Cancer, immune-based approaches
Current treatments for acute myeloid leukemia (AML) ensures long-term remission in only 40% of patients. Anti-CD33 monoclonal antibody therapy showed limited results and high toxicity. The CD33-targeting approach might be improved by the use of T cells tranduced with a CD33-specific CAR. To identify the optimal T cell population for clinical intervention, cytokine induced killer (CIK) cells, γ9δ2 T-cells and Epstein-Barr virus specific cytotoxic T lymphocytes (EBV-CTL) were generated. After efficient transduction with an anti-CD33-ζ CAR, cells showed, in 4hours-release assays, potent and comparable killing (>70% lysis at E:T ratio 20:1;p = 0.01) of different CD33 + -targets (HL-60, KG-1 cell lines and primary AML cells). Noticeably, in 6-days co-coltures of anti-CD33-CIK cells with a CD33 + -target at 0.01:1 E:T ratio, the leukemic cell recovery was negligible (4% residual HL-60 vs 80% with untransduced cells;p0.01). Moreover, anti-CD33 CIK cells presented 3-fold more INF-γ, 10-fold more TNF-α, 20-fold more TNF-β CD33-specific secretion (p = 0.05) compared to the other T cells. In addition, CIK cells displayed in the same culture period (15 days post-transduction) the highest expansion rate (average fold increase 19 vs 6,5 of γ9δ2 T-cells and 5,9 of EBV-CTL; p = 0.05). Our in vitro data suggest that, for their potent effector functions and capacity to expand in large quantities in a short-period of time, anti-CD33 CAR-CIK cells might the optimal T cell population for AML immunotherapy.
Development of a graft versus host disease and graft versus chronic myeloid leukemia mouse model
Session: Cancer, immune-based approaches
One of the major complications of allogeneic hematopoietic stem cell transplantation is Graft-vs-Host Disease (GVHD), caused by the recognition of host antigens by the donor's T cells. In contrast to the deleterious effect of GVHD, donor lymphocytes also generate a Graft-vs-Leukemia effect (GVL), which helps to control the leukemic relapse by destroying leukemic residual cells. With the purpose of studying factors that could modulate the GVHD/GVL reactions, we generated a chronic myeloid leukemia (CML) mouse model by means of the co-transplantation of allogeneic BM cells (C57Bl/6 Ptprca, H2b/b) with syngeneic lin- BM cells (B6D2F1, H2b/d) previously transduced with a retroviral vector carrying the BCR/ABL and the tNGFR marker genes. In all instances, B6D2F1 mice transplanted with these cells developed a CML-like pathology, and died on days + 28–72 after infusion. To investigate the GVHD/GVL effects produced by donor allogeneic T-lymphocytes in this CML mouse model, splenocytes from C57Bl/6 mice were infused with the BM co-transplantation. In contrast to the control group which developed CML in all mice, no CML cells were observed in mice receiving allogeneic T cells, although acute GVHD killed all the animals on days + 13–36 in this experimental group. Taking advantage of this mouse model we are currently investigating the influence of the infusion of mesenchymal stromal cells on the GVHD/GVL effect produced by allogeneic T cells.
University Vita-Salute San Raffaele Medical School, San Raffaele Scientific Institute , Italy
Session: Cancer, immune-based approaches
LN and MDP share senior authorshipTEMs are a subset of tumor-infiltrating myeloid cells that have nonredundant proangiogenic function in tumors (1) and can be exploited to deliver cancer biotherapeutics in a tumor-targeted fashion (2). In order to begin elucidating the lineage relationships between TEMs and other circulating or tumor-infiltrating myelomonocytic cells, we compared the gene expression profile of tumor-infiltrating TEMs with that of tumor-associated macrophages (TAMs) and circulating monocyte subsets, by qPCR-based gene arrays (3). We found that TEMs were highly related to TAMs; however, several genes were differentially expressed between the two macrophage subsets, highlighting a TEM gene signature consistent with enhanced proangiogenic/tissue-remodeling activity and lower proinflammatory activity. These findings suggest that TEMs represent a TAM subpopulation with strongly M2-skewed phenotype. Interestingly, when we profiled circulating monocyte subsets, we found that Gr1(negative) monocytes and tumor TEMs on one hand, and Gr1(positive) monocytes and TAMs on the other hand, expressed coordinated gene expression profiles, suggesting that the two blood monocyte subsets are committed to distinct extravascular fates in the tumor microenvironment. We then found that a prominent proportion of embryonic macrophages expressed distinguishing TEM genes. Together, these results suggest that embryonic macrophages, Gr1(negative) monocytes and tumor-infiltrating TEMs represent distinct developmental stages of a TEM lineage committed to execute proangiogenic and tissue-remodeling programs, which can be co-opted by tumors. This feature may be exploited for implementing tumor targeting strategies based on gene and cell therapy.
De Palma M. Cancer Cell
De Palma M. Cancer Cell
Pucci F. Blood
Session: Cancer, immune-based approaches
Our group provided evidence that the cancer testis antigen PRAME is a potential target for adoptive T-cell or vaccine therapy of many hematologic malignancies and solid tumors. PRAME-specific T cells can be detected in patients with hematologic malignancies and we have shown that they can be generated and expanded ex-vivo, using artificial antigen presenting cells (aAPC)1. So far, four PRAME-derived epitopes have been identified by a proteosome digestion, assay that may fail to identify of putative peptides in vivo generated. We have now adopted an alternative method that uses a library consisting of 135 synthetic pentadecapeptides, spanning PRAME protein, to evaluate if multiple immunogenic epitopes can be identified and used to generate polyclonal PRAME-CTL lines. CD8 + cells from 21 healthy donors and 7 patients with Chronic Myelogenus Leukemia (CML) were primed with dendritic cells loaded with PRAME-peptide library, and then expanded by stimulation with aAPC. We consistently generated PRAME-CTLs in 19/21 healthy donors and all 7 CML patients. These PRAME-CTLs were also able to target autologous CML cells when cultured with PRAME+ CML blasts, demonstrating that the same peptides were presented physiologically. A Cr51 release assay confirmed that the PRAME-CTLs were cytotoxic, lysing autologous-PHA blasts loaded with the peptides derived from the PRAME-library. Using pentadecapeptides sub-pools, we found that the responses of our expanded PRAME-CTLs were polyclonal, since they consistently released IFNγ in response to 1 to 6 pentadecapeptides pools. In conclusion, this novel approach should facilitate expansion of polyclonal PRAME-CTLs for adoptive transfer or after vaccine administration to patients with PRAME+ hematological malignancy.
Quintarelli C Blood
Session: Cancer, immune-based approaches
LYP (PTPN22) is a non-receptor protein tyrosine phosphatase involved in regulating T-cell receptor signal transduction. Recently, a missense mutation polymorphism in the gene PTPN22 (SNP rs2476601) has been associated with autoimune disease. The resulting variant protein, carrying substitution R620W has been described as a gain of function mutant. PTPN22 is also expressed in Natural Killer (NK) cells, at levels higher than T-cells. The anti-leukaemic capacities of NK cells make these cells a potential treatment modality. NK cells can be grown in vitro from peripheral blood mononuclear cell (PBMC) mass culture, however, the purity of the resulting NK cells varies from 95 to 10%, with the median around 50%. We investigated, wether the PTPN22 alleles affect NK cell/T-cell ratio in vitro. The SNP rs2476601 was genotyped using Applied Biosystems SNP genotyping assay by real-time PCR. PBMCs of 29 healthy donors were cultured using CellGro SCGM medium, IL-2 and OKT3 antibody. The cells were analysed by flow cytometry at days 1, 9, 15 and 21 on a Becton Dickninson LSR II. NK cell fractions were determined as CD3- CD56 + cells. Data analysis was performed on Prism 4 (GraphPad Software, Inc). We found that none of the analysed donors had two copies of the variant allele, (TT), although 38% (11/29) were heterozygous. The presence of the variant, autoimmunity-associated allele significantly reduced NK cell fraction in the culture, 15.4 (±11.1) vs 53.3 (±21.1) % , (mean ± 25% percentile), p = 0.0013 (Mann-Whitney test). We conclude that PTPN22 has a previously undetected function in regulating NK/T-cell ratio and the variant allele may reduce NK cell proliferation in culture.
Hematology and Oncology, University Medical Center Mainz , Germany
Session: Cancer, immune-based approaches
Immune suppression after allogeneic stem cell transplantation causes reactivation of cytomegalovirus (HCMV) and comes along with increased mortality in HCMV positive patients. The aim of cellular immunotherapy is the eradication of tumor cells by adoptive transfer of tumor antigen specific T lymphocytes. We pursue the strategy to generate bifunctional T cells which are reactive against well-defined tumor antigens as well as HCMV by T cell receptor (TCR) transfer. Therefore we utilize tumor antigen specific T cells as a recipient of HCMV-specific TCR genes as well as genetic equipment of HCMV-specific T cells with a tumor antigen specific TCR.We were able to introduce a p53.264–272-specific TCR into HCMV T cells by RNA transfer. The transgenic TCR p53 was expressed as well as the endogenous HCMV-specific TCR. Both TCRs showed efficient Interferon-gamma production in ELISA.Furthermore we transduced the human tyrosinase specific T cell clone with retroviral vectors encoding TCR HCMV or TCR MDM2. The expression of the transgenic TCR HCMV as well as TCR MDM2 resulted in a substantial downregulation of the endogenous tyrosinase (368–376) specific TCR. Therefore, to regulate transgenic TCR expression, we currently establish the inducible retroviral gene expression system Retro-X-Tet-On Advanced. We are also interested to generate novel pp65.495–503-specific TCRs. For this, we changed aminoacid residues in the CDR3 loops by site-directed mutagenesis of the TCR HCMV encoding genes. Importantly, the hybrid HCMV-specific TCR composed of the non-mutated TCRα chain and the mutated TCRβ chain elicited an improved functionality. Our results indicate the general possibility to generate bifunctional T cells and the importance to regulate their expression.
Non-viral tumor gene therapy with novel plasmids expressing the cytotoxic cytokine TNF-α
Session: Cancer, immune-based approaches
Tumor necrosis factor TNF-α plays a key role in the control of tumor growth. Systemic application of recombinant TNF-α is hampered by its high systemic toxicity dictating the need to target TNF-α activity selectively to the tumor. Here we demonstrate localized antitumor activity of TNF-α after systemic injection of a non viral, biodegradable gene carrier (G3-HD-OEI) exhibiting intrinsic tumor affinity. A novel plasmid vector with CpG free backbone and an optimized promoter-enhancer combination led to sustained and high TNF-α transgene expression in vitro on Neuro2a murine neuroblastoma cells. Secreted TNF-α induced high cytotoxicity on L929 indicator cells and increased endothelial cell permeability in a trans-well system. After tail vein injections into mice bearing subcutaneous Neuro2a tumors, transgene activity was found only in tumor tissue for at least one week with luciferase encoding plasmid. When applying TNF-α encoding plasmid in a similar way, inhibition of tumor growth was seen concomitant with decreased vessel density (CD31 expression). To harness the vessel permeabilizing properties of TNF-α, gene delivery was combined with liposomal doxorubicine (Caelyx). Pretreating mice with TNF-α polyplexes improved tumor accumulation and tissue distribution of Caelyx as compared to control polyplexes. This suggests a TNF-α mediated increase of tumor blood vessel permeability augmenting the antitumor effect of liposomal chemotherapeutics.
Cell Biology and Gene Therapy, Max-DelbrÜck-Center of Molecular Medicine , Germany
Session: Cancer, immune-based approaches
At present there is limited clinical success in treating metastatic renal cell carcinoma (RCC) by immunotherapy, although antigenicity of this tumor entity has been described. The adoptive transfer of genetically engineered T cells carrying redirected antigen specificity is a new option for the treatment of cancer. We used tumor-infiltrating lymphocytes (TIL) of a human RCC to establish a T cell clone that recognized an HLA-A2-restricted, shared tumor antigen. From this clone, the T cell receptor (TCR53) α- and β-chain genes were isolated, modified by codon optimization and murinization, and retrovirally transduced into peripheral blood lymphocytes. TCR53 gene-modified T cells recapitulated the specificity pattern of the original TIL population and recognized tumor cells in an HLA-A2-dependent fashion. They secreted different effector cytokines (IFN-γ, TNF-α, IL-2, MIP-1β) and were cytotoxic towards tumor cells, while ignoring primary kidney cells. TCR53 genes in combination with the gene encoding the human CD8α chain were transduced into the mouse hybridoma cell line B3Z to generate an indicator cell line (B3Z-TCR53). Based on the IL-2 secretion after antigenic stimulus, B3Z-TCR53 cells were employed to screen 122 tumor and normal cell lines, including 20 primary kidney cells, for the distribution of the antigen recognized by TCR53. We found shared recognition of TCR53 peptide: MHC ligand in more than 60% of RCC cell lines and in 26% of other tumor entities, while normal tissue cells were not recognized. Based on the HLA-A2-restricted recognition of a shared tumor antigen, TCR53 could be a candidate for TCR gene therapy of RCC.
Applied Virology and Gene Therapy, Georg-Speyer-Haus, Germany
Session: Cancer, immune-based approaches
Gene therapeutic approaches for a safe exploitation of T cell based adoptive immunotherapy to treat graft rejection and control relapse after stem cell transplantation have received increasing attention. The genetic modification of donor T cells with suicide genes allows the specific elimination of T cells eliciting unwanted anti-host reactivity and thus counteracts life-threatening Graft-versus-Host Disease. The safety and feasibility of the suicide gene approach has successfully been demonstrated in clinical trials using the Herpes Simplex virus thymidine kinase gene, however, several limitations have become apparent. In this study, we combined the human B cell surface antigen CD20/Rituximab approach with the truncated version of CD34 (tCD34). Based on a gamma retroviral expression vector, co-ordinate expression of CD20 and tCD34 was demonstrated in primary human T cells favoring both efficient tCD34-based immunomagnetic selection and high susceptibility to Rituximab-mediated cell death. In addition, primary T cells partially retained their alloreactive potential and their CD4/CD8 ratio after transduction and expansion. Moreover, sustained CD20 and tCD34 expression could be demonstrated for up to 17 weeks in transduced murine T cells adoptively transferred to Rag-1-/- animals. Following Rituximab infusion, CD20 expressing cells were fast and efficiently depleted from peripheral blood and secondary lymphoid organs, thus warranting further steps toward a clinical application.
Human Gene Therapy Division of Medical Genetics, Faculty of Medicine Akdeniz University , Turkey
Session: Cancer, immune-based approaches
Programa Gens i Malaltia, Centre de Regulacio Genomica , Spain
Session: Cancer, oncolytic approaches
Replication-competent adenoviruses armed with TK combine the concepts of virotherapy and suicide gene therapy. Moreover TK-activity can be detected by noninvasive PET imaging, what could potentially facilitate viral monitoring in vivo. Here, we report the generation of a novel oncolytic adenovirus that incorporates the Tat8-TK gene under the control of the Major Late Promoter in an ICOVIR-5 backbone that provides selectivity by targeting the retinoblastoma pathway. The selective oncolytic TK virus, termed ICOVIR5-TK-L, showed reduced potency compared to a non-selective counterpart. However the combination of ICOVIR5-TK-L with GCV induced a potent antitumoural effect similar to that of wild type adenovirus in a preclinical model of pancreatic cancer. Although the treatment with GCV provoked a reduction in the viral yield, both in vitro and in vivo, a two-cycle treatment of virus and GCV resulted in an enhanced antitumoral response that correlated with high TK-activity, based on microPET measurements. Thus, TK-expressing oncolytic adenoviruses can be traced by PET imaging providing real time information on the activity of the virus and its antitumoral potency can be optimized by GCV dosing.
LBPA and CliniGene, Ecole Normale Superieure de Cachan, France
Session: Cancer, oncolytic approaches
A synthetic EGF-responsive transcription element has been designed and introduced in HIV-1 and Fr-MuLV based vectors with a reporter gene (EGFP) enabling viral selectivity which resuls in tumour-specific expression of the transgene. Different human carcinoma cell lines have been tested from colon and breast (HCT116, SKBR3, H466B, MCF7, MDA, MB231), pancreas, lung and bladder establishing that the EGF responsive synthetic promoter can selectively drive expression only in cancer cells overexpressing EGF and EGFR. We have further introduced a HSV-tk and HFV-envelope bicistronic cassette as potential therapeutic genes. In ex vivo study, a fusiogenic effect is documented as HFV-envelope is expressed at the membrane which results in specific cancer cells death. A delayed by-stander effect is observed without GCV (from day 12–15) condioned by the sole HFV-derived sequences. Transduced-control cells are not affected although being PCR-positive. These vectors (i) mediate efficient & selective cytoxicity in a wide range of EGF-EGFR overexpressing carcinoma cells and (ii) are believed to improve safety in preventing undesirable expression in- and downstream destruction of non-target cells. In order to further evaluate the oncolytic potential of these new vectors we have engaged into a collaboration with Amos Panet, in order to test these vectors in organ cultures of mouse and human colon and breast carcinomas. Comparison of viral infectivity, transgene expression and toxicity in the normal and carcinoma tissue ex vivo should enable to better delineate the clinical potential of these vectors. Work performed with support of EC-DG research FP6-NoE, CLINIGENE: LSHB-CT-2006–018933
Proteasome inhibition during oncolytic infection improves antitumoral immunity and inhibits metastatic tumor growth
Session: Cancer, oncolytic approaches
Growing evidence exists that infection of a dying cell by a pathogen dictates the immune response against intracellular antigens. However, the impact of tumor cell death during oncolytic viral infection on the induction of antitumoral immunity is unknown. Recently, ER-stress due to disruption of the unfolded-protein-response (UPR) has been implicated in the bypass of immune tolerance mechanisms. Since viruses and proteasome inhibitors induce accumulation of misfolded proteins, we investigated ER-stress mediated apoptosis and antitumoral immune responses in mouse models of HCC after treatment with the proteasome inhibitor Bortezomib and the oncolytic adenovirus hTert-Ad. Bortezomib/hTert-Ad infection disrupted the UPR by elimination of Grp78/BiP, and by inhibition of elF2a-phosphorylation leading to significantly enhanced ER-stress-mediated apoptosis of HCC cells and improved oncolysis in xenograft in nude mice. In immunocompetent HCC mouse models, Bortezomib decreased antiviral immune responses, whereas ER-stress-induced apoptosis of HCC resulted in caspase-dependent triggering of antitumoral immunity. Importantly, Bortezomib combined with oncolytic therapy effectively eliminated non-infected HCC lung metastases in immunocompetent tumor models, but not in immunodeficient mice, demonstrating the systemic therapeutic efficacy of ER-stress induced antitumoral immune responses. Conclusion: Proteasome inhibition during oncolytic therapy disrupts virus-induced UPR and leads to enhanced ER-stress-induced apoptosis, improved local oncolysis and systemic antitumoral immunity in mouse tumor models. ER-stress induced apoptosis appears to be a relevant immunological danger signal that should be considered for immunotherapeutic strategies against tumors.
Cancer Gene Therapy Group, Uni. of Helsinki and Helsinki Uni. Central Hosp ., Finland
Session: Cancer, oncolytic approaches
Transfer of prodrug activation systems into tumors by using replication-deficient viruses has been suggested to be an effective method for selective elimination of tumor cells. However, most current suicide gene therapy strategies are still hindered by the poor efficiency of in vivo gene transfer, inefficient tumor penetration, limited bystander cell killing effect and need of large prodrug doses. We hypothesized that by combining virus replication to prodrug activation system we could overcome these limitations. Therefore, we generated a transductionally and transcomplementationally targeted oncolytic adenovirus (Ad) expressing the fusion suicide gene FCU1. FCU1 encodes a bifunctional chimeric protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil (5-FU) and 5-fluorouridine-5'monophosphate (5-FUMP), thus bypassing the natural resistance of certain human tumor cells to the chemotherapeutic 5-FU. We examined the feasibility and efficacy of Ad5/3-Δ24FCU1 and Ad5/3-FCU1 alone as well as of combining with 5-FC in human pancreatic (Panc) cancer and head and neck squamous cell carcinoma (HNSCC). Gene expression was confirmed by Western blot and enzymatic conversion levels in vitro and in vivo were determined by HPLC separation. Significant antitumor effect was observed in vitro and in vivo in subcutaneous model of HNSCC. Even though we observed a decrease in viral DNA copy number in vitro and in tumors treated with Ad5/3-Δ24FCU1 and 5-FC, suggesting an effect on virus replication, the highest antitumor effect was observed for this combination.In summary it seems feasible and efficacious to combine virus replication to the FCU1 prodrug activation system.
Cancer Gene Therapy Group, Uni. of Helsinki and Helsinki Uni. Central Hosp ., Finland
Session: Cancer, oncolytic approaches
Despite improvements in standard therapies, the high mortality and morbidity associated with treatment of advanced Head and Neck squamous cell cancer (HNSCC) has encouraged the pursuit of novel therapeutic strategies. Therefore, we investigated combination of oncolytic adenoviruses with HNSCC standard therapies: cetuximab, radiotherapy and chemotherapy in HNSCC primary clinical explants. We determined which adenoviral capsid modifications allows best gene transfer and cell killing of HNSCC clinical tumor explants in vitro and in vivo. The two most promising oncolytic Ads were afterwards combined with 5-FU and/or to cisplatin; irradiation and/or cetuximab in vitro and in vivo. In vitro, gene transfer was determined by luciferase expression analyses and cell killing by MTS assay. In vivo, antitumor effect was determined by tumor growth follow-up in a subcutaneous HNSCC tumor model. Cryo-stainings performed on tumor relapsing after cetuximab treatment revealed increased proportion tumor initiating cells in comparison to EGFR+ cells. Ad5 chimeras with Ad3 knob fiber, RGD or pK7 capsid modifications resulted in significantly increased transduction and oncolysis of HNSCC substrates in comparison to Ad5 based agents. Ad5/3 and pK7 modifications allowed best gene transfer and cell killing effects in vitro. However, only pK7 modification presented a significant tumor volume reduction in vivo. Combination of HNSCC standard therapies with oncolytic adenovirus resulted in a synergistic increases in cell killing effect in vitro and complete eradication of tumors in vivo. In summary, our preclinical data suggest that it is feasible and efficacious to combine oncolytic adenovirus into a multimodality treatment regimen for clinical testing in HNSCC patients.
Biocentre, Pro-Cure Therapeutics Ltd, United Kingdom
Session: Cancer, oncolytic approaches
Verapamil enhances the antitumoral efficacy of oncolytic adenoviruses
Session: Cancer, oncolytic approaches
The therapeutic potential of oncolytic adenoviruses is limited by the rate of adenovirus release. Based on the observation that several viruses induce cell death and progeny release by disrupting intracellular calcium homeostasis, we hypothesized that the alteration in intracellular calcium concentration induced by verapamil could improve the rate of virus release and spread, eventually enhancing the antitumoral activity of oncolytic adenoviruses. Our results indicate that verapamil substantially enhanced the release of adenovirus from a variety of cell types resulting in an improved cell-to-cell spread and cytotoxicity. Such effect is mediated by the calcium-channel blocking activity of the drug, since the blockage of calcium entry through independent mechanisms also results in enhanced adenovirus release. Furthermore, the combination of the systemic administration of an oncolytic adenovirus (ICOVIR-5) with verapamil in vivo greatly improved its antitumoral activity in two different tumor xenograft models without affecting the selectivity of this virus. Overall, our findings indicate that verapamil provides a new, safe and versatile way to improve the antitumoral potency of oncolytic adenoviruses in the clinical setting.
Translational Research Laboratory, IDIBELL-Institut Catala Oncologia , Spain
Session: Cancer, oncolytic approaches
Successful virotherapy requires efficient virus spread within the tumor. We tested whether the expression of hyaluronidase, an enzyme which dissociates the extracellular matrix, could enhance tumor distribution of an oncolytic adenovirus and improve its therapeutic activity. As a proof of concept, we demonstrated that intratumoral coadministration of hyaluronidase in xenografted mice improves the antitumor activity on an oncolytic adenovirus. Next, we constructed a replication-competent adenovirus expressing a soluble form of the human sperm hyaluronidase (PH20) under the control of the major late promoter (AdwtRGD-PH20). The intratumoral treatment of melanoma human tumor xenografts with AdwtRGD-PH20 resulted in degradation of hyaluronan, enhanced viral distribution and induced tumor regression in all treated tumors. Finally, the PH20 cDNA was inserted in an oncolytic adenovirus which selectivelly kills pRb pathway-defective tumor cells. The antitumoral activity of the novel oncolytic adenovirus expressing PH20 (ICOVIR-17) was compared to that of the parental virus ICOVIR-15. ICOVIR-17 showed more antitumor efficacy via intratumoral and systemic administration in nude mice with pre-stablished tumors, along with an improved spread of the virus within the tumor mass. Importantly, a single i.v. dose of ICOVIR-17 induced tumor regression in 60% of treated tumors. These results indicate that ICOVIR-17 is a promising candidate for clinical testing against many human cancers.
The oncolytic adenovirus Ad-i-REP2 utilizes p53-dependent RNA interference for tumor-selective replication
Session: Cancer, oncolytic approaches
RNA-interference (RNAi) is an evolutionary conserved mechanism and a potent tool for specific gene regulation. Since functional p53-deficiency represents a preferential target for molecular therapies we investigated whether p53-dependent RNAi-mediated gene silencing is feasible to restrict adenoviral replication to p53-dysfunctional tumors. We therefore developed an oncolytic adenovirus for conditional replication in p53-dysfunctional tumor cells that uses p53-selective expression of a microRNA-network directed against essential adenoviral genes. Compared to a control virus that expressed a scrambled microRNA-network, antiviral RNAi-input selectively attenuated viral replication in cells with transcriptionally active p53, but not in p53-dysfunctional tumor cells where both viruses replicated equivalently. Compared to ONYX-015, a putatively p53-selective oncolytic vector actually tested in clinical trials, our RNAi-controlled adenovirus displayed significantly improved selectivity for p53 dysfunctions, leading to drastically reduced intrahepatic load of viral DNA in virotherapeutic settings in vivo. We have generated a broadly applicable, RNAi-controlled adenovirus for selective recognition and destruction of p53-dysfunctional tumors and could thereby demonstrate that vector-based antiviral RNAi-networks represent an efficient and versatile tool for targeted restriction of viral replication. The use of inhibitory RNAi-networks to control viral replication can be applied to all transcriptionally regulated DNA-viruses and thus provides a means to generate highly specific vectors for clinical applications.
Department of Cell Biology, Centre of Oncology Medical University of Warsaw , Poland
Session: Cancer, oncolytic approaches
Conventional therapies of lung cancer patients remain unsatisfactory. As a consequence of a need for novel approaches, more research is focused on gene therapy, improving ways of administration and therapeutic genes used in gene preparations. Adeno-associated viral vectors have many properties that make it attractive and promising for gene therapy. In combination with an appropriate way of administration (i.e. intranasal intervention either with aerosol or liquid gene preparations) it could exert a therapeutic effect. Examinations were performed in vivo on Balb/c mice. In order to obtain mice with L1 (fibrosarcoma) tumors in lungs, L1 cells were cultured in vitro and then injected iv to the mice. After three weeks rAAVs encoding reporter gene (rAAV/LacZ) were administrated to mice by intranasal instillation. Two different doses of rAAV2 were applied (4.92E + 05 or 4.92E + 06 gc/mice). Five days later the mice were sacrified and the lung tumors were taken for further studies. The transgene expression in the tumors was tested by quantitative real-time PCR. It was shown that rAAV/LacZ effectively infect L1 tumors (1.2E + 02 to 0.9E + 04 of AAV2 gc were found). The tumor cells revealed an ability to uptake the rAAV/LacZ vectors and express β-gal gene. There was no virus sequence detected in control samples (non-infected L1 tumors). We observed that the infection efficiency depends on the virus particle number administrated intranasal to mice. These preliminary data demonstrate that gene therapy using rAAV vectors administrated by instillation into airway conduct, provides efficient transgene expression in the murine L1 tumors in lungs. This could provide potential therapeutic approach to clinical gene therapy of cancer.
Klinikum rechts der Isar, Technical University Munich , Exp. Oncology, Germany
Session: Cancer, oncolytic approaches
Recent evidence supports the presence of cancer stem cell populations that contribute to tumor progression through drug resistance, angiogenesis, invasion and metastasis. Comprehensive studies have shown that the human cellular factor YB-1 is an oncogenic transcription/translation factor that is associated with the development of cancer with a drug resistant phenotype. In addition it has been shown that YB-1 is involved in the epithelial-mesenchymal transition (EMT). We have demonstrated that the human cellular factor YB-1 by targeting the adenoviral E2-late promoter plays an important role in the adenoviral life cycle (Holm et al., JBC 2002). This knowledge results in the development of a YB-1 based virotherapy. We now demonstrate that YB-1 is highly overexpressed in glioma derived cells with stem cell characteristics. In contrast, in normal human stem cells YB-1 is barely detectable. Due to these findings we investigated the cell killing capacity of a YB-1 dependent oncolytic adenovirus Ad-Delo3-RGD in cancer stem cells. We show convincingly that Ad-Delo3-RGD replicates and kills cancer cells with stem cell chararacteristcs which are resistant against temozolomide (TMZ). Hence, YB-1 based virotherapy might represent a promising treatment strategy against glioma. Moreover, the significant over expression of YB-1 in glioma derived cancer stem cells suggest a predominant role of YB-1 in cancer development.
Biochemistry- IMRIC, The Hebrew University-Hadassah Medical School , Israel
Session: Cancer, oncolytic approaches
Advance melanoma tumors are resistant to all current therapies and therefore there is an urgent need to find new treatments. We have compared the oncolytic activity of a new strain of NDV (HUJ) on early and advanced melanoma primary cells, derived from melanomas of different patients. To our surprise, advance melanoma primary cells were much more sensitive to the killing effect of NDV as compared to normal and early melanoma cells. The oncolytic activity of NDV on these highly metastatic cells is dependent on the intrinsic pathway of apoptosis. Further investigations to understand the differential sensitivity of early and advance melanomas to NDV have revealed the involvement of a unique cell protein, termed Livin, that is known to regulate the apoptotic cascade. Advance melanomas over-express the Livin protein. Furthermore, NDV-HUJ infection specifically induced a limited proteolysis/truncation of Livin and thus converts it into a potent pro-apoptotic protein, termed t-Livin that accelerated apoptosis in the advance melanomas. Knock-down the expression of Livin in cells resulted in a loss of sensitivity to the oncolytic effect of NDV. These original observations demonstrate the ability of NDV to exploit an apoptosis regulatory protein-Livin to specifically destroy tumors. The data presented here indicate the potential of oncolytic NDV-HUJ in the treatment of melanomas that are otherwise extremely resistant to all current treatments.
Bioquimica i Biologia Molecular, Universitat de Barcelona , Spain
Session: Cancer, oncolytic approaches
Replication-competent adenoviruses used as anticancer agents are unable to overcome intra-tumor barriers and disseminate efficiently. Arming the replicative virus with a transgene that increases viral spread through the tumor could solve this limitation. We have generated replication-competent adenoviruses (wt and ICOVIR-5 backbones) with p53 or tat8-p53 under the control of the adenoviral major late promoter (MLP) to minimize the impact of p53 on viral cell cycle. In vitro and in vivo assays have been performed to assess the effects of these p53-armed adenoviruses as monotherapy or in combination with gemcitabine. Cell viability assays have shown that p53 and tat8-p53 expression only increases the citotoxicity of ICOVIR5 adenoviruses. Interestingly, in the xenografts, a single intratumoral administration of 109 vp of all p53-armed viruses resulted in a more marked antitumor effect than their corresponding non-armed replicative adenoviruses. Gemcitabine treatment enhances the antitumor effects. In conclusion, a more potent antitumoral action can be achieved on human pancreatic tumors when viral replication is combined with MLP-driven p53 expression and gemcitabine.
Application of the meganuclease I-Sce I as self-destruction switch to increase selectivity of oncolytic adenoviruses
Session: Cancer, oncolytic approaches
Several mechanisms have been developed to increase selectivity of oncolytic adenoviral vectors for cancer therapy. Nonetheless, break through mutants escaping regulation principles frequently can be observed. Here, we present oncolytic adenoviruses for conditional replication in p53-altered tumor cells that harbor a nuclease function for selective vector destruction in normal cells to overcome this limitation. Adenoviral vectors were constructed with a recombinant E1 gene region comprising an accessory expression cassette where the nuclease gene I-Sce I is controlled by the artificial p53-dependent promoter prMin-RGC. As target sites, two I-Sce I-cleavage sequences were inserted upstream of the E1A and downstream of the E1B gene. In p53 positive cells, these viruses exhibited a higher level of repression than corresponding EGFP-control viruses. Specific adenoviral degradation products were detected that did not lead to increased induction of apoptosis by DNA stress. In an advanced concept, the meganuclease mechanism was combined with an additional transcriptional regulator. prMin-RGC controlled the expression of the transcriptional repressor Gal4-KRAB and I-Sce I linked by an IRES; for this purpose, the E1A-controlling promoter was complemented with Gal4-KRAB binding sites. These vectors showed an increased restriction of replication compared to I-Sce I-only vectors demonstrating the potential combination of several regulation mechanisms. Importantly, all constructed viruses showed a similar lytic potency in p53-impaired cells.In summary, we established tightly regulated replicating adenoviruses that combine transcriptional regulation and vector destruction mechanisms to improve safety and efficacy of virotherapeutic treatment of solid tumors.
Pharmacology of Cancer Treatment, German Cancer Research Center , Germany
Session: Cancer, oncolytic approaches
Gene therapy of chronic myeloid leukemia (CML) can be of relevance to overcome conventional therapy-resistance. Unfortunately, for gene transfer into CML cells, most current vector systems either lack sufficient transduction efficiency or an acceptable safety profile. Conventional adeno-associated virus (AAV) based vectors have an advantageous safety profile, yet lack the required efficiency. We screened pseudotyped recombinant adeno-associated viruses of the serotypes 2/1 to 2/6 (rAAV2/1 to rAAV2/6) on human CML cell lines as well as on primary CML cells to determine their gene transfer efficacy. Additionally, double-stranded self complementary rAAV (scAAV) were used to determine possible second strand synthesis limitations. On CML cell lines, generally rAAV2/2 and rAAV2/6 were most efficient. For both, an interesting difference in transduction efficiency between the imatinib-resistant LAMA84-R and imatinib-sensitive LAMA84-S cells was observed. On primary human CML cells, rAAV2/6 proved to be significantly more efficient than the other tested vectors (4.6% ± 5.3% GFP-positive cells, p = 0.011). Additionally, the transduction efficiency could be significantly increased by using scAAV vectors (scAAV2/6: 43.1% ± 25.9% GFP-positive cells, p < 0.001 vs rAAV2/6). Our data suggest that both the conversion of the single stranded AAV genome to double stranded DNA, as well as cell binding/entry are rate limiting steps in efficient transduction of primary human CML cells with AAV vectors.
Department of Pharmacy, Mahidol University Bangkok , Thailand
Session: Cancer, oncolytic approaches
We examined the efficacy of the oncolytic adenovirus dl520 associated with magnetic nanoparticles (MNPs) upon magnetic field (MF) guided infection in multidrug-resistant cancer cells in vitro. The virus particles (VPs) was self-assembled with selected core-shell MNPs. Optimized virus binding, sufficiently stable in 50% FCS, was found at MNP-to-VP ratios of 5–20 fg Fe per physical VP resulting in 4000 to 5000 MNPs associated per VP. Electron and atomic force microscopy data showed structurally intact viruses surrounded by MNPs. Viral uptake into cells at a given virus dose was enhanced 10-fold compared to non-magnetic virus when infections were carried out under the influence of a MF resulting in a 10-fold enhancement of the oncolytic potency (or 10-fold decreased dose required for killing 50% of the target cells IC50) and an enhancement of four orders of magnitude in virus progeny formation at equal input virus doses compared to non-magnetic viruses. The full oncolytic effect developed within two days post-infection compared with six days in a non-magnetic virus. Plotting target cell viability versus internalized VPs for magnetic and nonmagnetic virus showed that the inherent oncolytic productivity of the virus remained unchanged upon association with MNPs. Hence, we conclude that the mechanism of boosting the oncolytic effect by magnetic force is mainly due to the improved internalization of magnetic virus complexes resulting in potentiated virus progeny formation.
Conditionally replicating adenovirus armed with TIMP for the treatment of ovarian cancer
Session: Cancer, oncolytic approaches
Conditionally replicating adenoviruses (CRAds) are designed to selectively lyse cancer cells. In clinical trials, CRAds have exhibited limited efficacy, and thus need to be armed with therapeutic transgenes to increase their antitumor efficacy. One target for therapeutic transgenes is the tumor microenvironment. The degradation of the extracellular matrix (ECM) is an essential step in tumor growth. Matrix metalloproteinases (MMPs) are endogenous proteases that degrade ECM components and are upregulated in numerous tumors. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of MMPs that can limit tumor growth. Our TIMP-armed CRAd will target the progression of ovarian cancer in two ways: (i) viral replication will directly lead to tumor cell lysis and (ii) TIMP production from infected cells will inhibit cancer progression.We have constructed CRAds (Ad5/3-CXCR4-TIMP) in which the viral E3B region has been replaced with TIMP-1, −2, or −3 to give high levels of expression, late in the viral cycle. Selectivity of replication is conferred by the CXCR4 promoter, which is highly active in cancer cells but not in the liver. Transductional selectivity is conferred by replacement of the Ad5 knob with that of the Ad3 knob, so that viral infection is mediated by binding to the Ad3 receptor, which is overexpressed on ovarian cancer cells. To evaluate the expression profile of the armed CRAds, ovarian cancer cells were infected with each Ad5/3-CXCR4-TIMP. Analysis showed that TIMP was expressed as a late protein, at levels similar to the native E3B proteins and did not interfere with expression of other E3 proteins. More studies are currently ongoing to evaluate the oncolytic potency of the CRAds in vitro and in vivo.
Helmholtz-University Group Oncolytic Adenoviruses, DKFZ and University Hospital Heidelberg , Germany
Session: Cancer, oncolytic approaches
Oncolytic adenoviruses are a promising new tool for treatment of cancer by tumor-specific viral replication, cell lysis and spread of progeny viruses. However, clinical trials have demonstrated that adenoviral oncolysis needs to be improved to achieve therapeutic benefit in patients. Towards this end our work aims at combining adenoviral oncolysis with the induction of systemic and sustained anti-tumor immunity. Here we focus on the investigation and manipulation of immune cell recruitment by adenoviral oncolysis. We analyzed the profile of chemokines expressed by melanoma cell cultures before and after infection with the melanoma-targeted oncolytic adenovirus Ad5/3.2xTyr. Moreover, we established precision-cut tissue slices of melanoma biopsies to investigate the production of immune modulators in context of the whole tumor microenvironment after infection with adenoviruses. In addition, we performed migration assays to analyze the effect of oncolysates on the recruitment of immune cells. Our data showed that infection of cultured melanoma cells with Ad5/3.2xTyr does not induce significant changes in chemokine production and immune cell recruitment in comparison to non-infected cells. Furthermore, we identified chemokines that complement oncolytic adenovirus for immune cell recruitment. Additional data with primary biopsy material will be presented. Based on these results we develop oncolytic adenoviruses expressing recombinant immunomodulators to induce the recruitment and activation of immune cells. We hypothesize that infection of melanomas with such genetically engineered oncolytic adenoviruses facilitates anti-tumor immune activation for an effective immuno-virotherapy of cancer.
Synergistic antitumoral efficacy of SST2 and TRAIL mediated by oncolytic adenovirus for pancreatic cancer
Session: Cancer, oncolytic approaches
Despite the introduction of new cytotoxic agents and intensive protocols, pancreatic cancer remains the most lethal cancer. With the characters of self-perpetuation, controlled lysis and spreading not only within the tumor but also to distant micrometastases, oncolytic virus has emerged as an exciting approach for cancer treatment. The somatostatin receptor subtype 2 (hSSTr2) gene was lost in 90% pancreatic carcinoma. Therefore, we hypothesized whether oncolytic adenovirus-medicated hSSTr2 restoration in TRAIL-resistant pancreatic cells might potentiate antitumor effect. We constructed oncolytic adenovirus, ZD55, with deletion of E1B 55-kDa gene. In vitro and in vivo antitumoral efficacy was evaluated with combined or single treatment of ZD55-hSSTr2 and ZD55-TRAIL. Restoration of hSSTr2 mediated by ZD55 increases TRAIL-induced apoptosis in association with up-regulated DR4 and down-regulated Bcl-2. Co-administration of both viruses achieved complete tumor regression in 9 out of 10 mice with remarkably improved animal survival, while ZD55-TRAIL only exhibited limited antitumoral efficacy due to TRAIL resistance of BxPC-3 cells (P < 0.01). In conclusion, E1B55-deleted adenovirus bearing hSSTr2 gene enhance TRAIL-induced antitumor efficacy, which implied for the first time that this novel strategy of “gene virotherapy” approach may have great clinical potential in pancreatic cancer treatment.
Brain Korea 21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine , Seoul, Korea
Session: Cancer:oncolytic
Recently, gene-based cytokine treatment has been actively pursued as a new promising approach in treating cancer. In an effort to augment the efficiency of anti-tumor effect by cytokine-mediated immunotherapy, we selected both interleukin-12 and 4-1BB ligand as suitable cytokines to fully activate the type 1 immune response. Co-expression of interleukin-12 and 4-1BB ligand mediated by oncolytic adenovirus greatly enhanced the antitumor effect. Further, synergistic enhancement in interferon-γ levels were seen in mice treated with oncolytic adenovirus expressing both interleukin-12 and 4-1BB ligand. Next, to improve the overall antitumor immune response, we co-administered interleukin-12- and 4-1BB ligand-coexpressing oncolytic adenovirus with dendritic cells. Combination treatment of interleukin-12- and 4-1BB ligand-coexpressing oncolytic adenovirus and dendritic cells elicited greater antitumor and anti-metastatic effects than either treatment alone. Moreover, enhanced type 1 antitumor immune response and higher dendritic cells migratory abilities in tumors were also observed in the combination arms. The nature of the enhanced antitumor immune response seems to be mediated through the enhanced cytolytic activity of cytotoxic T lymphocytes and interferon-γ-releasing immune cells. Taken together, these data highlight the potential therapeutic benefit of combining interleukin-12- and 4-1BB ligand-coexpressing oncolytic adenovirus with dendritic cells and warrants further evaluation in the clinic.
Long term stability of aqueous plasmid DNA solutions
Session: Cell and vector manufacturing
Physical and chemical stability of plasmid DNA is a requirement for the development of DNA-based pharmaceuticals capable of being stored, shipped and applied even under critical environmental conditions.The influences of different factors on the long term stability of aqueous plasmid DNA so-lutions were examined with two model plasmids: a 4472 bp plasmid (PF138) and a 6233 bp reporter gene vector. The DNA concentration was adjusted at 0.5 mg/mL, 1.0 mg/mL and 5.0 mg/mL. The plasmids were dissolved in saline, TE buffer or WFI. The filling volume of the micro tubes was 30 uL per vial. Changes in the topology distribution were measured densitometricly after agarose gel electrophoresis and more accurate with the capillary gel electrophoresis technology (CGE). The latter method allows separation and identification of the most im-portant plasmid topologies like ccc-monomer, ccc-dimer, oc-forms and linear forms. The examinations of the long term stability of aqueous plasmid DNA solutions started 2.5 years ago. In this period of time plasmids have remained stable during storage at −20°C and that independent from the DNA concentration and buffer conditions. The distribution of the main topologic forms determined by CGE was nearly unchanged. At 4°C PF138 shows a slight shift from the ccc-forms of the monomer and dimers to the open circular forms (oc-forms). After two years of storage at 4°C the examinations had to stop due to the evaporation of the samples. The results achieved from the reporter gene vector were very similar.
Transfusion Medicine, Hannover Medical School , Germany
Session: Cell and vector manufacturing
Long term cultivation of pluripotent, induced pluripotent or embryonic stem cells (ESCs) share one keyplayer - mouse embryonic feeder cells (MEFs). Despite their inevitable use there is very little known about the interaction of stems cells and MEFs. Although a few groups describe long term feeder-free cultures, the majority of researchers still utilize MEF based culture systems. Lately, our group demonstrated differences in growth behavior of ESCs between Mitomycin C treated and γ-irradiated MEFs (Fleischmann et al., 2009). Next to inactivation, cryopreservation greatly influences the quality of MEF. We speculate that many inconsistent results from different ESC researchers are caused by quality differences of feeder cell preparations. To address this problem, we established a “bench-top” quality control panel to ensure consistent feeder cell grade, including mycoplasma PCR control, different cryoprotective agents and freezing profiles, metabolic activity assay (MTT), survival and re-attachment assay via flow cytometry (FACS) and a semi-quantitative gene expression assay for marker genes important for intact feeder layer formation over time. A 30 gray irradiated NMRI MEF preparation which 1) is Mycoplasma free (PCR), 2) is frozen with 10% DMSO at 1 K/min freezing rate should have the quality of 3) has 90% cell survival (87.5% re-attachment) 4) has expression levels of the genes FN-1, Itgb-1, Col4a4 ≥ 50% and of Col-1 ≥ 25% at day 1 after thawing vs. b-actin, 5) has a metabolic activity of ≥ 1.6 fold compared to untreated MEFs and 6) will provide for ≥ 16 days an intact cell layer for ESC culture. This “bench-top” MEF quality assessment will help to reduce experimental variations and contradictory results in ESC culture and differentiation.
Fleischmann G., Cloning Stem Cells,
Session: Cell and vector manufacturing
Replication-defective adenovirus vectors based on human adenovirus type (HAdV)-5 are widely used for gene delivery and therapy. However, their efficacy is limited by poor transduction, pre-existing immunity and promiscuous tropism. Hopefully, some of these problems could be overcome by no-human adenovirus vectors. We have been developing bovine adenovirus (BAdV)-3 based vectors for vaccination and gene therapy. However, Short of efficient cell lines for isolating recombinant BAdV-3 hampered the progress in developing such vectors. Here, first we demonstrate that wt BAdV-3 infection or BAdV-3 genome trannsfection of cotton rat lungs fibroblast(CRLFB) cells produces progeny virus. Secondly, we established a novel BAdV-3 circular genome transfection system, in which the I-Sce I endonuclease recognition sites were engineered into the ITRs outside of the BAdV-3 genome in a circular plasmid, and the engineered genome was directly transfected into CRLFB cells expressing I-Sce I endonuclease. The virus is rescued through an enzymatic cleavage of full length BAdV-3 genome mediated by an intron-encoding rare endonuclease I-Sce I. This process of enzymatic rescue of circular molecular full length BAdV-3 genomic clones a) increased the viral genome transfection efficiency, b) shortened the viral rescue time and c) significantly decreased the DNA amount required. Thus, this method will facilitate a high throughput creation of a repertoire of BAdV-3 based vectors for vaccination and gene delivery in future.
Micro-freezing suitable for stem cell cryopreservation
Session: Cell and vector manufacturing
The current axioma in cryopreservation is that cells exhibit an optimal survival after freezing within a defined cooling/thawing regime. This is a specific challenge if storage of stem cells is considered. Because the process parameters differ among different cell types, they have to be optimized for each cell type of interest. Parameters that particularly need to be optimized are the cooling and thawing rate and the type and concentration of cryoprotective agents. Such parameter optimization studies however require a systematic approach to find the local optimum in cell survival for which a lot of samples have to be tested simultaneously which is problematic especially for rare and expensive cells like stem cells. Because commercially available freezing devices have limited possibilities to perform such parameter optimization studies, we developed a new device. The developed new freezing device [1] allows accurate control of freezing and thawing rates in a 12 by 9 matrix with 96 sample positions. Only a small sample volume of 200 μl is necessary and up to 12 different freezing protocols can be programmed and operated simultaneously with 8 samples each in parallel. Controlled thawing studies are also possible. With this device cryopreservation of mesenchymal stem cells (MSC) as a model system is performed. In previous studies [2] a systematic parameter optimization was established. By using this experimental setup, the MSCs are frozen with different concentrations of DMSO. Various combinations of cooling rates in two step freezing protocols are investigated. Different viability assays and testing for differentiation ability show the success of the respective cryopreservation protocol. Acknowledgements Funded by Cluster of Excellence “REBIRTH”
F. Evertz Cryobilogy
I. Bernemann Kaelte Luft Klimatechnik
Session: Cell and vector manufacturing
Human stem cells have potential use in clinical therapy and regenerative medicine. One of the major challenges regarding the application of these cells is the development of an efficient cryopreservation protocol, since currently used methods exhibit poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling/thawing rate and the presence of a cryoprotective agent (CPA) in a sufficiently high concentration. The most widely used CPAs, dimethyl sulfoxide (DMSO) and glycerol, however are toxic at high concentrations and have detrimental effects on the biological functioning of the stem cell as well as side effects for a stem cell transplanted patient. Therefore, it is of great interest to develop new cryoprotective strategies to replace the currently used CPAs or to reduce their concentration.This is especially important for particular (i.e. transgene) stem cells that will be provided as a medical device. Hereby it is necessary to avoid additives such as serum because of the required well-defined composition of all components used.For this we investigated different combinations of the non-cytotoxic biocompatible osmolytes ectoine and proline as CPAs in a systematic parameter optimization set up [1]. The findings were compared to those gained with a commercial freezing medium (Biofreeze, Biochrom). A freezing medium containing a low proline and a higher ectoin content showed promising results but so far the highest survival rate was achieved with the biofreeze medium. Acknowledgement: We thank Claudia Marx for her outstanding technical support.The project is supported by LOEWE (Landes-Offensive zur Entwicklung wissenschaftlicher und ökonomischer Exzellenz) and AIF (Pro Inno II)
Bernemann I. et al. Int J Artif Organs
Session: Cell and vector manufacturing
Cell therapy products often involve small cell samples mostly to be used in patient-specific manner. And not for chemotherapy, have a distinct characteristic of time limitation from development to administration into patients and should be performed within short period. Pharmacopoeia sterility testing are not applicable for cell therapy products. The purpose of this study was to establish of appropriate sterility test for cell therapy product. We investigated various sample size, media amount and inoculation amount of pharmacopoeia sterility testing and suggested minimum sterility test suitable for pharmacopoeia sterility testing suitable for cell therapy products under 2 samples, 2 ml media and 1% inoculating condition. Established appropriate sterility test for cell therapy products was compared with the pharmacopoeia sterility testing by 19 strains, including 6 strains of the test micro-organisms suitable for use in the growth promotion test and the validation test. Method validation was performed to demonstrate specificity and precision of established sterility testing by 19 strains. Detection limit for 19 organisms was 10–25CFU/ml by established sterility test. Compared to the pharmacopoeia sterility testing(10CFU/ml), detection limit of suggested sterility test was more insensitive but time to detection of organisms was equivalent. Our data demonstrate that the assay system is a good tool for supporting the diagnosis of bacterial contamination.
A. Mirjalili Biologicals
F. Cobo Cell Biol Int
H. M. Khuu Transfusion
Session: Cell and vector manufacturing
Baculoviruses can express transgene under appropriate promoter in a wide range of vertebrate cells including primary and stem cells. However, transgene expression is dependent on culture conditions. We have studied mechanisms behind the remarkable effect of cell culture medium on the BV transduction efficiency in different cell lines. Vimentin, a major component of type III intermediate filaments, was reorganized in the optimized medium, which associated with enhanced nuclear entry of BVs. In accordance with this, phosphorylation pattern of vimentin was altered in the studied cell lines. These results thus suggest that vimentin has a role in the baculoviruse entry into vertebrate cells. Interestingly, enhanced gene delivery in the optimized medium was not restricted with BV but was also observed with other viruses. The results highlight the general importance of culture medium in gene delivery.
Development of a 293T-based producer cell by consecutive integration of vector components via RMCE
Session: Cell and vector manufacturing
Stable third generation γ-retroviral producer cells are commonly generated by consecutive stable transfections of expression vectors for the retroviral genes gag/pol and env followed by transduction or again transfection of the retroviral vector to be produced. Although the split genome method leads to safe producer cells, stable transfections have the disadvantage that the expression vectors often integrate into loci that provide suboptimal conditions for their expression. As a consequence screening for suitable clones delivering the necessary expression level is very time consuming and expensive. In order to overcome this potential problem, recombinase mediated cassette exchange (RMCE) technology offers the opportunity to integrate the expression vectors as single copy in a previously well defined and stable locus of the genome. We demonstrate the establishment of 293T based packaging, into which single copies of expression constructs for MLV gag/pol and GALV glycoprotein (env) were integrated. The FLP-recombinase mediated targeted exchange of a previously tagged chromosomal locus lead to activation of a resistance marker (gpt). After selecting suitable clones the FRT site, the marker and the LTR of the tagging vector is removed from the locus by CRE recombinase mediated excision. Thus, the design of the tagging and targeting constructs allowed multiple rounds of exchange/clean-up reactions for consecutive integrations of MLV-gag/pol, GALV-env expression vectors as well as the retroviral transfer vectors. The new producer cell 293 gpG-tagIII allowed stable production of the tagging vector of around 6 × 106 IP/ml of GALV pseudotyped retroviral vectors. Production data of this cells after introduction of LTR and SIN-vectors will be presented.
Effect of culture condition on multiplication of embryonic cells from early stage in vitro derived buffalo embryos
Session: Cell and vector manufacturing
The present experiment was conducted to study the effect of different culture condition on derivation of ES cell from IVF derived early stage embryos in buffalo. Early stage 16–32 cell stage IVF derived embryos were treated with proteinase-K for zona removal. After complete dissolution of zona pelucida, the clumped blastomere were cultured on mitomycin-c inactivated buffalo embryonic feeder in ES cell medium supplemented with different percentage of fetal bovine serum. The media used were, Group-1: DMEM + LIF + ITS + 10%FBS, Group-2: DMEM + LIF + ITS + 15%FBS and Group-3:DMEM + LIF + ITS + 20%FBS. The clumped blastomere were cultured at 38°C, 5% CO2 and 90% relative humidity in CO2 incubator. The blastomere growth and establishment of embryonic stem cells if any were observed in each group. The results indicated that the clumped blastomere were attached to the feeder within 24 hrs and started multiplication. In Group-1, 9 embryos were cultured and the multiplication of cells was arrested on day 3 of culture. In Group-2, 8 embryos were cultured and again the growth was arrested on day 4 of culture. In Group-3, 20 embryos were cultured and 12 ES cell clones were formed. The ES cell clones could be propagated upto 2nd passage. In some case, when the blastomere were cultured, there were formation of blastocyst like structure with distinct inner cell mass but no trophectodermal cell. When the inner cell mass from these blastomere were mechanically isolated and cultured on inactivated buffalo feeder cells, no primary ES cell clones were formed. The results indicated that 20% serum supplementation support the multiplication of buffalo early stage blastomere cells better than 10 or 15% serum.
Progress in minicircle manufacturing and performance testing
Session: Cell and vector manufacturing
Plasmid DNA is commonly used in vaccination, gene- or cell therapy but also as a basic substance in viral vector production.The dissemination of antibiotic resistance genes, as well as the uncontrolled expression of backbone sequences present in such plasmids may have profound detrimental effects. Therefore, an important goal in vector development is to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle DNA. Our minicircle production technology facilitates the large scale production of highly pure minicircle DNA for applications in gene therapy and vaccination as well as e.g. virus production. The production technology is based on two processes: 1) An inducible, sequence specific, and very efficient in vivo recombination process; 2) A chromatographic purification technology for the isolation of the minicircle DNA. The resulting minicircle DNA consists of the gene of interest and a residual sequence stretch from the recombination. The chromatographic purification results in an exceptional purity, proven by various QC tests. For first efficacy studies, reporter genes for different types of analyses within various tissues, cells, animals and for testing the mode of administration (e.g. electro gene transfer, sonoporation, lipofection, magnetofection etc.) have been used. Also biodistribution studies using these constructs have been performed. Additionally, minicircle constructs for vaccines, virus production, etc. are currently investigated. The results demonstrate a significant increase of gene expression of minicircle DNA in comparison to a standard plasmid. As a result of the work presented here, PlasmidFactory has launched the first commercially available minicircle DNA product now.
A new optical method for analysing effects of ice formation on the cryopreservation success of human stem cells
Session: Cell and vector manufacturing
Stem cells are the centre of interest in biomedical research, whereas their isolation and culturing are not standardised yet and the comparability of protocols is still restricted. Therefore, cryobanking of human stem cells is one of the fundamental premises to support the success in stem cell based cell therapy [1], since researchers have always access to a well defined stock of therapeutic relevant cells. But efficient cryopreservation has to be adjusted to the specific characteristics of each cell type. An optimal cooling rate, for example, depends on cell size and corresponding active osmotic volume. Consequently, cryopreservation of biological samples containing cells of variable size, like many adult stem cell populations, is much more challenging compared to homogenous cell samples. For human embryonic stem cell colonies, mainly the three-dimensionality and the co-cultivation of mouse embryonic fibroblasts avoid optimal heat dissipation and permeation of cryoprotectants to the inner part of the colonies. But a suboptimal cooling rate results in an increased risk of ice formation within the cells or colonies and a disruption of cell-cell contacts [2]. To understand processes in frozen samples and to find possibilities that prevent intracellular ice formation, a standard cryomicroscope was combined with multi-photon-laser-scanning microscopy. This technique enables the detection of ice crystals between and inside cells or colonies as well as a defective membrane integrity with a high spatial resolution, penetration depth and photo quality. Using this new set-up, effects of different surfaces and cryoprotective agents on the performance of variable adherent stem cells like hESCs and MSCs during cryopreservation have been observed.
Glyn N. Stacey Advances in Tissue Engineering 229–254 (2008)
Peter Mazur Exp Cell Res
Session: Cell and vector manufacturing
For current research within the field of cell based therapies including Tissue Engineering different kinds of stem cells are used. Amongst others, murine embryonic stem (mES) cells serve as model cells for studies of regenerative medicine. These applications require a great quantity of undifferentiated mES cells that must be produced by large scale cultivations. At this point, a great amount of Leukemia Inhibitory Factor is needed which maintains their pluripotentiality. Here we describe a production process for recombinant human Leukemia Inhibitory Factor (hLIF) in E. coli. Soluble protein was expressed in fusion with N-terminal thioredoxin. After metal chelate affinity chromatography the fusion protein was cleaved with recombinant Tobacco Etch Virus (TEV) protease releasing hLIF. Subsequent purification of hLIF was performed by the use of cation exchange chromatography resulting in 95% purity. The biological activity was demonstrated by cultivation of ES cells in media supplemented with the purified cytokine throughout several passages. Our protocol for the production of recombinant hLIF is very suitable for the production of low soluble proteins through expression in fusion with the solubilizing partner thioredoxin. Additionally, in-house produced TEV protease makes this process extremely cost effective and thus applicable to numerous other cytokines produced with this expression system.
Institute of Multiphase Processes, Leibniz UniversitÄt Hannover , Germany
Session: Cell and vector manufacturing
Mouse embryonic fibroblast feeder cells (MEFs) are used as support layers to culture embryonic stem cells (ESCs). The quality of the MEFs influences long term growth and proliferation of undifferentiated ESCs. Two procedures greatly affect MEF quality before use: (1) Inhibition from proliferation by γ-irradiation and (2) cryopreservation. In this study, cryomicroscopy and Fourier transform infrared spectroscopy (FTIR) were used to investigate why survival rates after cryopreservation of MEFs are so highly variable. The effect of different cryoprotective agents (CPAs) including Me2SO, glycerol and proline was also tested. Slow cooling protocols resulted in cellular dehydration. In the presence of CPAs, dehydration occured more gradual. In the absence of CPAs, rapid freezing resulted in intracellular ice formation (IIF), which occurred almost directly after extracellular ice formation. In the presence of CPAs, IIF was not observed directly after extracellular ice formation but was delayed. Membrane phase behavior of γ-treated and control cells during freezing with controlled nucleation was studied by FTIR. Membranes displayed a highly cooperative fluid to gel lipid phase transition upon ice nucleation at −2°C, which was not observed with stochastic nucleation at −12°C. The freezing induced fluid to gel phase transition with nucleation at −2°C directly correlates with the cellular dehydration that was observed by cryomicroscopy under similar freezing conditions. Gamma treatment was found to have little effects on the membrane phase behavior of the cells, suggesting that gamma irradiation does not cause major alterations in membrane phase properties of the cells.Source of funding: Cluster of Excellence “REBIRTH” (DFG).
Molecular Biotechnology, Helmholtz Centre for Infection Research , Germany
Session: Cell and vector manufacturing
To ensure quality and safety of human mesenchymal stem cell expansion for advanced cell therapeutic purposes, a disposable cell culture bag system for GMP compliant adherent cell cultivation was developed. We have previously shown that the closed cell culture bag is suitable for the cultivation of suspension cells but did not support growth growth of adherent cells. To enhance adherent cell growth, the polymer surface was functionalized by an atmospheric-pressure plasma process whereby the inner bag surface is selectively modified through film forming agents to create an amino group (APTMS, DACH) or silane (TMOS) containing coating. Biocompatibility of the surface treated cell culture bag was assessed using human bone derived mesenchymal cells and SAOS-2 cells. Cellular responses were evaluated in terms of cell morphology, attachment (fluorescence microscopy, scanning electron microscopy), cell proliferation (WST-1) and osteogenic (Alizarin red staining) and adipogenic differentiation (Nile red staining). All modified surfaces showed initial cell adhesion in contrast to the cell repellent properties of the unmodified cell culture bag. Proliferation on the other hand was only observed in case of APTMS- and DACH- in contrast to TMOS-treated surfaces where after initial cell adhesion took place, no further increase in cell number could be observed. Both osteogenesis and adipogenesis of the MSC could be induced on the modified surfaces, comparable to classic polystyrene culture vessels. Further, we successfully cryopreserved adherent cell layers in situ on the modified culture bag surface whereby the cells retained their proliferative potential after thawing.
RDIF-MSYS, Helmholtz Centre for Infection Research , Germany
Session: Cell and vector manufacturing
So far, production of HIV-derived lentiviral vectors (LVs) is based on transient transfection protocols in 293T cells. To meet the requirements for gene therapy applications LV production from characterized stable and safe producer cell line (PCL) is mandatory. We took advantage of well characterized tagged chromosomal loci of HEK293-based cell lines for LV expression towards the generation of stable LV PCL, thereby employing Flp mediated cassette exchange. Unexpectedly, production of LV from all tested loci was severely impaired. We followed various approaches to rescue virus production from a stably integrated single copy LV. However, neither suppression of RNAi-based antiviral mechanisms employing viral interferon antagonists (e.g. VP35) nor co-expression of lentiviral accessory proteins could overcome this block in virus production. Finally, we evaluated the impact of the expression of large T antigen protein from SV40 virus (TAg). Expression of TAg upon transient transfection of HEK293-based targeted cells did not affect virus production. However, upon stable transduction of TAg in HEK293-based LV targeted cells, a 200fold increase in LV titers was observed. This suggests that long term expression of TAg, rather than TAg as such, affects the cellular ability to produce LVs.Currently, the molecular mechanism(s) underlying these unexpected phenomena is being investigated towards the establishment of a modular cell line for LV vector production.
Molecular Virology and Gene Therapy, Catholic University of Leuven , Belgium
Session: Cell and vector manufacturing
Institute for Clinical Transfusion Medicine , StÄdtisches Klinikum Braunschweig gGmbH, Germany
Session: Cell and vector manufacturing
One approach in cancer immunotherapy are cell based therapies. For possible clinical applications of these therapies standardised culture conditions and minimization of risk of contamination are crucial. For this reason we designed a flexible modular closed bag system for cultivation of human monocyte derived dendritic cells (DC) as a potential tool for cellular immunotherapies. The closed bag cultivation system integrates ex vivo selection/enrichment, culture, differentiation, genetic modification and cryopreservation of these cells under GMP-compliant conditions using sterile docking for all addition or splitting steps. CD14 positive monocytes were enriched by leukapharesis (Cobe Spectra, GambroBCT) and positively selected by magnetic cell sorting (CliniMACS-System, Miltenyi). Differentiation of monocytes to immature dendritic cells was induced by GM-CSF and IL4 under serum free conditions. Immature dendritic cells were transduced by adenoviral vectors very efficiently. After 5 days of culture maturation of DC was induced by addition of IL1beta, IL6, prostaglandine E2 and TNFalpha. On day 7 of cultivation mature dendritic cells were harvested, analyzed and cryopreserved. Yield, maturation, transgenic expression and stimulatory capacity of DC differentiated and modified in the closed bag system were comparable to cell culture flask generated DC. The closed bag system is an easy to handle system for GMP-compliant generation and modification of dendritic cells under clinical conditions and could easily be adapted for cultivation of other nonadherently growing cells.
Institute for Clinical Transfusion Medicine , StÄdtisches Klinikum Braunschweig gGmbH, Germany
Session: Cell and vector manufacturing
There are increasing numbers of studies investigating the application of therapeutic cells. A major problem for these studies is the safe and standardized generation of these cells under GMP-compliant conditions. One approach is the generation of cells in a closed bag system as we have shown for non-adherently growing dendritic cells. We wanted to exploit the advantages of the closed bag system to other aspects of cell generation: 1) growth of adherent cells, 2) side specific genetic modifications and 3) cell selection. For this purpose we used dielectric barrier discharge which allows the chemical modification of the inner bag surface by plasma at atmospheric pressure.We used different chemical precursors for the primary modification of the bags by plasma. The primary modification enabled the culture of adherently growing cells on such treated plastic surfaces, but it also allows secondary coupling of additional molecules for cell modification and selection. As a model for the specific localized genetic modification of cells, we used adenoviral vectors encoding for the EGFP- or dsRed-Protein. These vectors were linked to the surface by biotin-streptavidin based mechanisms and allowed localized transduction of target cells. This system was also used for direct coupling of antibodies to cell surface molecules. By using Protein G we could further enhance the load of antibodies on the surfaces and preliminary data shows that this modification may enable specific selection of target cells.
Department of Pharmaceutics, University of Kuopio , Finland
Session: Cell and vector manufacturing
The complexity of gene delivery tools leads to far more challenging manufacture and storage schemes than with traditional small molecule pharmaceuticals. Developing gene delivery vehicles with extended shelf- lives is crucial in making the life span of these products more cost effective and environmentally sustainable. Freeze drying is a widely used method for drying pharmaceuticals, especially protein drugs, to achieve enhanced stability and longer shelf life. In a well designed freeze drying cycle, the product forms elegant solid cake in which degradation reactions, like protein unfolding, can be avoided. Designing of freeze-drying cycle requires knowledge of the critical formulation properties such as glass transition temperature of the maximally freeze-concentrated solute (Tg') and collapse temperature of formulation (Tc). In this study we have determined these critical temperatures for two types of gene delivery vehicles: cationic polymer (PEI) pDNA complexes and adenoviral vectors. Different sugars and carbohydrate polymers were used as lyoprotectants in the studied formulations. Tg' was measured with Differential Scanning Calorimetry (DSC) and it was detected as a shift in the heat flow signal. For Tc analysis Freeze-Dry Microscopy (FDM) which simulates actual freeze drying conditions, was used and it took place during sublimation drying. After determination of critical temperatures for all formulations, adequate freeze drying cycles were designed and samples were freeze dried. The freeze dried products were thereafter analyzed for their physical properties and activity, and after two month storage, activity of pDNA/PEI complexes was well preserved.
Biochemistry, University of Medicine and Pharmacy Victor Babes , Romania
Session: Clinical trials, cell and gene therapy
The study describes the influence of different inclusion criteria in angiogenic gene therapy using plasmidial vectors that contain the genes VEGF165 and HGF in severe chronic ischemia of the lower limbs. The study group includes 20 patients divided into two groups - group A that were administered only one injection of gene plasmidial construct and group B that were administered two injections (initial injection followed by a second four weeks later). The second injection, four weeks after the first one, significantly increases the success rates of the therapy. Results analysis after 12 month of periodic controls show that are some inclusion criteria that influence more significant the outcome of therapy. Therapy failure was noted in patients with advanced disease and in patients with multi-level lesions. The moment of the treatment seems to be very important: patients that experienced rest pain for more than 12 weeks and/or the values of ankle-brachial index ≤ 0.2 on admission had a worse prognosis.The final conclusion shows that an earlier therapy has considerably successful rates. The more important admission criteria which influence the success rate of angiogenetic gene therapy (no trophic ulcers, intermittent claudication, rest pain less than 12 weeks and ankle-brachial index > 0.2) are correlated with the earlier stage of the disease.
Cancer Gene Therapy Group, University of Helsinki , Finland
Session: Clinical trials, cell and gene therapy
Granulocyte-macrophage colony stimulating factor (GMCSF) can mediate anti-tumor effects by recruiting natural killer cells. Also, it can induce tumor-specific cytotoxic T-cells through antigen presenting cells (APC). Oncolytic tumor cell killing can produce a potent co-stimulatory danger signal and release of tumor epitopes for APC sampling. Therefore, an oncolytic adenovirus coding for GMCSF was engineered and shown to mediate tumor specific immunity in an immune competent syngeneic hamster model. Subsequently, 20 human patients with advanced solid tumors refractory to standard therapies were treated with Ad5-D24-GMCSF. Overall, objective evidence of anti-tumor activity was seen in 12/19 evaluable patients (63%). Responses were frequently seen in injected and non-injected tumors. Treatment was well tolerated and resulted in induction of both tumor-specific and virus-specific immunity as measured by ELISPOT and pentamer analysis. This is the first time that oncolytic virus mediated anti-tumor immunity has been demonstrated in humans.
A Phase I study of DWP418, a relaxin-expressing oncolytic adenovirus, in patients with recurrent solid tumors
Session: Clinical trials
Oncolytic virotherapy is the use of genetically engineered viruses that specifically target and destroy tumor cells via their cytolytic replication cycle. An important issue in replicating virus-based cancer gene therapy that has received little attention is the limited distribution such viruses have been able to achieve in solid tumors. With the aim of improving viral distribution and tumor penetration for cancer gene therapy, we have developed a novel relaxin-expressing oncolytic adenovirus, DWP418. We carried out a phase I study of DWP418 in patients with recurrent solid tumors. DWP418 was administered via ultra-guided injection in to recurrent solid tumors every 3 weeks until tumor progression. Five cohorts were investigated with a dose escalation from 1 × 1011 – 1 × 1013 viral particles per treatment. Toxicity was assessed using NCIC criteria, and tumor response was assessed by clinical and radiological measurement. Blood samples were taken to detect adenovirus DNA and neutralizing antibody to adenovirus. Tumor biopsies were taken to detect viral replication and expression of relaxin in tumor tissues. Treatment was well tolerated, with the main toxicity being injection site pain and mild flu-like symptoms. Dose-escalation proceeded up to the 1 × 1012 viral particles so far. Dose-limiting toxicity was not reached up to the dose of 1012 viral particles. All patients treated so far had preexisting neutralizing antibodies to adenovirus, and all patients developed increased antibody levels after treatment. Immunohistochemisty analysis showed viral replication as well as expression of relaxin in 6 out 8 available patients treated. Using modified WHO criteria, 1 out of 9 patients showed PR and 4 out of 9 patients showed SD. More interestingly, when tumor response was assessed by PET-CT, 4 out 6 available patients showed tumor shrinkage. These preliminary results show that intratumoral administration of DWP418 is feasible, well tolerated, and associated with biological activity.
Centro de Biologia Molecular e Engenharia Genetica, Universidade Estadual de Campinas-UNICAMP , Brazil
Session: Clinical trials, cell and gene therapy
The low efficiency of plasmid DNA (pDNA) delivery to the cells nucleus is a major concern in gene therapy and DNA vaccination studies using non viral vectors. This is mainly due to the fact that during their traffic to the target cell nucleus, pDNA vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery taking advantage of molecular motors, like dynein, for the transport of cargos from the periphery to the interior (centrosome) of mammalian cells. Here, a fusion protein containing an N-terminal dynein binding domain from human pericentrin was fused to a synthetic DNA binding sequence at its C-terminus. Cloning was successfully performed in pET28 a expression vector. Expression studies indicated that the fusion protein was correctly and best expressed in E. coli Rosetta strain. After evaluation of the protein secondary structure using circular dichroism, gel mobility shift assays confirmed the ability of the recombinant protein to interact and condense plasmid DNA. Preliminary transfection assays in HeLa cells using the recombinant proteins in combination with a model pDNA vector (pVAX1GFP) and Lipofectamine indicated an improvement in transfection level. By performing systematic transfection studies, we expect to confirm the capacity of these constructs to interact with dyneins and facilitate intracellular pDNA trafficking. The development of recombinant shuttle proteins able to take advantage of strategies used by virus to infect mammalian cells may, in the near future, provide new potent tools for gene therapy and DNA vaccination studies.
Cardiothoracic Surgery, Deutsches Herzzentrum Berlin - BCRT, Germany
Session: Clinical trials, cell and gene therapy
Myocardial cell therapy using autologous marrow cells is currently being evaluated in clinical trials. It is known that age-related changes influence their proliferative and regenerative capacity, but the clinical impact of stem cell ageing on myocardial regeneration is unclear. We tested the hypothesis that age impairs the clinical efficacy of cardiac cell therapy. A total of 55 patients (age 14–81 years) with impaired heart function (45 with ischemic and 10 non-ischemic heart disease) were treated by intramyocardial marrow cell injection (CD133 enriched or MNC). In 45 patients cell therapy was done as part of coronary artery bypass surgery, and in 10 patients at the time of mechanical cardiac assist device implantation. Echocardiography and cardiac MRI were used to monitor LV function during follow up. A matched group of patients undergoing routine CABG or VAD surgery served as a control. There was negative association between CD34 +/CD133 + cell yield and patient age, but not for total cell count. The relative gain in LV contractile function (LVEF) 6 months postoperatively was 17% (-21 - 67). By linear regression, an inverse correlation between the gain in LV contractility and patient age was identified (r = 0.48, p = 0.03). LVEF improved by >10% in 64% of the patients aged < 60 years but only in 28% of those >75 years (p = 0.01). In the control group, no association between age and LV function change was found. Cardiac cell therapy with autologous marrow cells is less effective in elderly patients. The design of future clinical studies should take this into account, and strategies to enhance the function of aged marrow cells are needed.
Department of Veterinary Clinical Sciences, Royal Veterinary College University of London , UnitedKingdom
Session: Clinical trials, cell and gene therapy
Muscle-targeted gene therapy (MTGT) with insulin and GLP-1 constructs shows promise as diabetes mellitus (DM) treatment. However, gene therapy prejudices could influence acceptance of such therapy. This study aimed to record ease of acceptance of MTGT amongst people with DM. Patients attending a Dutch diabetes clinic were asked to complete a survey, consisting of a lay introduction to MTGT and multiple choice questions. 66 people completed the survey (37(56%) male, various educational backgrounds, age 54 + /−17 years). 24(36%) had type 1 and 42(64%) type 2 DM. 62(94%) injected insulin and 60(91%) suffered from DM-related complications. 54(82%) stated to have a positive attitude towards gene therapy in general, 10(15%) neutral and 2(3%) negative. 57(86%) answered to be prepared to undergo gene therapy if this would aid their DM and 59(89%) if this would cure their DM. 42(64%) reported to be “certainly” prepared to receive an intra-muscular DNA injection every 2 months; 37(56%) even if daily drugs were still needed; 48(73%) when “if you would not need to inject yourself (anymore)” or “if you would not need other medication” was added; 52(79%) when “if it would reduce the risk of DM complications” was added. 49(74%) answered they would not refuse MTGT if attenuated virus was used. In the studied population a minority showed marked concerns about the application of MTGT to treat their DM. Nevertheless, it would be prudent to involve people with DM in the implementation process.
Ratanamart Current Gene Therapy
Mahmoud Diabetologia
Shaw Journal of Endocrinology
Session: Clinical trials, cell and gene therapy
The aim of suicide gene therapy in allogeneic hemooietic cell transplantation (HSCT) is to control GvHD without imparing immunoreconstitution (IR). We studied 28 patients with high-risk acute leukemia treated with donor lymphocytes expressing HSV-TK after HSCT from a haploidentical donor (TK007). The control cohort comprised 40 patients receiving an unmanipulated graft and rapamicin as GvHD prophylaxis (TrRaMM). TrRaMM and TK007 reached IR (circulating CD3 + > 100/μl) at day + 30 and + 90, respectively. In TK007, TK cell engraftment was required for sustained IR. In both trials, IR was mainly composed of Th1/Tc1 lymphocytes with an inverted CD4/CD8 ratio. While in TrRaMM IR was biased towards naive, central memory and regulatory T cells, in TK007 activated effector T cells were dominant. Protective frequencies of CMV-specific responses were observed 3 months after HSCT in both trials. GvHD occurred in 10 TK patients (36%) and 16 TrRaMM (40%). While in TrRaMM, CMV-specific immunity was hampered by the immunosuppressive treatment of GvHD, in TK007, ganciclovir administration resulted in control of GvHD without impairing virus-specific immunocompetence. Accordingly, in TrRaMM 80% of viral reactivations occurred after treatment of GvHD. In TK007, no significant differences were observed before and after ganciclovir treatment. These results confirm that suicide gene therapy allows selective GvHD control while preserving IR.
Vector silencing in a gene therapy trial for X-CGD and strategies to improve long-term transgene expression
Session: Clinical trials, cell and gene therapy
Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency characterized by recurrent, often life threatening bacterial and fungal infections due to a functional defect in the microbial-killing activity of phagocytic neutrophils. In 2004 two adult X-CGD patients were treated with gene modified CD34 + cells after a mild myelosuppressive regimen. A long terminal repeat (LTR)-driven gammaretroviral vector encoding for the cDNA of gp91phox was used for the transduction of patients CD34 + cells in this Phase I/II clinical study. Two to four years after transplantation, high levels of gene marking were detected in peripheral blood cells of both patients. However, the level of superoxide production by gene corrected cells decreased constantly with time. Quantitative RT-PCR analysis over time revealed a transcriptional down regulation of gp91phox expression due to CpG methylation at the viral LTR promoter. Notably the enhancer region within the LTR was never found to be methylated. Therefore the enhancer could still mediate transcriptional activation of cellular genes at insertion sites leading to the accumulation of non-functional gene transduced cells. We established an in vitro silencing prediction assay in a murine embryonal carcinoma cell line and compared vector performance in terms of protein expression and promoter CpG methylation. In this setup promoter/enhancer combinations resistant to CpG methylation were identified.
Getting a glimpse in the in vivo dynamics during immune reconstitution after gene therapy treatment in ADA-SCID patients
Session: Clinical trials, cell and gene therapy
Senescence of hematopoietic cells has been recently pointed out as a concern for the long term outcome of BMT. At HSR a successful clinical trial for the gene therapy (GT) of ADA-SCID is ongoing, but an in depth analysis of the in vivo dynamics of the new immune progeny is mandatory. We measured telomere length and performed cell cycle analysis on peripheral blood (PB) and bone marrow (BM) samples collected from GT patients, using pediatric and adult normal donors (ND) and BMT patients as controls. The latter is important for assessing any effect introduced by the transduction protocol on the BMT procedure. We measured the telomere length in CD15, and in naive and memory T cells in the PB, and in the total mononuclear cells and CD15 in the BM. For the cell cycle analysis we also dissected central and effector memory, and TEMRA cells in the PB. We observed no telomere shortening in the BM compartment and in granulocytes in comparison with NDs, while in the PB the naive T cells from both GT and BMT groups showed a telomere length in the range of adult controls. Cell cycle analysis revealed that cells from the GT group are the most active, in particular at early FU in the CD4 compartment. In conclusion we only observed signs of senescence in the PB, suggesting a correct cellular dynamic at the level of progenitor cells. This could be the consequence of a poor thymic performance, typical of primary immunodeficiencies, and to the stress of peripheral expansion driven by the initial low cellularity in the PB. Our data are overall encouraging in considering GT a valid long term treatment for ADA-SCID.
Telethon Institute for Gene Therapy HSR-TIGET, Scientific Institute Hospital San Raffaele , Italy
Session: Clinical trials, cell and gene therapy
Hematopoietic stem cell gene therapy (GT) for ADA-SCID leads to reconstitution of lymphopoiesis, metabolic correction, and clinical benefit. Treated patients show increase in naive cells, TREC and thymic volume after GT, but immune reconstitution is slower than in standard allogeneic bone marrow transplant (BMT). To study the contribution of homeostatic expansion to Tcell reconstitution we performed cell cycle analysis in peripheral blood (PB) CD4 + and CD8 + cells (naive, central and effector memory, TEMRA) in GT patients compared to pediatric recipients of allogeneic BMT and healthy controls. Our results revealed naive and memory cells as the most cycling at early time points after GT (median value in CD4 + naive 4.6%), suggesting an important contribution of peripheral expansion to immune reconstitution, and decrease later (0.48%) toward values observed after BMT (0.32%), but higher than controls (0.14%). There was no evidence of clonal expansion by analyses of TCR repertoire and vector integration profile. To assess if the increased cycling could result in accelerated cell senescence, we measured telomere length by qPCR. We found that PB naive T cells from GT patients had shortened telomere compared to healthy children, but not different from BMT patients and adult controls. Furthermore, we didn't observe telomere shortening in BM and PB granulocytes, suggesting appropriate cellular dynamics at the level of stem/progenitor cells, even after the ex vivo transduction procedure.
Applied Virology and Gene Therapy Unit, Georg-Speyer-Haus Frankfurt, Germany
Session: Clinical trials, cell and gene therapy
In our gene therapy trial of patients with X-linked chronic granulomatous disease (X-CGD) we demonstrated reconstitution of oxidative burst capacity and elimination of preexisting severe infections in two treated patients. However, an unexpected expansion of gene marked myeloid progenitors occurred five months after transplantation, which was triggered by insertional activation of MDS1/EVI1, PRDM16 or SETBP1. We monitored patients for clinical health, hematological reconstitution, phagocyte function, gene marking, clonal fluctuation, cytogenetic and DNA methylation status. After the initial resolution of bacterial and fungal infections, both patients developed a myelodysplastic syndrome (MDS) caused by insertional activation of MDS1/EVI1 followed by clonal progression and the gradual loss of chromosome 7. P1 died 27 months after gene therapy of MDS in combination with severe septicemia, the latter resulting from loss of bacterial killing activity in transduced cells. P2 underwent allogeneic stem cell transplantation. Forced overexpression of MDS1/EVI1 or EVI1 in human cells disrupted normal centrosome duplication, linking MDS1/EVI1 activation to the development of genomic instability. Gene therapy for X-CGD can provide considerable clinical benefits. Advances in vector design that avoid protooncogene activation and promoter methylation will enable the safe and effective application of this strategy for the long-term correction of X-CGD.
Centro de Biologia Molecular e Engenharia Genetica, Universidade Estadual de Campinas, Brazil
Session: Physicochemical approaches
Non viral gene therapy has been considered as an outstanding alternative to an easier and safer delivery of plasmidial DNA to eucariotic cell nucleus. However, the feasibility of this method depends on the development of delivery vectors with high targeting and delivery efficience. In the present study, we aimed to exploit cells microtubule network to facilitate plasmidial DNA traffic through cells citosol to the nucleus via molecular motors like dynein. Dynein light chains Lc8, Tctex-1 and rp3 (Tctex-3) were cloned in pET28a (Novagen) together with four different N-terminal DNA binding domains, consisting of different combinations of basic aminoacids. These recombinant light chains are supposed to interact and condense plasmidial DNA in vitro through it s N-terminal and interact with the dynein motor complex in vivo, allowing the plasmid to be carried to cell s nucleus. The recombinant Lc8 and rp3 light chains with and without the four different DNA binding domains were successfully expressed in BL21 (DE3) and BL21(DE3) Rosetta Escherichia coli strain, respectively. Both, Lc8 and rp3 were purified by affinity chromatography using Ni-NTA resin. The secondary structure was evaluated by circular dichroism for all eight constructs and preliminar condensing capacity was accessed by gel retardation assay (using a model pDNA vector, pVAX1GFP) for the Lc8 light chain and indicated capacity to interact and condense DNA even without the DNA binding domain. Further analysis using dynamic light scattering, gel retardation assay, circular dichroism and transfection assays will allow us to analyse the best construct for DNA condensing and transfection, hopefully adding new information on non viral delivery mechanisms.
Development, Cellectis Genome Surgery, France
Session: Eye, brain, nerves, skin
The majority of current anti-viral treatments are based on the prevention of productive viral replication through the utilization of agents that inhibit essential virally encoded proteins. In the majority of cases these treatments eventually become ineffective due to the generation of viral mutations that result in drug-resistance. Many chronic viral infections are due to double-stranded DNA viruses or viruses that involve a double-stranded DNA intermediate during their replicative cycle. Thus, an attractive alternative antiviral strategy is to specifically cleave and either partially excise or eliminate viral DNA from infected cells and thus render them virus free.Meganucleases are endonucleases that recognize large cleavage sites (>12bp) with a high specificity. We have shown that the expression of the meganuclease I-SceI, either before or after infection with a modified Herpes Simplex Virus (HSV) containing a meganuclease recognition site, results in a dramatic reduction of viral DNA. Meganucleases specific for viral DNA could thus represent a novel class of agents for the treatment of viral infections that could cleave and either partially excise or eliminate viral DNA from infected cells, rendering them virus free. Using a semi-rational approach, we have used a two step strategy to produce meganucleases cleaving several different viral genomes. We will present data concerning the use of virus specific meganucleases for the development of a new anti-viral approach and therapeutic strategies for certain persistent infections, particularly those caused by HSV-1 in ocular herpes infections.
Unit of Gene Therapy and Stem Cell Biology, Jules-Gonin Eye Hospital , Switzerland
Session: Eye, brain, nerves, skin
In the retina, the balance between pro- and anti-angiogenic factors is critical for angiogenesis and is also involved in cell survival and maintenance. We previously reported an upregulation of VEGF after light-damage (LD). Furthermore, systemic delivery of PEDF, as well as lentiviral gene transfer of an anti-VEGF antibody rescued photoreceptors (PRs) from cell death. We herein describe the effect of anti-VEGF local delivery by lentiviral gene transfer.We constructed a lentiviral vector encoding an anti-VEGF antibody (LV-anti-VEGF) and tested its efficiency in a model of choroidal neovascularization (CNV) induced by laser injury. Animals treated with LV-anti-VEGF showed reduced CNV compared to control groups, demonstrating the good efficiency of the vector. The LV-anti-VEGF was simultaneously assayed in LD model. Evaluation of functional parameters showed that LV-anti-VEGF treatment rescued the retinal function compared to control groups. Furthermore, PR survival correlates with the level of anti-VEGF expression. These results suggest a deleterious role of VEGF in PR death. We next investigated the effect of high levels of VEGF in the retina after LD. Analyses of protein expression level showed that the VEGF pathway is disturbed. Concomitantly, we observed a transient permeabilization of the RPE cells, which could indirectly contribute to the deleterious effect of VEGF observed during LD-induced PR apoptosis. This study further involves VEGF in LD and highlights the prime importance of the angiogenic factor balance in PR survival. Moreover, our investigations to elucidate the mechanism of action of VEGF in this context will provide more precise targets for a gene therapy deprived of potential side-effects due to unspecific inhibition of VEGF.
Responsiveness of stem cell-like brain tumor cells to differentiation-inducing reagents
Session: Eye, brain, nerves, skin
Malignant brain tumors are amongst the most lethal solid tumors. The standard treatment consists of surgical resection followed by consecutive radiochemotherapy. Recurrence is essentially not avoidable. It has been proposed that brain tumor stem cells may represent the source of the therapy-resistant tumor cell subpopulation, as they may escape from chemo- and radiotherapy, self-renew and initiate tumor regrowth. Induction of terminal differentiation of NSC-like tumor cells may constitute an attractive, new strategy in brain tumor therapy. We analyzed the possibility to induce cell cycle exit and differentiation of stem cell-like brain tumor cells. All tumor biopsies harboured Nestin-positive neural stem cell-(NSC-)like cells, which self-renewed in culture. These cells exhibited a substantial heterogeneity with respect to morphology and growth behaviour. This was reflected by the expression levels of the stemness factor Oct4, and in particular of Sox2. All primary cell lines responded to increasing amounts of serum with a proliferation stop, which was paralleled by downregulation of Nestin and the stemness factors. Additionally, application of retinoic acid (RA) decreased the proliferation in the presence of cAMP and downregulated Nestin and the stemness factors. The efficacy with which FCS and RA/cAMP could mediate these effects was cell line-specific. Proliferation stop, however, did not concomitantly induce neural differentiation, as only a subfraction of the NSC-like cells differentiated into glial and neuronal derivatives, respectively. In summary, these data indicate that RA/cAMP and a yet unknown serum-derived factor are capable to block the proliferation of NSC-like brain tumor cells in a cell type-specific fashion.
REMEDI, NUI Galway, Ireland
Session: Eye, brain, nerves, skin
Following spinal cord injury, a highly inhibitory environment for axonal regeneration develops. One of the main sources of this inhibition is the glial scar which is formed after injury by reactive astrocytes. The inhibitory environment is mainly due to chondroitin sulphate proteoglycans (CSPGs). NG2, one of the main inhibitory CSPGs, is upregulated following injury. shRNA was designed to target NG2 and cloned into a lentiviral vector. The neurotrophic factor NT-3 was also cloned into a lentiviral vector. In vitro assessment of these vectors using a co-culture system of dorsal root ganglia neurons and Neu7 inhibitory astrocytes was carried out. The Neu7 cell line is a rat astrocyte cell line which overexpresses NG2, thereby mimicking the inhibitory atmosphere following spinal cord injury. These experiments showed that both the knockdown of NG2 via shRNA and overexpression of NT-3 can significantly increase neurite growth. Lentiviral vector expressing NT-3, shRNA to NG2 or a combination of both vectors were directly injected into contused rat spinal cord at T9 one week post injury. Animals were sacrificed two or six weeks post injection of virus and examined by immunohistochemistry for changes in expression of NG2 and NT-3. Motor and sensory function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale, Von Frey filaments and Hargreaves apparatus Immunohistochemical staining showed significantly reduced levels of NG2 and increased levels of NT-3 following lentiviral vector treatment in vivo.
Biological Systems Group, Oxford BioMedica, UnitedKingdom
Session: Eye, brain, nerves, skin
Oxford BioMedica has developed a range of recombinant Equine Infectious Anemia Virus (EIAV) vectors expressing therapeutic genes for the treatment of a variety of diseases. One of these vectors, ProSavin, is currently undergoing evaluation in a Phase I/II clinical trial for the treatment of Parkinson's disease; this is the first trial in which a lentiviral vector has been directly administered to patients. Lentiviral vectors can transduce a broad range of dividing and non-dividing human cell types, including ocular cells. Oxford BioMedica has developed a vector, RetinoStat, for the treatment of 'wet' or exudative age-related macular degeneration (AMD); AMD is the principle cause of blindness in the developed world and around 20% of patients are affected by the wet form, caused by the formation of aberrant blood vessels from the choroid (neovascularisation). RetinoStat vector expresses human endostatin and angiostatin which have potent angiostatic activity which leads to the suppression of neovascularisation in vivo. We have previously shown that sub-retinal administration of RetinoStat targets gene expression to the target cell layer, the retinal pigmented epithelium (RPE). The key to success of this therapy will lie in achieving therapeutic levels of these proteins with acceptable ocular tolerance after vector delivery in patients. We are evaluating protein production and ocular tolerance in a rabbit and NHP dose-ranging study, using RetinoStat vectors configured with the photoreceptor-specific VMD2 promoter and the constitutive CMV promoter. Results from these in vivo expression and toleration studies will be presented.These data further support progressing RetinoStat toward a first in Man study.
Therapeutic Gene Regulation, Sangamo BioSciences, United States of America
Session: Eye, brain, nerves, skin
Glial cell line-derived neurotrophic factor (GDNF) is a potent trophic factor for the dopaminergic neurons of the substantia nigra, whose loss is responsible for Parkinson's Disease (PD). Increasing GDNF levels is therefore an attractive strategy for the treatment of PD but methods that permit the appropriate magnitude and duration of GDNF expression remain elusive. Engineered zinc-finger protein transcription factors (ZFP-TFs), which can be designed to induce more physiological expression levels of virtually any gene by acting on a natural gene promoter, offer a potential solution to this problem. However, the efficacy of ZFP TFs in the brain has yet to be demonstrated. To explore a neurotrophic factor-based therapy for PD, a panel of GDNF-targeted ZFP TF activators was designed, assembled, and tested for endogenous GDNF up-regulation at the level of mRNA and protein. A ZFP TF that significantly activates GDNF expression (>20 fold) in cultured rat striatal neurons was identified. Adeno-Associated Virus (AAV) expressing the GDNF activator (AAV-GDNF.ZFP) was infused using convection enhanced delivery into the striatum of rats lesioned with 6-hydroxydopamine (6-OHDA). Intrastriatal delivery of AAV-GDNF.ZFP drove a modest increase in GDNF levels that nonetheless provided statistically significant functional improvements in forelimb akinesia, sensorimotor neglect and amphetamine-induced rotations, suggesting that ZFP-driven physiological increase of GDNF expression is sufficient to confer functional neuroprotection in the rat 6-OHDA model. These results establish the utility of ZFP TF-driven gene regulation in the brain and support the further development of the GDNF ZFP TF activator as a potential treatment for PD.
Department of Neurology, University Hospital of Zurich , Switzerland
Session: Eye, brain, nerves, skin
Intracerebral experimental gliomas attract hematopoietic progenitor cells (HPC) in vivo . HPC are therefore promising vehicles for a cell-based delivery of therapeutic molecules to malignant gliomas. Before therapeutic application, however, it is important to investigate the time-frame of homing and the spatial distribution of the HPC within the glioma as possible systemic distribution of HPC after intravenous injection. Time lapse two photon laser scanning microscopy (2PLSM) and positron emission tomography (PET) are two powerful techniques to visualize cellular migration in living glioma-bearing mice. We prepared a cranial chronic glass window and stereotactically implanted green fluorescent protein-positive glioma cells into the neocortex of mice. Human CD34 + HPC isolated from granulocyte-colony stimulating factor-mobilized peripheral blood or murine lineage-depleted Sca1 + Kit +(LSK) bone marrow cells were stained with PKH26 and injected five days later intravenously to assess the glioma tropism by 2PLSM. Furthermore, we implanted glioma cells into the right striatum. Fourteen days later we injected mutant H. simplex tyrisone-kinase (Hsv-tk)-positive HPC into the tail vein. PET analysis with the tracer [F-18]FHBG, a ganciclovir-analogue, was used to determine the biodistribution of injected Hsv-tk-positive HPC by small animal PET imaging. The combination of these imaging modalities enabled us to characterize both, the time-frame of homing and the systemic distribution of HPC after intravenous injection in living glioma-bearing mice. We will present the data and discuss conclusions for the therapeutic application.
Deptartment of Pathobiology Institute of Virology, University of Veterinary Medicine Vienna , Austria
Session: Eye, brain, nerves, skin
The lack of success of clinical studies with non-replicating MLV-based vectors delivering therapeutic genes into the patients with malignant gliomas is most likely due to low rate of tumor cell transduction. Recently, MLV-based replication-competent retroviral (RCR) vectors had been developed which are able to spread within the tumor mass and thus lead to an improved therapeutic effect. In this study described here, we have analysed the spread of RCR vectors expressing the EGFP marker gene in a panel of human and rodent glioma cell lines in vitro and in vivo in mouse orthotopic tumor models. Upon in vitro infection with low MOI, virus spread was observed in 13/15 cell lines. Infection rates of > 95% were obtained within 14 days in 8/9 human and in 2/6 rodent cell lines. Different degrees of virus spread were detected in mouse intracranial human brain tumor xenografts. The therapeutic potential of an RCR vector harbouring the yeast cytosine deaminase gene was demonstrated after administration of clinically relevant doses of the prodrug 5-FC to vector-transduced glioma cells. In the immune competent mouse model carrying intracranial gliomas, overall survival time of treated animals was substantially longer than in control mice (56 vs. 16 days). Our data reveal that this gene therapeutic approach represents a versatile and promising option for the treatment of malignant brain tumors. However, further investigations on RCR vector biodistribution and shedding are mandatory.
Otolaryngology, ENT research Center , Iran
Session: Eye, brain, nerves, skin
Bone morphogenetic proteins (BMPs) belong to the Transforming Growth Factor (TGFβ) superfamily. The Bone Morphogenetic Protien (BMP) pathway in the ear regulates the hair-cell generation through regulated differentiation of hair-cell progenitors. BMP4 in early development of hair cells remains intriguing. Exogenous BMP4 was able to differentiate in hair-cell precursors. This study was carried out to evaluate effect of BMP4 on hair cell differentiated period. For the induction of progenitor cells, passage 3–5 mesenchymal stem cell (MSC) were trypsinized and transferred to coated-flask at 5 × 104 cells/ml. Cells were cultured for 5–7 days, and then cultured in serum-free medium composed of DMEM/F12 1:1 containing N2/B27 supplements. In experimental group for progenitor cell pre-induction, we used a combination of BMP4 (3 ng/ml), EGF (20 ng/ml) and IGF-1(50 ng/ml) for 2 weeks followed by the addition of bFGF (10 ng/ml) plus the other growth factors for an additional 2 weeks was N2. In control group BMP4 was omitted. The samples were obtained from experimental and control groups for semiquantitative RT-PCR & immunocytochimical studies. Expression of specific hair cell genes were evaluated in experimental group and compared with control group at the same time. In the cells at the end of the period cytoplasmic and nucleus hair cell markers (Brn3.c & Myosin VIIa) were showed by immunocytochimical technique.Analysis of gene expression shows that there was difference between genes expression in two groups.We found evidence that BMP4-treatment affacts differentiation period.
Improvement of dyspigmentated scar by multi-skin cells inclusive of melanocyte in hydrogel suspension
Session: Eye, brain, nerves, skin
Dyspigmentation, in the form of hyperpigmentation or hypopigmentation, is often a serious psychological problem for the self-image of patients. This is especially the case after a burn injury. The most common alteration in skin color occurs as the result of changes in the epidermal melanin. Therefore, the melanocyte reservoir is essential to obtain the repigmentation by medical therapies. To apply the cell therapy for dyspigmentaion due to burn and traumas, multi-skin cells co-culturing method was developed. In this study, keratinocyte, melanocyte and fibroblast were isolated from < 1 cm2 single donor site and co-cultured by regular ratio. Co-cultured multi-skin cells were suspended with hydrogel, gelled above 20°C and grafted on full-thickness wound of Balb/c nude mice. In contrast separately cultured cell, the co-cultured method was cultured without PMA and serum. Melanin synthesis of co-cultured melanocyte was detected by tyrosinase assay. The grafts were inspected 1 and 3 weeks after the application. At after 1week, on average 97.47 per cent of the neodermis was covered with a fragile regenerated epithelium. Result of immunohistochemistry, melanocyte was detected until 3weeks. This study suggests that multi-skin cells inclusive of melanocyte should be considered as an alternative method for treatment of dyspigmentaion due to burn and traumas.
San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute , Italy
Session: Eye, brain, nerves, skin
Intracerebral gene therapy for Globoid Cell Leukodystrophy (GLD) requires efficient distribution of the β-galactocerebrosidase (GALC) enzyme in the CNS. This currently requires multiple vector injections. In this work we targeted the external capsule (EC), a white matter region enriched in neuronal projections, with the aim of obtaining maximal transgene biodistribution from a single injection site. The use of bidirectional (bd)LV (bdLV.GFP.NGFR, mock vector; bdLV.GFP.GALC, therapeutic vector) allowed monitoring simultaneously transgene distribution and enzyme reconstitution. A single injection of either bdLV (2 × 106 total TU) in the EC of early symptomatic Twitcher mice resulted in robust transduction of neuronal and glial cells and relevant expression of the GALC protein in the bdLV.GFP.GALC-treated mice. Also, numerous GFP + ve projections ran rostrally and caudally to the injection site, in both brain hemispheres. While bdLV genome and transgenic GALC mRNA were detected only at the injection site, a detailed biochemical analysis showed relevant levels of a functional GALC enzyme in the brain and spinal cord of bdLV.GALC-treated Twitcher mice, with GALC activity globally rescued to 50–100% of wt levels. These results indicate robust synthesis and efficient transport of the GALC protein following a single intracerebral LV injection in a highly interconnected CNS region. This mechanism is amplified by cross-correction, as suggested by the presence of GFP-ve GALC + ve neurons. Histopathology performed at postnatal day 42 showed moderate decrease of activated astroglia and microglia. We are currently testing whether LV injection in the EC of neonates may delay/prevent neuropathology development.
A highly efficient laser based retinal gene delivery system
Session: Eye, brain, nerves, skin
Applied Virology and Gene Therapy, Georg-Speyer-Haus, Germany
Session: Eye, brain, nerves, skin
Malignant glioma is the most frequent primary brain tumor and still has a very poor prognosis despite advances in neurosurgical resection and adjuvant radio- and chemotherapy. A promising new approach is the use of vesicular stomatitis virus (VSV)-based systems for glioma-targeted oncolytic virotherapy and/or gene therapy. To improve glioma specificity and to abrogate the VSV G-mediated VSV-inherent neurotropism, we pseudotyped VSV with the LCMV glycoprotein (LCMV GP). Retargeting to glioma cells while sparing healthy neurons was successfully achieved in vitro with a panel of different brain tumor cell lines in comparison to primary neurons. Furthermore, we investigated different systems to address viral spread and microdistribution of LCMV GP-pseudotyped VSV within the tumor. Besides the generation of a replication-competent LCMV GP pseudotype rVSV(GP) as a pure virotherapy approach, we were able to show that tumor-infiltrating adult stem cells can be used as a delivery system and packaging cell line for non-cytopathic, replication-deficient VSV vectors encoding the HSV-TK as suicide gene. Efficacy of rVSVΔG-TK(GP) producing progenitor cells in mediating tumor killing after ganciclovir treatment was shown in vitro in a G62 spheroid model. If the reduced neurotoxicity can be confirmed in our current studies in vivo, rVSV(GP) would be an extremely promising candidate for oncolytic virotherapy of brain cancer.
Transfection of neural precursors using proneuronal genes increases the number of DARPP-32 positive neurons in vitro
Session: Eye, brain, nerves, skin
Unit of Gene Therapy and Stem Cell Biology, Jules-Gonin Eye Hospital , Switzerland
Session: Eye, brain, nerves, skin
Lentiviral vectors (LV) are among the most efficient gene transfer tools for both dividing and non dividing cells, and are, for instance, of particular interest to deliver a transgene to retinal pigmented epithelial (RPE) cells for the treatment of various retinal diseases, such as Retinitis Pigmentosa or Leber Congenital Amaurosis (LCA). One of the latest developments in the field of LV, which represent a significant biosafety advance, consists in the use of non-integrative LV (NILVs). These newly described tools were already used in various tissues, including the RPE. They allow prolonged transgene expression as far as transduced cells do not divide or divide slowly. However, they were also shown to induce transgene expression less efficiently than their integrative counterparts. Further investigations are thus needed to improve their potential. With the aim of developing an efficient and safe vector for gene transfer in the RPE, we used NILVs with different integrase mutations and compared their transduction efficiency and the residual integration frequency of the vector genome. In a second part of this work, we evaluated the use of these various vectors to transfer genes in vivo into the RPE of mice. Gene transfer was performed with Mokola pseudotyped NILVs both in newborn and in adult mice. We evaluated the efficiency of transgene expression and the specificity of the vectors for RPE cells. NILVs appear as suitable tools for gene transfer in the retina, particularly to transduce RPE cells. They can be advantageously used, for instance, to develop neuroprotective strategies aimed at rescuing photoreceptors from death in various retinal diseases or for the treatment of diseases directly affecting RPE, such as LCA.
Inserm U745, Faculty of pharmaceutical Sciences Paris, France
Session: Eye, brain, nerves, skin
Metachromatic leukodystrophy (MLD) is a lethal CNS demyelinating storage disorder due to the deficiency in arylsulfatase A (ARSA), a lysosomal enzyme involved in the degradation of sulfatides. Because of highly progressive neurological deterioration, patients with infantile form of MLD could benefit from rapid delivery of ARSA enzyme, through intracerebral injections of AAV encoding ARSA. Using an AAV5-ARSA vector, we demonstrated a correction of the MLD mouse phenotype. We further demonstrated in non-human primate that intracerebral delivery of AAV5-ARSA vector resulted in a significant spread of the expression and bioactivity of ARSA. Before engaging towards potential clinical application, we evaluated AAVrh.10 vector, recently shown to better diffuse in the brain than AAV5. We compared intra-striatal injections of AAVrh.10 and AAV5 in symptomatic MLD mice. AAV-rh.10 treated mice display a better diffusion and higher number of vector genome copy, stronger expression and diffusion of the ARSA and improved correction of the brain pathology. Despite AAV transduction is theoretically limited to neurons in vivo, we demonstrate in AAVrh.10-treated mice the presence of ARSA in oligodendrocytes and prove the correction of specific sulfatide accumulation in these cells as soon, as 2 months after injection. Using AAVrh.10 vector results in a substantial improvement of our gene therapy strategy. Final preclinical steps using a GLP-grade AAVrh.10 vector are currently on going.
Cell transplantation of mesencephalic neurons in a rat model of Parkinson's disease
Session: Eye, brain, nerves, skin
Parkinson s disease is a neurodegenerative disorder caused by a progressive loss of dopaminergic (DA) neurons. Exogenous cell replacement results in functional improvements but is limited by supply of donor cells and low survival of grafted cells. Further development of our previous work is focused on higher amounts of DA cells and their genetic modification to over-express neurotrophic factors. First, we produced SV40 large T antigen (SV40Tag) immortalized cell lines derived from embryonic ventral midbrain. Immortalized progenitor cells are nestin-positiv, and do not display markers for neurons or astrocytes. However, treatment with dbcAMP and GDNF induced differentiation into both cell types. Although this treatment induced low amounts of TH- and DAT-transcripts (markers for DA-neurons), no TH-immunoreactive cells were detected. Similarly, transplantation of SV40Tag cells into the dopamine-denervated rat striatum failed to induce DA differentiation. In a second attempt neural progenitor cells of the ventral midbrain were transfected with a non-viral EGFP expression plasmid, which resulted in high transfection efficiency and strong EGFP signal for at least 3 weeks in vitro. Furthermore, after grafting those cells into neurotoxin lesioned rat brains, a strong EGFP signal was also detected after 2 weeks in vivo. Current experiments in which EGFP is replaced by different neurotrophic factors will study their effects on differentiation and survival of DA cells.
Timmer Neurobiol. Dis.
Cesnulevicius Stem Cells
Session: Eye, brain, nerves, skin
Optimum wound healing requires a well-orchestrated interaction of growth factors and keratinocytes (KC). Keratinocyte function is impaired by factors from chronic wound environment. Stimulating factors secreted by adipose-derived stem cells (ASC) may reactivate keratinocyte function. We evaluated the effects of chronic and acute wound fluids on ASC and KC behavior. ASC/KC were harvested and chronic/acute wound fluids were extracted from standardized patient's wounds. Proliferation, migration and protein/gene expression patterns in ASC/KC were measured after the cell incubations with either CWF or AWF. Wound-healing competence of KC was measured in an in vitro wound-healing model. Total protein content in CWF and AWF was comparable (27,27 vs. 31,73 mg/ml). Both tested cell types showed changes in proliferation and migration behavior under the influence of AWF/CWF. Gene/protein analysis showed changes in VEGF, FGF as well as MMP2 + 9 expressions in both cell types. KCs competence to close a defined defect in vitro was impaired by CWF, which on the other hand stimulated migration rates of ASC and KC. In conclusion: both AWF and CWF affected the behavior of human ASC and KC in vitro. The subsequent decrease of KC migration and proliferation might be responsible for delayed wound healing in chronic wounds. ASC are not negatively influenced by CWF or AWF. In the future, growth factors expressed by transplanted ASC can possibly re-activate impaired KC function.
Effects of acetylcholinesterase overexpression in murine embryonic stem cells and on their differentiation
Session: Eye, brain, nerves, skin
It has long been acknowledged that acetylcholinesterase (AChE) also displays a large set of non-cholinergic functions. These functions have been attributed to both developmental and degenerative processes. Our previous gene and protein expression studies demonstrated that AChE and other components of the cholinergic system are expressed in murine embryonic stem cells (ESCs). To investigate the function of AChE during early embryonic neurogenesis, we examined the expression of this protein and its role in proliferation and differentiation of murine ESC cells into neural precursor cells (NPC). We stably transfected the D3-stem cell line with a plasmid encoding the synaptic form of mouse AChE. Cells from the same passage were also transfected with a mutated form of AChE (which leads to the ER retention of the cholinesterases) or GFP and used as experimental control. We examined at the stem cell stage parameters as cell vitality and proliferation. AChE overexpression led to a significant decrease of cell proliferation, but didn't affect cell vitality. Stably transfected cells were differentiated toward neural precursor cells following an ITSF protocol and their differentiation was compared with differentiation of control cells. Expression of stem cell and neuronal markers was examined by RT-PCR, qPCR and immunohistochemistry. Interestingly, the AChE stem cells expressed all tested stem cell markers for an extended period during the differentiation protocol. We also found that AChE overexpression led to a more efficient differentiation of stem cells into neuronal cell type, by comparing expression of nestin, beta3-tubulin, N-CAM.
Department of Human Genetics, University of Aarhus , Denmark
Session: Eye, brain, nerves, skin
Age-related macular degeneration (AMD) is the primary cause of vision loss in the Wester World, and increased levels of vascular endothelial growth factor (VEGF) is a key factor in the disease development. In order to inhibit VEGF production in the eye we are developing AAV2/8-vectors with shRNA targeting the VEGF gene. The efficacy of ten different shRNAs was analyzed in transfected RPE cells, and three of these revealed potent silencing of VEGF. On this basis a microRNA cluster for expression of multiplexed anti-VEGF RNAs is being engineered. The efficacy AAV-vectors are analyzed in a pig model following intravitreal administration. Studies of the potential shedding of the AAV-vectors show the presence of vector genomes in injected eyes only, whereas uninjected eyes and other organs (liver, kidney, skin, brain, lung, muscle and heart) were found not to contain traces of AAV. Finally, trafficking of intravitreal administered AAV is investigated in isolated pig eyes.
Keratinocytes from different compartments of human skin-defining conditions for stem cell protection
Session: Eye, brain, nerves, skin
Transplantation concepts for chronic wounds and burns using a high portion of quality controlled autologous keratinocyte stem cells (SC) would deliver clonogenic cells with high proliferative potential and unlimited life span. Up to now, no concept exists which will deliver defined subsets of keratinocyte SC, nor are the culture conditions for isolation, serial propagation, and protection of stem cell properties defined. We isolated keratinocytes (KC) from different compartments of human skin (epidermal basal membrane (BM), outer root sheath (ORS), scalp skin (SS)). Cell adhesion, cell kinetics, expression of SC-markers, label retention as well as clonal analysis were investigated during serial culture in dependence on different keratinocyte media (DMEM/F12, KGM, KGM-2, CnT57/07), serum, feeder cells, and coating substrates. Follicular KC from SS showed highest proliferation and holoclone expansion compared to BM- and ORS-KC; however, lost their SC-properties already during early passages. Whereas addition of serum and feeder cells accelerated differentiation and dramatically reduced proliferation and clonality, defined KC-media as well as fibrin as coating substrate may protect SC properties and enhance proliferative capacity. Follicular KC from SS seem to be a favourable cell source to deliver adult SC for reconstruction of epidermal defects by tissue engineered concepts.
Molecular Medicine, Catholic University Leuven , Belgium
Session: Eye, brain, nerves, skin
Human NT2 neural precursor-derived tumor-infiltrating cells and glioblastoma gene therapy
Session: Eye, brain, nerves, skin
Engrafted mouse neural stem and precursor cells (NSCs) or primary and immortalized NSCs isolated from human brain tissues are capable of spreading through the existing migratory pathways in healthy brain as well as non-typical routes when gliomas are present. These NSCs are able to deliver a therapeutic gene to tumor sites distant from the NSC implantation site, suggesting a powerful therapeutic approach of using NSCs to target the main tumor mass, tumor outgrowths and even distant tumor microsatellites. In an attempt to identify a cell source for human NSCs other than human brain tissues, we tested whether the well-established human NT2 cell line, which share many characteristics of neural precursor cells, can be used to derive a cellular vehicle with glioma tropism. After treating NT2 cells with retinoic acid for 2 weeks, we isolated using Boyden chambers a group of NT2 cells that migrated toward human U87 glioblastoma cells. In mice, these cells could home in on intracranial U87 glioblastoma xenograft following systemic administration into the tail vein. To test the feasibility of using these tumor-infiltrating cells for targeted glioma therapy, we introduced the herpes simplex virus thymidine kinase gene into the cells and injected them into the brain side contralateral to a site pre-inoculated with U87 cells. Following ganciclovir injection, we observed inhibition of tumor growth and prolonged survival of tumor-inoculated animals. NT2 cells are stable, easy to cultivate and amenable to scale-up for cell production, thus holding great potential to produce a cellular delivery vehicle with good manufacturing practice quality.
Nuclear Medicine, Medizinische Hochschule Hannover, Germany
Session: Eye, brain, nerves, skin
Center of Advance Studies in Veterinary Physiology, Indian Veterinary Research Institute , India
Session: Heart and vessels
Most of the embryonic stem cell lines have been derived from inner cell mass of blastocyst and have been extensively studied in different aspects including generation of organ specific cells. However no detailed studies have been made on cardiac specific gene expression, immunohistochemical and electrophysiological characterisation of cardiomyocytes generated from morula derived embryonic stem cells in mice. In the present study, five new embryonic stem cell lines were derived from very early stage embryos of 129SV/Pass strain of mice. In vitro differentiation of cell line-1 and 2 readily generated cardiomyocytes, which expressed different cardiac specific genes viz GATA-4, α-MHC, β-MHC, cTnT, Nkx2.5 and MLC2 v in a temporally regulated manner. In cell line-1, GATA-4 and Nkx2.5 expression were started on Day-4, β-MHC and MLC2 v from day-5 onwards while α-MHC, cTnT on Day-7 onwards. In cell line-2, GATA-4 was expressed on day-5 onwards, while α-MHC, β-MHC, cTnT were expressed on day-7 onwards. MLC2 v was expressed on little later of differentiation i.e. Day-8 onwards. The cardiac cells also expressed sarcomeric proteins and showed normal electrophysiological properties. The results indicated that cardiomyocytes could be generated from ES cells derived from early stage embryos and can be transplanted for cardiac regeneration.
Department of Molecular Medicine, A.I.V Institute University of Kuopio , Finland
Session: Heart and vessels
Adventitial vasa vasorum plays a role in the pathogenesis of vascular occlusive diseases. Vascular endothelial growth factors (VEGFs) and their receptors are key molecules in the regulation of angiogenesis and lymphangiogenesis. VEGF-E is a group of proteins encoded by several Orf viruses that binds specifically to VEGF receptor (VEGFR)-2. VEGF-ENZ7 is 22.7% and 27.3% identical to human VEGF-A165 and placental growth factor (PlGF)-1 respectively, but is reported to have a high antigenicity in experimental animal. Novel VEGFE/PlGF chimera genes have recently been developed with VEGF-E functions and reduced antigenicity. Using an established model of periadventitial gene delivery, we performed VEGF-E chimera gene transfer to the periadventitial spaces of rabbit carotid arteries and investigated the role of VEGFR-2 in adventitial angiogenesis and neointimal formation.
Angiogenesis laboratory, Russian Cardiology Research Centre , Russia
Session: Heart and vessels
Recently it has been established that a heart contains a reservoir of stem and progenitor cells (c-kit + , sca-1 + , isl-1+) having the capability for differentiation toward the vascular and cardiac lineages and showing cardiac regeneration potential in vivo after administration in the infracted myocardium. The aim of the present study is to characterize cardiac stem cells (CSC) in the tissue of human chronic left ventricular aneurysm. We analyzed 15 samples of aneurysm tissue immunohistochemically and by flow cytometry. The amount of c-kit positive cells was about 3300/cm3 of tissue. Co-staining experiments demonstrated that c-kit positive cells were of non-hematopoietic origin, since they didn't express CD34 or CD45. Flow cytometry confirmed that c-kit positive cells in aneurysm tissue were largely CD34-, and also negative for a cocktail of blood lineage markers Lin1. However, we identified two populations of c-kit positive cells: c-kit + CD45 + (about 60%), which might populate the heart via circulation, and c-kit + CD45-, which might contain distinct human resident CSC. C-kit positive cells localized mainly nearby large vessels. Majority of c-kit positive cells expressed another stem cell marker MDR1, though they were negative for marker of cell proliferation Ki67, nor did they express markers of differentiation in cardiac (Nkx 2.5, Gata-4, Mef2c, alpha-actinin), endothelial (Ets1, CD105, vW) and smooth muscle (Gata6, SMA) lineages. About 20% of c-kit positive cells expressed cycline-dependent kinase inhibitor p21. Using magnetic cell sorting c-kit positive cells could be successfully isolated from aneurysm tissue and expanded in vitro. It thus appears that aneurysm tissue could be an alternative source of autologous CSC.
Herz und kreislaufphysiologie, University of DÜsseldorf , Germany
Session: Heart and vessels
We have analyzed the potential of unrestricted somatic stem cells (USSCs) to differentiate into cardiomyocytes in culture and investigated the fate of USSCs after transplantation into rat heart in-vivo. After 7 days of coculture with neonatal rat cardiomyocytes, expression of cardiac specific transcription factors (GATA4 & Nkx2.5) and cardiac markers (TnT & [alpha]-actinin) were found. FACS showed 43% (n = 3) of the cells acquired a cardiac phenotype and some USSCs contracted spontaneously and synchronously. In a next step we delivered eGFP + USSCs transcoronarily into immunosuppressed rat hearts via a minimal invasive catheter-based technique. We found 79% (n = 4) of the cells deposited in the myocardium 2 hours after transplantation and USSCs adhered to the vascular wall and migration across the endothelial barrier was observed. After 7 days, 19.8 % (n = 5) of the transplanted eGFP + USSCs were well integrated into the host myocardium. USSCs were morphologically elongated and expressed cardiac specific markers, while some eGFP + USSCs were stained positive for smooth muscle actin and constituted the vascular wall. In addition, we found some cells stained positive for activated-Caspase 3 (apoptosis) and paralleled by the massive infiltration of CD11b + cells into the myocardium. After 21 days only a small fraction of USSCs were found in the myocardium (0.13%, n = 4), however, the remaining cells clearly exhibited a sacromeric structure ([alpha]-actinin) similar to mature cardiomyocytes. Identical results were obtained in nude rats. In summary, USSCs can differentiate into beating cardiomyocytes, after coronary transplantation in-vivo, however, long term survival of differentiated USSCs was rather low despite a high initial fraction of trapped cells.
GATA transcription factors are essential for angiogenesis in vitro
Session: Heart and vessels
Six GATA transcription factors were identified in vertebrates (GATA 1–6). GATA factors are important regulators of basal and stress-induced gene-expression in different cells and tissues. Although the expression of endothelial genes like EDN1 (endothelin 1), Pecam1 and Nos3 were previously reported to be regulated by GATA factors, the expression patterns and function of GATA factors in endothelial cells are still mostly unknown. In primary human venous (HUVEC), arterial (HUAEC) and cardiac microvascular endothelial cells (HCMEC) GATA2 and GATA6 are the most abundant GATA factors (Real-time PCR, Western blot). Stimulation of HUVECs with endothelial growth medium (EGM) resulted in nuclear translocation of GATA2 and GATA6. Upon EGM stimulation, ChIP assays from HUVECs revealed enhanced GATA6 binding of the EDN1, Pecam1 and Nos3 promoters. Knock-down of GATA2 or GATA6 in HUVECs and HCMECs resulted in markedly impaired capillary-like tube formation, endothelial cell proliferation and migration. Antithetically, adenoviral overexpression of GATA2 or GATA6 in HUVECs and HCMECs lead to enhanced cell migration. Interestingly, tube formation was only enhanced by GATA2 or GATA6 overexpression in HCMECs, but not in HUVECs.
Cardiovascular Research Group, The University of Manchester , UnitedKingdom
Session: Heart and vessels
Cardiovascular repair mechanisms become impaired with age resulting in an increased susceptibility towards vascular pathology. Endothelial progenitor cells (EPCs) have gained particular interest in the field of regenerative biology as they play a key role in vascular repair. Cell surface heparan sulphate (HS) proteoglycans, which are essential for EPC function, undergo age-associated structural changes. We aim to establish if structural alterations of HS on EPCs underlie the impaired function of these cells in the ageing vasculature. The number and function of EPCs in patients with systemic lupus erythematosus (SLE), an autoimmune disease associated with accelerated vascular ageing, was compared to age-matched healthy controls (52 patients and 30 controls; mean age 52 and 50 years respectively). To enumerate EPCs, peripheral blood mononuclear cells were labelled with CD133 and CD34 and analysed by flow cytometry. The formation of colony-forming units (CFUs) after 7 days in culture was used as a measure EPC function. The overall mean percentage of EPCs in SLE was similar to controls. However, an impairment in EPC function with vascular ageing was evident from the significantly reduced mean number of CFU [7(SD = 5) vs 17(SD = 18), P = 0.01], and fewer large CFU [17% vs 40%; P < 0.05], in patients compared with controls. Current work is focused on the molecular mechanism underlying the deterioration of EPC function. Differences in HS structure, as determined by high performance liquid chromatography and flow cytometric analysis, on EPCs isolated from young and old healthy subjects are being investigated. How such changes affect EPC function including migration, proliferation and integration into vascular structures will be assessed.
Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO, Hannover Medical School , Germany
Session: Heart and vessels
Medizinische Klinik I, Klinikum Grosshadern, LMU, Germany
Session: Heart and vessels
Angiopoietin-2 (Ang-2), a co-factor in postnatal angiogenesis interacting at the endothelial Tie-2 receptor, is a potential target for therapeutic angiogenesis. The impact of long lasting Ang-2 expression on cardiac function has not been characterized. We analysed the impact of chronic Ang-2 overexpression and interaction with Ang-1 on myocardial morphology and function using a AAV2.9-vector overexpressing Ang-1 (rAAV-Ang-1).
Determination of cardiotoxicity by verapamil and valproic acid on human embryonic stem cells derived cardiomyocytes
Session: Heart and vessels
Human embryonic stem cells (hESCs) can be differentiated into specific cell types, including cardiomyocyte like cells. These cells express cardiac-specific markers. In addition, hESC-derived cardiomyocytes have the potential to be extremely useful in various in vitro applications and to provide the opportunity for using them as an effective tool for drug screening. Verapamil, an L-type calcium channel blocker is used in the treatment of hypertension, angina pectoris, and cardiac arrhythmia and does not appear to be significantly teratogenic. Whereas, Valproic acid, an anticonvulsant, used to control seizures and juvenile myoclonic epilepsy was found to be teratogenic. In this study we used these drugs in different concentrations for treatment. We show that cardiotoxic drugs in different concentrations affect the beating frequency of hESC derived cardiomyocytes. It has been recommended that activation of genes and signaling pathways is an early response of the heart to toxic abuse, and that examination of these genes and pathways will facilitate an understanding of cardiotoxicity. Studies indicate that measuring changes in gene expression could assist in the ranking of compounds before they are tested further during drug discovery. The objective of these studies were to determine the effect of various doses of these drugs on expression of cardiac markers.
Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO, Hannover Medical School , Germany
Session: Heart and vessels
G.K. and I.G. contributed equally
Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine , Germany
Session: Heart and vessels
The generation of functional somatic cells from pluripotent cells is a critical step in the development of cell-based regeneration therapies. Recently, we have obtained germline-derived pluripotent stem (gPS) cells from adult mouse unipotent germline stem cells. The objective of our study was to test whether functional endothelial progenitor cells (EPCs) suitable for the promotion of angiogenesis and arteriogenesis in ischemic diseases can be derived from this new source of pluripotent stem cells. EPCs were derived from gPS cells. Using fluorescence-activated cell sorting (FACS) analysis it was demonstrated that the cells expressed the endothelial cell-specific markers PECAM-1 (CD31), von Willebrand Factor, Tie2, Flk1, and vascular endothelial-cadherin. These results were confirmed by immunofluorescence staining and RT-PCR. The cells were successfully maintained in in-vitro culture for many passages, without a significant loss in expression of the endothelial cell markers. Furthermore, the cells were capable of forming capillary-like structures when cultured on Matrigel. Dil-conjugated acetylated low-density lipoprotein uptake confirmed the functionality of the gPS-derived EPCs. Furthermore, the integration of transplanted gPS cell-derived EPCs into growing collateral arteries in a mouse hindlimb ischemia model evaluated their functionality in vivo. Our results provide evidence that EPCs can be obtained from gPS cells, which display characteristics typical of endothelial cells. Therefore, gPS cells can serve as a new source of stem cells for endothelial cell-based cell therapies, and their use would not be limited by the ethical considerations associated with the use of embryonic stem cells.
Institute of Neurophysiology, University Hospital of Cologne , Germany
Session: Heart and vessels
Institute of Neurophysiology, University Hospital of Cologne , Germany
Session: Heart and vessels
Department of Cardiology, University of Munich-Grosshadern , Germany
Session: Heart and vessels
Faculty of Basic Medicine, Moscow Lomonosov State University , Russia
Session: Heart and vessels
Critical lower limb ischemia can be treated by intramuscular injections of naked plasmid DNA encoding vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). Previous data suggests that HGF and VEGF may have an additive effects in vitro. We report that these factors amplify angiogenesis in ischemic skeletal muscle after plasmid co-transfer using a novel PC4 W vector. Constructed phHGF and phVEGF expression activity was tested in vitro using western-blot and ELISA. In vivo tests were performed using hind limb ischemia model in C57/Bl6 male mice (n = 56). Animals were randomly assigned to groups treated with phHGF, phVEGF, combination of phHGF + phVEGF. Controls were sterile saline, pβ-gal and empty PC4 W. Limb perfusion was assessed using Laser Doppler on days 0, 7, 14 and 21. Reverse-transcription PCR using specific primers was used to show transcript expression in skeletal muscle up to 14 days after injection. Both phHGF and phVEGF had higher perfusion in all time points compared vs NaCl (p < 0.05). Mice from phVEGF group at D21 had higher perfusion than pβ-gal (55.3 ± 4.9% vs 33.8 ± 5.1%; p = 0.02) and PC4 W (55.3 ± 4.9% vs 37.4 ± 4.2%; p = 0.04).Mice from pHGF +pVEGF group had higher perfusion than phHGF solely at D7 (44.9 ± 6.2% vs 25.4 ± 4.0%; p = 0.02), D14 (64.5 ± 3.4% vs 50.7 ± 5.7%; p = 0.04) and D21 (70.5 ± 2.9% vs 50.9 ± 5.1%; p = 0.03). Perfusion in pHGF + pVEGF group was higher compared with phVEGF at D14 (64.5 ± 3.4% vs 47.7 ± 4.3%; p = 0.01) and D21 (70.5 ± 2.9% vs 55.3 ± 4.9%; p = 0.05). No significant edema was observed in phVEGF group. Given data suggests that novel plasmid constructs with phVEGF and phHGF render significant angiogenic effect in ischemic muscle and that co-administration of phHGF and phVEGF can amplify the angiogenic effect.
Department of Heart Surgery, University of Rostock , Germany
Session: Heart and vessels
MicroRNAs (miRNAs) modulate stem cell development pathway. The aim of this study is to investigate which miRNA is essential and could influence the endothelial progenitor cell lineage decision during the human CD133 + cell differentiation to mature endothelial cells. Human bone marrow derived CD133 + cells were differentiated into mature endothelial cells, which was confirmed by CD31 expression and AcLDL uptake. The miRNA expression profiling from undifferentiated and differentiated CD133 was analyzed by real-time PCR and QuantiMir MicroRNA qPCR array. The differentiated cells revealed over 2-folds downregulation of the miRNAs: 200c, 23b, 122a, 125a, 125b, 126, 132a, 132b, 155, 181a, 181c and 206. Only the miRNA 92 was significantly upregulated. In conclusion, the investigated miRNAs might take part in the regulation of endothelial differentiation of CD133 + stem cells. It is of great therapeutically importance to further determine the direct influence of miRNA on the endothelial differentiation pathway and the combination of different miRNAs.
Improvement of stem cell biodistribution in myocardial restoration. A quantitative analysis using fluorescent microspheres
Session: Heart and vessels
Successful stem cell delivery and optimized biodistribution are critical factors for efficient stem cell therapy. We used fluorescent microsphere particles to simulate cell retention and biodistribution after cardiac regenerative therapy. Viscose carrier substances were evaluated to augment particle retention. Fluorescent microspheres (5,0 × 105; d = 10 um) were transplanted into 21 mice hearts in 4 groups (A =injection in PBS into explanted hearts; B-D = injection after LAD ligation in situ; B = PBS; C = matrigel; D = fibrinogen). Ten minutes after injection hearts were explanted and fluorescent bioimaging was performed using a Xenogen IVIS Lumina-System. Hearts were then homogenized, filtered and aliquots were analysed using fluorometry and particle counting. Compared to ex situ microsphere injection, the in situ delivery in PBS showed a 90% decline in particle retention (3,2 × 105 vs. 0,3 × 105; p < 0,01) indicating immediate loss during injection, mainly through venous drainage into the lungs. Particle retention could be improved using matrigel (0,7 × 105; p = 0,07) or fibrinogen (1,1 × 105; p < 0,05) as a carrier substance. Most adequate quantitative values were obtained by macroscopic fluorescence bioimaging and counting-chamber analysis (p < 0,05).We introduce a reliable tool to analyse retention and biodistribution of cell-like particles after injection into infarcted myocardium. Beating heart application techniques are associated with a massive loss of fluorescent microspheres. Fibrinogen improves cardiac microsphere retention and should be considered the carrier of choice for clinical intramyocardial stem cell delivery.
Rhesus monkey derived “cardiospheres” (CSps) for myocardial restoration in a mouse infarct model
Session: Heart and vessels
Resident cardiac stem cells have been isolated and expanded in vitro. They form large cell clusters termed “cardiospheres” (CSps), differentiate into cardiomyocytes in vitro and might be a promising autologous cell source for cardiac restoration. The aim of this study was to determine cell survival and functional effects of Rhesus monkey cardiosphere derived cells (CDCs) in a mouse myocardial infarction model (MI). Cardiospheres derived from Rhesus monkeys were expanded in vitro and either CDCs (1,0 × 106) or placebo were injected intramyocardially into immunodeficient SCID beige mice after LAD ligature (each group N = 10). All animals underwent conductance catheter (CC) evaluation to determine functional cardiac outcome (each group N = 5). LAD ligature resulted in a significantly reduced myocardial function compared to non-infarct controls (LV-EF 64% vs. 23%, p < 0,05). CDC grafts could be identified in all animals after cell transplantation. Cell proliferation was detectable until one week as determined by immunohistochemical staining for Ki67. The characterization of the CDC grafts (e.g. cardiospecific markers) is currently under investigation. Functional parameters determined by CC evaluation tended to improve with cell therapy after 4weeks (LV-EF placebo 21% vs. LV-EF CDC 30%; n.s.).For the first time, Rhesus CDCs have been used to restore infarcted myocardium in an animal model. The cells proliferated in vivo, survived until 4 weeks after grafting and apparently improved heart function as determined by conductance catheter evaluation. Cardiosphere derived cells appear to be a promising cell source for myocardial regeneration. Further investigation is warranted to develop therapeutic applications in patients.
FLK1+ progenitor cells derived from mouse induced pluripotent stem (iPS) cells for cardiovascular repair
Session: Heart and vessels
Induced pluripotent stem (iPS) cells are a novel source of stem cells obtained from mammalian somatic cells by reprogramming the genome to a pluripotent state. These iPS cells are almost indistinguishable from embryonic stem cells and offer the possibility to generate autologous patient-specific pluripotent stem cells. The aim of the present study was to characterize the potential of murine iPS cell-derived FLK1 + progenitor cells to give rise to cardiovascular cell types in vitro and in vivo. FLK1 + progenitor cells were FACS-sorted from differentiating murine iPS cells and either reaggregated and further cultivated on gelatin-coated dishes or injected into the acute ischemic hearts of LAD-ligated SCID-beige mice. Cultivated murine iPS cell-derived FLK1 + progenitor cells gave rise to self-beating cardiac troponin T expressing cardiomyocytes, α-smooth muscle actin positive cells and CD31 expressing endothelial cells. Using an iPS cell clone ubiquitously expressing the YFP variant, Venus, in the nucleus we were able to detect cell derivatives of transplanted FLK1 + progenitor cells in infarcted myocardium of SCID-beige mice. The characterization of these cell derivatives and possible effects on cardiac function is currently under investigation. In conclusion, iPS cell-derived FLK1 + progenitor cells are able to differentiate into cells of cardiovascular lineages. This source of cells might therefore be used for cardiovascular regeneration approaches.
Institute of Neurophysiology and CMMC, University of Cologne , Germany
Session: Heart and vessels
Heart failure due to the loss of functional cardiomyocytes is one of the most frequent cardiovascular diseases. Understanding the genetic network that leads to functional cardiomyocytes is the first step to develop future therapies. A global transcriptome analysis yielded up-regulated genes in cardiomyocytes, which were derived from murine embryonic stem cells (Doss et al., 2007). We were interested in a fast screen for the functional role of transcripts with unknown function (TUFs) for an intact activity of the heart. Therefore we searched for homologues in the zebrafish genome and performed a morpholino-based knockdown approach. We tested 11 TUFs of the zebrafish and found most of them to be expressed in the cardiovascular system. Morpholino-oligonucleotide injections caused highly specific cardiovascular defects in the majority of them such as altering of heart morphology, vascular defects or accumulation of blood cells to a different extent and penetrance. This pilot approach thus shows the potential of the zebrafish to identify TUFs in the cardiovascular system.
Department of Biotechnology and Molecular Medicine, AIV Institute and Ark Therapeutics Oyj , Finland
Session: Heart and vessels
Vascular endothelial growth factors are considered as key molecules inducing angiogenesis and lymphangiogenesis during embryogenesis and maintaining vasculature in adults. Because of their importance in angiogenesis, VEGFs have been suggested as potential therapeutic agents to improve compromised blood flow. In our previous studies, we found out that the substitution of Cys-25 residue at the dimer interface of mature VEGF-D by various amino acids enhanced the formation of disulfide-linked dimers and improved the activity of the mature VEGF-D. To further characterize our most potent mutant, C25L, we performed receptor affinity measurements, phosphorylation assays and protein stability tests. The results showed that C25L mutant had slightly increased affinity to VEGFR-2 and VEGFR-3 and correspondingly it also induced receptor phosphorylation at lower concentrations. In addition, C25L mutant was highly stable compared to the native VEGF-D. Our preliminary in vivo studies showed that the C25L mutant did not induce acute vascular permeability which is a major side effect of VEGFs when used to induce therapeutic vascular formation. To conclude, we have created a novel VEGF-D variant, C25L, with increased stability and enhanced receptor activation properties. The increase in stability and efficacy could be useful in gene therapy when sustained angiogenic stimulus is required.
Department of cardiac surgery, University of Rostock , Germany
Session: Heart and vessels
Medizinische Klinik I, Klinikum Grosshadern, LMU, Germany
Session: Heart and vessels
We have used murine embryonic endothelial progenitor cells (eEPCs, Tie-2+, c-Kit+, Sca-1+, Flk-1 low, MHC-1−) in a pig model of acute ischemia (I, 1h LAD occlusion)/reperfusion (R, 24 h). When 5 × 106 eEPCs were infused retrogradely at the end of ischemia (at 55 min), acute I/R injury was significantly attenuated, unless Wortmannin was co-applied (Kupatt et al., Circ 2005). Screening the eEPC transcriptome for paracrine factors, which are capable of activating PI3K-AKT, Thymosin ß4 (TB4) was identified. Indeed, retrograde application of TB4 peptide was diminishing infarct size to a similar extent as the eEPC retroinfusion (Hinkel et al., Circ 2008). In a different pig model of chronic hibernating myocardium (reduction stent implantation), regional myocardial dysfunction depends solely on malperfusion, not infarcted myocardium. Since eEPC retroinfusion improved hibernating myocardium significantly, we tested the paracrine potential of forced TB4 overexpression to induce therapeutic neovascularization, utilizing a current cardiomyotropic AAV 2/9 vector for longterm expression. The AAV 2/9 TB4 vector or a LacZ vector was retroinfused at the 4 week timepoint. At 8 weeks, collateral count and perfusion were significantly improved in the AAV 2/9 TB4, sufficing to improve myocardial function of the hibernating region. We conclude that an AAV 2/9 based gene therapeutic approach utilizing paracrine effectors of progenitor cells, e.g. TB4, may complement cell therapy in cases where viability, homing or survival of progenitor cells from patients with manifest atherosclerosis are limited.
Medical Biochemistry, Max F. Perutz Lab.-Medical University of Vienna, Austria
Session: Cardiovascular system
Somatic stem cells of the heart have been isolated in past years by several groups using different strategies. These cell populations had different morphology but in common that they differentiate only to cells residing in the heart and that they could not be maintained as clonal stable cell lines so far. We have isolated and cloned stable cardiac stem cell lines from murine hearts and demonstrated that they express stemness genes such as Oct4, Nanog, Sox2, and Tert1, and marker genes for primitive mesoderm and early cardioblasts, such as Brachyury, Mesp1, Desmin, Nkx2.5, Mef2C, and Isl1 at the same time. They self-renew in a LIF dependent manner for at least 87 passages without loosing their potential to spontaneously differentiate to cardiomyocytes, smooth muscle cells and endothelial cells. Cardiomyocytes develop to mature pacemaker cells and atrial and ventricular cardiomyocytes expressing Connexin 43. Bmp2 promotes early cardiomyogenesis via activation of the Nkx2.5 gene, whereas LIF inhibits cardiomyogenesis in cardiovascular progenitor cells. Our results demonstrate successful isolation and long-term culture of clonal cardiovascular progenitor cells within a micro-environment accurately recapitulating a minimal stem cell niche.
Dept. of Cardiology, University Hospital Heidelberg , Germany
Session: Heart and vessels
The dystrophin protein plays an important role in muscle contraction linking the intracellular cytoskeleton of striated muscle cells to the extracellular matrix. Mutations in the gene that encodes dystrophin can lead to a complete loss of the protein causing Duchenne muscular dystrophy (DMD) frequently associated to severe cardiomyopathy. Mutations that permit production of a truncated, less functional protein are usually associated to the milder Becker muscular dystrophy clinical phenotype. While clinical phase-I-studies for therapy of skeletal muscle are performed, trying to convert DMD in the milder Becker form, therapies for cardiac muscles have barely been evolved. The aim of our study is to establish an efficient long term treatment for DMD-associated cardiomyopathy in a mouse model for dystrophin-deficiency (mdx). Mdx mice were treated with a microdystrophin-cDNA (mDys), delivered by adeno-associated-viral vectors (AAV9) specifically into cardiac muscle. mDys, under the control of the CMV or CMV enhanced myosin light chain promoter (CMV-MLC), was inserted into AAV9 and 4xE11 or 1xE12 viral genomes were administered to mdx mice via tail vein injection. Four weeks after injection, heart and quadriceps femoris muscle (MQF) were dissected and analysed by immunofluorescence for expression of mDys. We found (1) a maximum transduction-efficiency of 60% of all cardiomycytes and (2) no expression of mDys in the MQF. This demonstrates a high specificity and efficiency of the chosen therapy for cardiac muscle.In conclusion, we established an efficient and specific AAV9-mediated gene transfer of microdystrophin-cDNA in mdx hearts, which may represent a promising tool to develop treatment strategies for DMD-associated cardiomyopathy.
Clinic for Cardiac, Thoracic, Transplant and Vascular Surgery, LEBAO, Hannover Medical School , Germany
Session: Heart and vessels
Induced pluripotent stem (iPS) cells and their dervatives may represent a favoured cell source for future regenerative therapies. In this study we analysed cardiomyocytes that were differentiated from human cord blood derived induced pluripotent stem cells (CBiPS-CM) in comparison with human ES cell derived cardiomyocytes (hES-CM). RT-PCR analyses of typical cardiac markers like Nkx2–5, TBX5, TBX20, MLC2a, αMHC as well as immunostainings for the cardiac proteins α actinin, αMHC, troponin t and CX43 revealed similarities of CBiPS-CM to hES-CM. qRT-PCR for αMHC show comparable efficiencies of cardiac differentiation between CBiPS-CM and hES-CM. Important for their potential use in pharmacological screening systems, the level of hERG mRNA relative to αMHC expression was higher in CBiPS-CMs than in hES-CMs and similar to human ventricular myocardium. The functional coupling of the CBiPS derived CM and the existence of a functional sarcomeric reticulum in the CBiPS-CM was demonstrated by measurement of Ca2 + transients and caffeine-sensitive Ca2 + storage. Multi-electrode array (MEA) and patch clamp experiments show a cardiotypic phenotype with ventricle- and pacemaker-like cells as well as sensitivity to isoproterenol and E-4031. We were able to demonstrate the functionality of differentiated human iPS cell derivatives: CBiPS cells are capable of generating CMs, which are functionally comparable to their ESC-derived counterparts. Our results establish an important basis for the development of iPS cell-based therapies with high relevance for the treatment of pediatric patients.
Derivation and functional characterization of α-smooth muscle actin expressing cardiomyocytes derived from mouse embryonic stem cells
Session: Heart and vessels
Alpha-smooth muscle actin (Acta2) is transiently expressed during the cardiovascular development in early mouse embryos. In the adult, Acta2 is expressed in smooth muscle cells and not in cardiomyocytes. To date, the nature and phenotype of the cardiomyocytes expressing Acta2 during the early stages of development have not been characterized. This is mainly due to the practical difficulties in isolating Acta2 + expressing cardiomyocytes during early embryonic development in vivo. One exciting approach to identify and characterize early Acta2-expressing cardiomyocytes is offered by murine ES cells. We have developed a novel approach for the derivation of the Acta2 + cardiomyocytes from transgenic Acta2 ES cell line. Applying a specific embryoid body (EB) differentiation protocol, functional beating Acta2 + cardiomyocytes expressing several genes and proteins characteristic of cardiomyocytes, as detected by semi-quantitative RT-PCR and immunostainings could be isolated from Acta2 ESCs. Acta2 promoter-driven EGFP expression allowed us to monitor the onset of Acta2 expression, which occurred in parallel with the appearance of early beating cardiomyocytes. Addition of puromycin at this time point resulted in a high yield of cardiomyocytes. Patch-clamp electrophysiological characterization demonstrated that over 75% of the Acta2 + cardiomyocytes displayed ventricular-like action potentials similar to the fetal ventricular cardiomyocytes and responded similarly to muscarinic receptor analogue carbachol. This is the first report showing the differentiation of functional early Acta2 + ventricular-like cardiomyocytes and also Acta2 + SMCs from the same Acta2 ESC line.
Department of Biotechnology and Molecular Medicine, AIV Institute and Ark Therapeutics Oyj , Finland
Session: Heart and vessels
VEGFs are considered as potential therapeutics to induce blood vessel formation when normal blood flow is compromised. The mature form of VEGF-D has been found to be one of the strongest growth factors for inducing angiogenesis by adenoviral gene transfer. Most VEGFs exist as covalently bound dimers, but the mature VEGF-D is predominantly a non-covalent dimer even though the cysteine residues forming the intersubunit disulfide bridges in the other VEGFs are also conserved in VEGF-D. We studied the structure of VEGF-D protein by homology modeling and mutational studies. Our model revealed that VEGF-D bears an extra cysteine residue (Cys-25) at the dimer interface, located close to cysteines forming the intersubunit disulfide bridges in other VEGFs. We hypothesized that this extra cysteine residue may hinder the formation of the intersubunit disulfide bridge, and the three cysteines at the subunit interface were mutated to alanines. The two conserved cysteines were found to be essential for the function of VEGF-D, but the substitution of Cys-25 was found to stabilize the dimer and improve activity. In further analysis, substitutions to hydrophobic amino acids Ile, Leu and Val, equivalent to those found in other VEGFs in homologous position, affected most favorably the activity of VEGF-D. These VEGF-D variants with improved biological activity are potential therapeutics for ischemic diseases administered either as recombinant proteins or using gene therapy vectors.
Small interfering RNA-directed knockdown of MHC6 causes loss-of-function of cardiogenesis in EB derived from mES cells
Session: Heart and vessels
Embryonic stem (ES) cells are well recognized as stable lines of undifferentiated pluripotent cells isolated from early mouse embryos, today. Besides their basic central role in cell replacement modalities, ES cells also offer a unique system for the genetic manipulation to create knockout strains of mice through gene targeting which is essential to know the gene functions. During the differentiation, cardiac-specific genes including the transcription factors, proteins, receptors, and essentially the ion channels are expressed in a developmental coherent of progression, which assures developmental pattern of cardiogenesis. Failing to do so can be due to non functionality of cardiac related genes, rendering its critical role in heart diseases. In the present study we have identified such a gene which has an implication on cardiogenesis. In this perspective, we have generated stable knockdown clone of mouse ES cells expressing a shRNA construct against annotated gene MHC6 (in-house designation), which also has a green fluorescent protein (GFP) reporter to track the silencing effect. The GFP expressing clones were induced to differentiate by forming EBs, and their developmental continuum was monitored via PCR, IHC and western blotting along with end point analysis by checking the functional beating cells. In many repeat experiments we found that the knockdown clones were not bearing the ability to form functional cardiomyocytes. In addition these EBs failed to show characteristic expression of cardiac markers such as cardiac troponin T, myosin heavy chain and calcium channel. Our results show the importance of MHC6 gene in driving the maturation for contractile functions of cardiogenesis.
AG MÜller, Innere III (Kardiologie), UniversitÄtsklinik Heidelberg , Germany
Session: Heart and vessels
Knock-out mice often show a severe phenotype early on in development limiting breeding and analysis of adult mice. Several strategies have emerged to surpass these problems, including inducible promoters and Cre-recombinase (Cre)-mediated gene-activation/inactivation. These strategies currently require time-consuming breeding of several transgenic strains. Aim of our study was to establish a novel and simple system for cardiac gene activation and more noticeably inactivation by means of expression of a Cre-recombinase using recombinant adeno-associated viral (AAV) vectors of serotype 9. AAV9-vectors showed an efficient cardiac gene transfer in adult mice after systemic vector application. We generated AAV9 vectors carrying the Cre-cDNA under control of the CMV-enhanced myosin-light-chain (MLC) promoter which revealed a high specificity for myocardium in previous studies. AAV9-Cre (1012 genomic particles) was injected intravenously into homozygous ROSA26-reporter mice. In these mice, Cre-mediated recombination of loxP sites activates lacZ-reporter expression in cells expressing Cre. After 4 weeks, lacZ expression could be detected in about 70% of cardiac muscle cells. Except for a strong lacZ signal in the liver, other tissues (spleen, kidney, lung, skeletal muscle, brain) revealed only few lacZ positive cells. In a second step, we examined whether this system could be used to knock down expression of serum response factor (SRF) in homozygous mice from the SRF-flex1 strain harboring a loxP-flanked exon. Five months after intravenous injection of AAV9-Cre (1012 genomic particles) in four months old mice, cardiac function was reduced as measured by fractional shortening in echocardiograms while control-injected mice did not show any alterations.
MSYS-Model Systems for Infection and Immunity, Helmholtz Centre for Infection Research , Germany
Session: Heart and vessels
Cells reflecting in vivo properties are of high interest for establishment of in vitro test systems and regenerative approaches. Existing cellular models are either lacking biological relevance or are not available in sufficient amounts. We have developed a flexible cell expansion strategy that allows strict and reliable control of cell proliferation. The system is based on a synthetic autoregulated module for transcriptional control of expansion genes, thereby supporting reversible expression. Using this system cells of various types and species were expanded. Importantly, strict control of proliferation was observed and even maintained when two independent expansion genes were simultaneously employed. Murine and human endothelial cell lines were evaluated in more detail with respect to their biological function. These studies revealed the expression of relevant endothelial specific markers as well as acLDL uptake. Further tube formation on Matrigel was displayed in vitro. The biological relevance of these cells was confirmed upon transplantation in mice: the cells form fully functional blood vessels interconnected with the host blood vessel system suggesting that the cell lines fully retain their angiogenic potential. We envision the controlled cell expansion strategy presented here to provide biologically relevant cells in sufficient numbers for cell based testing and screenings and in the long term for regenerative medicine approaches.
Model Systems for Infection and Immunity, Helmholtz Centre for Infection Research , Germany
Session: Hematopoiesis
A controlled and timely restricted expansion of hematopoietic stem cells (HSCs) without compromising their stem cell potential would be the ultimate solution to provide sufficient number of cells for cell replacement therapy in various diseases. In the last years we developed a reliable technology for expansion of fibroblast and endothelial cells from mouse and human. In this system an autoregulated Dox dependent regulatory module is used for tightly controlled expression of the proliferation gene(s). The focus of this project is laid on expanding hematopoietic stem/progenitor cells (HSCs) by means of conditional, i.e. reversible expression of proliferation genes. We explored if and in which way a timely restricted expression of these genes supports the maintenance of the in vivo potential of HSCs for differentiation and long-term repopulation. Upon infection of mouse lineage negative CD34-Sca1 + c-Kit + (LSK) cells with various Dox-controlled proliferator cassettes, LSK cell lines have been established that could be cultivated in vitro for up to ten months without loosing the stem cell markers. Moreover, they were able to differentiate and form colonies in Methocult media, indicating that the protocol does support differentiation. Transplantation is currently being followed to evaluate further the grafting potential of these cells. In order to genetically modify these cells in a predictable manner, we evaluate the exploitation of recombinase based methods for defined genetic modification of the immortalized hematopoietic (stem) cells. For this purpose, immortalized HSCs were generated that display a defined chromosomal tag supporting recombinase mediated cassette exchange (RMCE). Results from these studies will be presented.
Inserm U951, Genethon, France
Session: Hematopoiesis
The WASP protein is the product of the gene mutated in the Wiskott-Aldrich syndrome, therefore mechanisms regulating WASP cellular levels are of interest. Transcriptional control restricts WASP expression to hematopoietic cells. WASP levels are also regulated in resting cells by interactions with the WASP-interacting protein (WIP), thereby controlling proteolysis. Yet, little is known of the expression of WIP in human cells. This prompted us to examine the levels of WIP and WASP in a panel of human cell lines, in the steady-state or following gene transfer. VSVg-pseudotyped lentiviral vectors were used. While the PGK-WASP vector had been described, the PGK-WIP vector was newly-constructed by cloning a B cell WIP cDNA (1512 bp). Levels of WASP mRNA and protein were high in B-LCL compared to Jurkat or K562 cells and absent in HT1080 cells as expected. Gene transfer of WASP in these cells or in WASP-null B cells did not affect WIP. In the steady-state, WIP was high in B-LCL and lower in Jurkat, K562 or HT1080 cells. Except for B-LCL, gene transfer of WIP increased WIP mRNA by 2 to 14 fold and augmented WIP protein levels accordingly. In WASP-expressing cells, this increased WASP protein 2 to 5 fold without impacting on its mRNA levels. Thus, the levels of WIP and WASP are correlated in human hematopoietic cells. Variations in WIP levels regulate WASP at the post-transcriptional level and there is no reciprocity. Mechanisms regulating WIP expression should be investigated.
Gene Therapy Research Unit, Childrens Medical Research Institute , Australia
Session: Hematopoiesis
The development of leukaemia as a consequence of vector-mediated genotoxicity in gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) has prompted substantial research effort into the design and safety testing of integrating vectors. In this study we set out to explore the effect of promoter-enhancer selection on the efficacy and safety of HIV-1-derived lentiviral vectors in γc-deficient mice. We observed restoration of functional T- and B-cell compartments in mice transplanted with progenitors expressing γc from the EF1α promoter. Of concern, however, 4 of 14 mice reconstituted with this vector subsequently developed lymphoma. Extensive analyses including the identification of vector integration sites, analysis of gene expression at the site of vector integration and determination of transgene expression levels all failed to implicate insertional mutagenesis or γc over-expression as the underlying mechanism. In addition, frame-shift NOTCH1 gene mutations were identified in the malignant clones. These findings highlight the need for detailed mechanistic analysis of tumour read-outs in preclinical animal models, and point to the existence of other ill-defined risk factors for oncogenesis, such as replicative stress, in gene therapy protocols targeting the haematopoietic compartment.
CCU Molecular Oncology of Solid Tumors, German Cancer Research Center , Germany
Session: Hematopoiesis
Transplantation of retrovirally transduced hematopoietic stem cells (HSC) has been reported to be associated with clonal dominance and leukemia in host organisms. Although suitable methods to detect and track repopulating clones in vivo are described, which, when combined with quantitative real-time PCR, allow determining copy numbers of individual HSC clones, they lack the potential of a screening method for the detection of dominant or leukemic HSC. We conducted serial bone marrow transplantations (BMT) in a mouse model and investigated the long term effect of MGMTP140K-gene transfer followed by dose-intensive in vivo selection with alkylating agents. We analyzed clonality of hematopoiesis by combining LM-PCR and next-generation sequencing (Roche/454-system) and assessed proportions of individual HSC clones based on sequence read counts. While mice of the primary and secondary BMT cohorts were seen to polyclonally reconstitute, a mouse of the tertiary transplanted cohort showed monoclonal bone marrow, with a single HSC clone making up 99.5% of all transplanted cells. The dominant clone carried a vector insertion in the first intron of the Ubiquitin-specific peptidase 10 gene. We here describe a novel, reliable screening strategy to detect both, integration sites of vectors in the HSC genome and the associated HSC clone copy number. The method showed to reliably detect oligo/monoclonal outgrowth of genetically modified HSC after transplantation, which will be of great importance for future HSC gene therapy studies.
Experimental Hematology, Hannover Medical School , Germany
Session: Hematopoiesis
Congenital amegakaryocytic thrombocytopenia is a rare genetic disorder in which defects in Mpl (Thrombopoietin receptor) cause severe thrombocytopenia and aplastic anemia. With the aim to avoid severe adverse reactions induced by ectopic Mpl expression (uncontrolled expansion or exhaustion of hematopoietic cells), we developed self-inactivating lentiviral vectors with different lineage-specific promoters derived from human Mpl, murine Mpl and human GPIba. For comparison we used the ubiquitously active PGK1 promoter. Lineage-specific vectors drove expression in the megakaryocytic cell line HEL, in megakaryocytes (CD41+) in vivo and in hematopoietic stem cells with minor activity in other lineages and progenitor populations. All vectors corrected defective megakaryopoiesis of Mpl-/- cells in vitro. To monitor vector activity in vivo and assess systemic effects, we transplanted transduced Mpl-/- hematopoietic cells in conditioned Mpl-/- mice. Mpl expressed by the PGK1 promoter caused death 6 days post transplantation (6/7 mice) due to rupture of enlarged spleens (359–667 mg) with erythroid hyperproliferation. This underlines the extreme sensitivity of Mpl-negative hematopoiesis to ectopic Mpl expression. Early post-transplant complications were prevented when using the lineage-specific promoters, which restored platelet production in > 50% of transplanted mice (n = 21). Only the GPIba promoter conferred long-term correction rendering this vector a promising candidate for Mpl gene therapy.
Molecular Immunology, UCL Institute of Child Health , UnitedKingdom
Session: Hematopoiesis
Lentiviral vectors integrate their genetic payload into both dividing and non-dividing cells, making them useful tools for long-term gene delivery to stem cells. Mutant viruses are available that have a defective integrase protein, which maintain the ability to efficiently deliver genetic material as stable circular molecules, but cannot directly integrate it into target-cell chromosomes. Because no origin of replication is present, lentiviral backbones are lost as the host cell divides. We are utilising this characteristic to provide transient expression of a transgene in dividing cells, especially in hematopoietic progenitors. IDLV have been used successfully to express genes in non-dividing tissues such as retina and brain. However, we have found many variables affect IDLV efficiency, such as gene insert, target cell type and host species. We have optimised this technology to improve the production and efficacy of IDLV vectors. Development has been required to address areas including virus production, titration of vectors, variable expression in different species and low expression levels in hematopoietic stem cells.
RG Reprogramming, Hannover Medical School , Germany
Session: Hematopoiesis
Hematopoietic stem cells genetically modified to overexpress drug resistance genes have been advocated to overcome chemotherapy induced myelosuppression, and we have recently shown that CDD overexpression profoundly protects hematopoiesis from the cytostatic drugs cytarabine (ara-C) and gemcitibine (Rattmann, Blood, 2006). However, these studies also revealed lymphoid toxicity and the lack of long term in vivo selection following CDD gene transfer. Therefore, we have generated novel SIN lentiviral vector constructs, expressing the human CDD either from a spleen focus forming virus (SFFV) or a truncated elongation factor 1α (EFS) promoter/enhancer element. These vectors are able to profoundly protect myeloid (32D) and lymphoid cell lines (WBα-β-) against Ara-C as well as primary clonogenic hematopoietic cells against ara-C (up to 300 nM) and gemcitabine. To investigate the lymphoid toxicity of the novel CDD-vectors, a lymphoid differentiation model based on the coculture of lin-murine or CD34 + human hematopoietic cells on OP9/OP9-DL1 (delta-like 1) feeders has been established. In addition, we are going to investigate the in vivo selection potential of these new vector constructs in murine model systems employing optimised ara-C application schedules.
Rattmann I. Blood
Session: Hematopoiesis
Improvements in directing differentiation of embryonic stem cells (ESCs) to long-term repopulating hematopoietic stem cells (ESC-HSCs), in vitro, are pivotal to advance the use of pluripotent stem cells in cell replacement therapies. The CD41hickit+ population marks the earliest definitive hematopoietic progenitors (HPC) in the mouse embryo. Ectopic expression of HOXB4 enhances the expansion of CD41hickit+ cells emerging in differentiating embryoid bodies (EBs) of murine ESCs, and is required to promote their conversion into repopulating HSCs in vitro. However, the efficiency of their conversion into ESC-HSCs is extremely low. Determining the cellular identity of the ESC-HSC is crucial for the rational design of new strategies for maturation to fully functional bone marrow-like HSCs. Thus, we flowcytometrically characterized in detail the expression of fetal and adult HSC-associated surface markers (Tie2, CD34, CD150, CD48 and CD201) on hematopoietic cells that were generated in bulk cultures of dissociated day 6 EB HOXB4+ cells after cultivation in the presence of cytokines for 10 days, with and without OP9 stromal cell support. Cocultivation of HOXB4-expressing EB cells on OP9 stromal cells resulted in a 6-fold increased frequency of CD41hickit+CD34neg cells, a subpopulation known to contain the highest CFC activity. Moreover, OP9-supported expansion cultures contained 7-fold and 23-fold higher proportions of CD41hickit+CD34neg cells that also express CD150 or CD201, respectively. Together our results suggest that OP9 stroma cells provide signals that induce the acquisition of markers found on mature HSCs (CD150, CD201), which may prove valuable for the prospective isolation of ESC-HSCs and subsequent functional studies.
Experimental Paediatric Oncology and Haematology, University Frankfurt , Germany
Session: Hematopoiesis
Despite several shortcomings (e.g., slow and incomplete killing, unspecific toxicity of high ganciclovir (GCV) doses) the Herpex simplex virus thymidine kinase (HSVtk) gene together with its prodrug GCV has widely been used in cancer gene therapy and as a conditional safety device. We developed a codon-optimised HSVtk [coHSVtk] and modified this by introducing the A168H mutation [TK.007]. We assessed performance of the two new suicide genes as compared to splice-corrected wild-type scHSVtk in different preclinical models. We found strongly improved killing kinetics for TK.007 as compared to both other HSVtk's for retrovirally transduced haematopoietic cell lines and primary T cells in vitro. Next we transduced various tumour cell lines with LeGO vectors encoding one of the TK genes. Again, we discovered highest killing rates for TK.007 at significantly lower GCV doses as compared to controls. Moreover we observed a much stronger bystander effect for TK.007. Finally, the improved killing efficiency of TK.007 was confirmed in an in vivo adoptive immunotherapy model in Rag2-/-gc-/- mice. In fact, TK.007 transduced cells were killed significantly faster and to a greater extent, particularly at lower GCV doses (10 mg/kg) as compared to scHSVtk transduced T cells. In summary, the novel TK.007 gene has demonstrated superior performance in all assays executed. It therefore represents a promising alternative to currently used HSVtk's for suicide gene-based therapeutic strategies.
Transfusion medicine, University Hospital Essen , Germany
Session: Hematopoiesis
In adults an ongoing renewal of the different blood cell types is carried out by the hematopoietic stem and progenitor cells (HSPCs). These HSPCs are characterized by specific cell surface markers like CD34 as well as CD133 (Prominin-1) and reside in the stem cell niches. Stem cell niches are found in the bone marrow (BM) near the endosteum as well as in the vicinity of sinusoidal endothelial cells. In a non pathological situation, most HSPCs residing in their stem cell niches are quiescent. After profound blood loss, as it occurs in polytraumatic patients, enlarged numbers of HSPCs need to be activated to produce new progenitor cells and blood cells. As in healthy adults only a small percentage of HSPCs (CD34 + cells) are found outside their niches, we investigate whether polytraumatic events mobilize HSPCs to the peripheral blood system. Performing flow cytometric analysis we observed that approximately 0.14% (±0.05 SD) of the mononuclear cells of healthy blood donors express the stem cell surrogate antigen CD34 (n = 5). In polytraumatic patients the amount of CD34 + HSPCs is increased up to 0.25% (±0.21 SD) five days after injury (n = 5). Even though the total amount of the more primitive CD133 + CD34 + cells was increased as well, the ratio between CD133 + and CD133- cells did not notably change. While the mechanism of HSPC mobilization is not fully understood yet, influencing the number of circulating HSPCs may be an attractive route to improve regeneration after trauma.
Predicting disease dynamics in chronic myeloid leukemia- paving the way for model-based treatment optimizations
Session: Hematopoiesis
The treatment of chronic myeloid leukemia (CML) with the tyrosine kinase inhibitor imatinib mesylate (IM) represents a successful application of a molecularly targeted cancer therapy. Although IM induces rapid hematologic and cytogenetic responses in the majority of patients, the complete eradication of the malignant cells, characterized by the expression of the BCR-ABL fusion protein, is a rare event. The reasons for the long-term persistence of the malignant clone are currently not known and provide a major challenge for clinicians and biologists. Based on a mathematical, agent-based model we are able to consistently explain short- and long-term dynamics of BCR-ABL transcript dynamics in CML patients under IM mono-therapy[1]. In agreement with recent clinical 7 year follow-up data, the model predicts a continued decline in the median BCR-ABL transcript levels over time, suggesting the ultimate possibility of an eradication of the malignant clone. Whereas the average time to the elimination of the malignant clone is estimated to exceed 20 years in case of IM mono-therapy, the model predicts that this time can be considerably shortened by combination of the tyrosine kinase inhibitor with proliferation stimulating compounds. This model prediction will be discussed with respect to recent clinical data on the combination of IM with the cytokines G-CSF[2] and IFN-alpha which has recently been shown to activate dormant hematopoietic stem cells in the mouse[3].1. Roeder et al. Nat. Med., 2006, Vol.12(10):1181–4.2. Drummond et al. Leukemia, 2009, Vol.23(6):1199–201.3. Essers et al. Nature, 2009, Vol.458(7240):904–8.
Individual clone dynamics and the effect of clonal heterogeneity-lessons from a mathematical model analysis
Session: Hematopoiesis
Haematopoietic stem cells (HSCs) are characterized by the ability to generate mature cells of all haematopoietic lineages and to simultaneously maintain their own population of undifferentiated cells. This differentiation/self-renewing capacity is critical for the successful application of HSCs in treatment of haematologic diseases, as supportive therapy following marrow ablation, or in gene therapeutic applications. In all these settings, it is essential to control and possibly predict clonal (repopulation) dynamics. However, little is known about the regulatory mechanisms underlying the dynamic control of HSCs on the level of individual clones. A potentially major effector of these processes is clonal heterogeneity, i.e. differences in the properties and the behaviour of individual HSC clones. For example, it has been shown that HSC clones exhibit considerable, epigenetically determined differences with respect to their repopulation behaviour and their lineage contribution. Other studies convincingly demonstrated the heterogeneity of HSCs regarding cell cycling activity. In contrast to the above mentioned, clonally fixed heterogeneity, the proliferative activity of individual HSCs has shown to reversibly change and, therefore, to vary within individual clones. Using an agent-based mathematical model we investigated the effect of different levels of cellular heterogeneity on the behaviour of individual HSC clones. Additionally to an introduction of the conceptual ideas behind such a model analysis, I will discuss the impact of our modelling results with respect to the prediction of clonal development in the context of gene therapeutic settings and the treatment of haematologic malignancies, such as chronic myeloid leukaemia.
IMISE, University of Leipzig , Germany
Session: Hematopoiesis
The kinetics of label uptake and dilution in dividing stem cells, e.g., using Bromodeoxyuridine (BrdU) as a labeling substance, are a common way to assess the cellular turnover of all hematopoietic stem cells (HSCs) in vivo. The assumption that HSCs form a homogeneous population of cells which regularly undergo cell division has recently been challenged by new experimental results. For a consistent functional explanation of heterogeneity among HSCs, we propose a concept in which stem cells flexibly and reversibly adapt their cycling state according to systemic needs. Applying a mathematical model analysis, we demonstrate that different experimentally observed label dilution kinetics are consistently explained by the proposed model. The dynamically stabilized equilibrium between quiescent and activated cells leads to a biphasic label dilution kinetic in which an initial and pronounced decline of label retaining cells is attributed to faster turnover of activated cells, whereas a secondary, decelerated decline results from the slow turnover of quiescent cells. These results, which support our previous model prediction of a reversible activation/deactivation of HSCs, are also consistent with recent findings that use GFP-conjugated histones as a label instead of BrdU. Based on our findings we interpret HSC organization as an adaptive and regulated process in which the slow activation of quiescent cells and their possible return into quiescence after division are sufficient to explain the simultaneous occurrence of self-renewal and differentiation. Furthermore, we suggest an experimental strategy which is suited to demonstrate that the repopulation ability among the population of label retaining cells changes during the course of dilution.
Immunology, Hannover Medical School , Germany
Session: Hematopoiesis
T cell development in the thymus depends on continuous supply of T cell progenitors from bone marrow (BM). Several extra-thymic candidate progenitors have been described that range from multi-potent cells, to lymphoid cell committed progenitors and even largely T lineage committed precursors. However, the nature of precursors seeding the thymus under physiological conditions has remained largely elusive and it is not known whether there is only one physiological T cell precursor population or many. Here, we employed a competitive in vivo assay based on depletion rather than enrichment of classes of BM-derived precursor populations, thereby retaining near-physiological precursor ratios to assess the contribution of various extra-thymic precursors to T lineage differentiation. We found that under these conditions multiple precursors, belonging to both multi-potent progenitor (MPP) and common lymphoid progenitor (CLP) subsets have robust T lineage potential. However, differentation kinetics of different precursors varied considerably, which might ensure continuous thymic output despite gated importation of extra-thymic precursors. In conclusion, our data suggest that the thymus functions to impose T cell fate on any precursor capable of filling the limited number of progenitor niches.
Department of Human Genome, IRCCS Humanitas Clinical Institute , Italy
Session: Hematopoiesis
WAS is an X-linked immunodeficiency characterized by thrombocytopenia, infections, autoimmunity and lymphomas. Gene therapy with hematopoietic stem cells (HSC) could represent an alternative for patients lacking an HLA-identical donor. We first set up a transduction protocol for CD34 + cells isolated from normal donor bone marrow and mobilized peripheral blood. HSC were exposed once or twice to GMP grade lentiviral vector (LV) encoding WASp driven by its endogenous promoter, after a pre-stimulation with cytokines. qPCR performed on transduced cells showed a vector copy number per cell (VCN) of 0.4 ± 0.1 (1 hit), and 0.6 ± 0.2 (2 hits). The same protocol was used to transduce CD34 + cells from WAS patients resulting in a VCN of 0.7 ± 0.1 (1 hit) and 1.4 ± 0.3 (2 hits), with restored WASP expression in differentiated cells, including megakaryocytes. About 40% of CFC for 1 hit and 60% for 2 hits protocols were positive for integrated vector. Transduced CD34 + cells injected in sub-lethally irradiated Rag2-/-/gc-/- mice showed a normal biodistribution into hematopoietic organs (mean VCN 1.1 in BM), without evidence of vector shedding or germline transmission. Transduced HSC showed normal differentiation capacity into myeloid cells and lymphocytes (T and B).Together with the efficacy and safety studies previously performed in the mouse model, our study provides the preclinical basis for the implementation of a gene therapy trial for WAS in combination to a reduced intensity conditioning.
Dept. of Hematology and Oncology Internal Medicine, School of Medicine University of Frankfurt , Germany
Session: Hematopoiesis
X-linked Chronic Granulomatous Disease (X-CGD), a rare inherited disorder of phagocyte function, is caused by dysfunction of the gp91phox gene. Our Phase I clinical study with gene modified autologous stem cells showed substantial clinical benefit, but also severe adverse effects, caused by insertional mutagenesis. Also, transgene expression was lost due to DNA methylation at the viral LTR promoters. To improve safety and efficacy for gene therapy of X-CGD, we developed a series of enhancer-deleted gammaretroviral vectors, expressing gp91phox from constitutive as well as myeloid-specific internal promoters. From this series, the SERfes-vector, in which transgene expression is driven by the promoter of the human c-fes gene, was selected for further studies. Evaluation of the SERfes vector expressing either gp91phox or GFP was performed in human CD34 + hematopoietic cells obtained from X-CGD patients or healthy donors. Transduction efficiencies were 35–50% and 20–40% for SERfes-GFP and SERfes-gp91phox respectively. After in vitro differentiation, gp91phox-transduced cells from X-CGD patients showed effective superoxide production, at levels similar to those observed in wildtype cells. SERfes-GFP transduced CD34 + cells engrafted in NOD/SCID mice and bone-marrow cells, derived from these mice, showed expression of the transgene mainly in myeloid cells. Currently, SERfes-gp91phox-transduced CD34 + cells from X-CGD patients are being studied in vivo in NOD/SCID mice.
Division of Immunology-Hematology-BMT, University Children Hospital Zurich , Switzerland
Session: Hematopoiesis
Chronic granulomatous disease (CGD) patients have impaired NADPH oxidase function, resulting in poor antimicrobial activity of neutrophils including the inability to generate Neutrophil Extracellular Traps (NETs). Invasive aspergillosis is the leading cause of death in patients with CGD; it is unclear how neutrophils control Aspergillus species in healthy individuals. The aim of this study was to determine whether gene therapy (GT) restores NET formation in CGD by complementation of NADPH oxidase function, and whether NETs have antimicrobial activity against Aspergillus nidulans. Here we show that reconstitution of NET formation by GT in a patient with CGD restores neutrophil elimination of Aspergillus nidulans conidia and hyphae and is associated with rapid cure of pre-existing therapy refractory invasive pulmonary aspergillosis, underlining the role of functional NADPH oxidase in NET formation and antifungal activity.
Medical and Molecular Genetics, Kings College London, United Kingdom
Session: Hematopoiesis
Adenosine deaminase (ADA) deficiency leads to T, B and NK cell toxicity resulting in severe combined immunodeficiency (SCID). Correction of SCID-ADA has been reported in phase I/II clinical trials employing a conventional gammaretroviral vector to transduce haematopoietic stem cells (HSCs). However, the risk of insertional mutagenesis and proneness of LTR silencing has prompted the development of alternatives with improved efficacy and safety profiles. Furthermore, SCID-ADA patients suffer from hepatic, skeletal and neurological abnormalities in addition to immunodeficiency due to ADA being ubiquitously expressed. Therefore, a fully effective gene therapy for SCID-ADA requires systemic detoxification as well as correction of the immunological defect. We are addressing these issues with lentiviral vectors containing transcription units incorporating a combination of human β-globin locus control region (βLCR) elements (HS2/3/4) linked to a ubiquitously expressed housekeeping gene promoter (EF1α). The rationale is to enhance expression of ADA in the erythroid lineage to provide systemic detoxification whilst achieving adequate expression in lymphoid precursors to ensure correction of immunodeficiency. We found that the βLCR enhances expression of an EF1α-GFP cassette approximately 3-fold in murine erythroleukaemia cells upon induced terminal erythroid differentiation. We also find that the βLCR does not alter the expression of GFP in non-erythroid cell lines. Preliminary results in an ex vivo HSC transplant model in mice, show that the βLCR is capable of specifically enhancing GFP expression at least 7-fold in the TER119+ (erythroid) cell population. These encouraging results prompt further testing in relevant model systems of ADA-SCID.
Section for Transplantation Immunology, University of TÜbingen , Germany
Session: Hematopoiesis
A proteolytic microenvironment in the bone marrow comprising the proteases cathepsin G, neutrophil elastase and the matrix metalloproteinase MMP-9 can facilitate the trafficking of hematopoietic stem cells (HSC) towards the periphery. In mice lacking one or two of these proteases, however, stem cell trafficking is hardly affected suggesting the involvement of other proteases. In the present study, the expression and secretion of the cysteine protease cathepsin X by cells of the human bone marrow microenvironment was investigated. Osteoblasts as the major cell type of the endosteal stem cell niche secrete cathepsin X, whereas hematopoietic stem and progenitor cells (HSPC) do not synthesize this protease. Using the cathepsin X-selective substrate Abz-FEK(Dnp)-OH, the catalytic activity of cathepsin X could be detected in cell culture supernatants of primary osteoblasts. Activated cathepsin X is able to reduce cellular adhesive interactions between human CD34 + HSPC and adherent osteoblasts. The chemokine CXCL-12 which is secreted by osteoblasts and bone marrow stromal cells is a highly potent chemoattractant and important mediator of cell adhesion for HSPC. As a potential substrate for cathepsin X, CXCL-12 was incubated with the protease for different periods. MALDI-TOF analysis showed that CXCL-12 is readily digested by cathepsin X, as truncated CXCL-12 was detected in presence of cathepsin X. These data give rise to the following scenario: secreted and activated extracellular cathepsin X can proteolytically influence HSC trafficking in the bone marrow by digesting the chemoattractant CXCL-12. Whether cathepsin X additionally interacts with other proteins on the cell surface of HSPC in a non-proteolytic context is currently under investigation.
Applied Virology and Gene Therapy Unit , Georg-Speyer-Haus Frankfurt, Germany
Session: Hematopoiesis
Dysfunction of gp91phox (Nox2), the major subunit of the phagocytic NADPH oxidase complex, causes X-linked Chronic Granulomatous Disease (X-CGD), a rare inherited disorder of the phagocyte system. In a Phase I clinical trial we have demonstrated the therapeutic potential of gene modified autologous stem cells for the treatment of X-CGD. However, severe adverse effects were observed in two treated adult X-CGD patients due to insertional mutagenesis and the activation of growth promoting genes leading to the occurrence of haematological abnormalities. In addition, we observed downregulation of transgene expression caused by CpG methylation at the viral promoter. Optimized SIN gammaretroviral vectors have been shown to possess an enhanced safety profile and could be the vectors of choice for the next X-CGD gene therapy trial. From a series of enhancer-deleted gammaretroviral vectors expressing the gp91phox cDNA under the transcriptional control of either constitutive or myeloid-specific promoters, we selected a construct containing the promoter of the human c-fes gene for detailed pre-clinical studies. We used a mouse model of X-CGD for monitoring long-term repopulation, transgene expression and functional reconstitution of NADPH oxidase activity in primary and secondary transplanted X-CGD mice and could show that the SINfes vector performed as well or even better than the LTR driven vector used in the clinical trial. Also, our data indicate that the internal c-fes promoter is less prone to silencing than the internal SFFV promoter.
Division of Stem Cell Therapy, Center for Stem Cell and Regenerative Medicine, Institute of Medical science University of Tokyo , Japan
Session: Hematopoiesis
X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency characterized by functional defects in phagocytes. Although stem cell-gene therapy is considered a promising therapy option, successful long-term reconstitution of gene-corrected granulocytes has not been established. Using gp91phox mutant mice as a model of X-CGD, we sought to scrutinize the functional reconstitution in granulocytes after gene-therapy. Bone marrow hematopoietic stem/progenitor cells (HSPCs) from X-CGD mice were transduced with a retroviral vector harboring murine gp91phox cDNA together with Kusabira Orange (KuO) gene. These transduced cells were transplanted into lethally irradiated X-CGD mice. Levels of specific reactive oxygen species (sROS) production by APF dye staining assay were assessed in conjunction with KuO fluorescence intensities in Gra-1 + granulocytes of recipient mice. Interestingly, these reconstituted Gra-1 + cells were clearly separated into 2 fractions: KuObrightAPF-positive and KuOdullAPF-negative populations. We thought two possibilities to explain this result: 1) gp91phox transgene was not expressed at all in KuOdull populations, or 2) there was a threshold in expression levels of gp91phox for functional correction in X-CGD granulocytes, which was not reached in KuOdull populations. Although preliminary, our PCR and RT-PCR analyses on sorted granulocyte populations so far favored the above second possibility, as they demonstrated the presence of gp91phox transgene and also its expression in KuOdullAPF-negative populations. These results will have implications into improvement of gene therapy strategies for X-CGD by maximizing their efficacies in functional reconstitution at single cell levels. Detailed analyses are ongoing.
Div. of Immunology-Hematology-BMT, University Childrens Hospital ZÜrich , Switzerland
Session: Hematopoiesis
Mutations in p47phox are the second leading cause of Chronic Granulomatous Disease (CGD). The detection of p47phox expression is a mandatory requirement for p47phox gene therapy monitoring. Intracellular immunostaining of the cytoplasmic p47phox transgene product interferes, however, with respiratory burst activity. We report a new system for measuring p47phox expression using the FMDV Virus 2A oligopeptide co-expression technology. After retroviral transmission the translation of the ΔLNGFR-2A-p47phox open reading frame (ORF) generates two discrete products: cytoplasmatic p47phox and [ΔLNGFR-2A] on the cell surface synthesized in a 1:1 ratio. Cotranslational cleavage products and functional reconstitution could be detected in granulocytes ex vivo differentiated from transduced p47phox-/- murine HSC. Six weeks after reinfusion of transduced HSC we could detect functional reconstitution of NADPH oxidase activity in blood granulocytes in the p47phox-/- animal model. In all hematopoietic cell populations analyzed the SFFV promoter/enhancer driven expression of the ΔLNGFR-2A-p47phox ORF led to detectable ΔLNGFR surface marking of p47phox-trangenic cells as revealed by ΔLNGFR-surface/p47phox-intracellular staining. Functional reconstitution could also be shown in transduced human monocyte derived macrophages from p47phox-/- CGD patients in cell culture. This new marker system enables highly efficient, indirect, detection of cytoplasmic transgene products by FACS surface staining.
Institute for Transfusion Medicine, University Hospital Essen , Germany
Session: Hematopoiesis
Somatic stem cells are required to maintain homeostasis in different tissues. For this pupose stem cells have to give rise to committed cells, but also have to self-renew over a long period of time. Since both, an uncontrolled expansion or a loss of stem cells would be fatal for an organism, the mechanisms governing the decision of self-renewal versus differentiation needs to be tightly controlled. In this context there is good evidence that niches as well as asymmetric cell divisions are involved in governing the developmental fate of stem cells, at least for hematopoietic stem and progenitor cells (HSPCs). Our group has identified four proteins which segregate asymmetrically in about 20% of dividing human HSPCs (Beckmann et al. 2008). As two of these proteins (CD53 and CD63) are members of the tetraspanin family, these results suggest a putative role of tetraspanins for HSPC biology. To test possible influences of tetraspanins on HSPCs and to obtain potential markers allowing to identify asymmetrically dividing cells in vitro, we decided to stably transduce primary hematopoietic cells with several novel tetraspanin-eGFP fusion proteins. In preliminary studies, we transiently expressed these tetraspanin-eGFP fusion proteins and evaluated their subcellular localisation. The results of our transient expression experiments will be presented.
Department of Medical Biotechnology, Jagiellonian University Cracow , Poland
Session: Hematopoiesis
Recently, we identified in human cord blood (CB) a population of very small embryonic-like (VSEL) stem cells that are i) smaller than erythrocytes, ii) SSEA-4 + Oct-4 + CD133 + CXCR4 + Lin-CD45-, iii) express embryonic markers. Similar population from murine bone marrow (BM) reveals hematopoietic activity after co-culture over OP-9 cells and possess potential of long term repopulating hematopoietic stem cells (LT-HSC). To investigate hematopoietic capacity of CB- derived VSEL, we employed staining with Aldefluor detecting aldehyde dehydrogenase (ALDH)- the enzyme of primitive hematopoietic cells and we sorted CD133 + CD45-ALDHhigh (VSEL/ALDHhigh) and CD133 + CD45-ALDHlow (VSEL/ALDHlow) subfractions of VSEL from CB samples. Both freshly sorted VSEL populations did not grow hematopoietic colonies in vitro. However, when expanded over OP-9 cells, they exhibit hematopoietic potential and initiate colonies composed of CD45 + cells when replated into methylcellulose cultures. Furthermore, VSEL/ALDHhigh gave raise to such colonies faster than VSEL-ALDHlow. Real time RT-PCR revealed that freshly isolated VSEL/ALDHhigh express more hematopoietic transcripts (c-myb, 80.2 ± 27.4 fold difference), while VSEL-ALDHlow more pluripotent markers (Oct-4, 119.5 ± 15.5 fold difference) as compared to total CB cells. Moreover, both VSEL/ALDHhigh and VSEL/ALDHlow populations - freshly isolated or expanded on OP-9 cells - reconstituted hematopoiesis in lethally irradiated SCID mice after 1 month post transplantation.In conclusion, our data suggest that CB- derived VSEL that are CD133 + CD45-ALDHlow are enriched for most primitive population of LT-HSC. These cells may be responsible for long term CB engraftment and should be expanded into HSCs for potential clinical applications.
Immune response versus immune tolerance following minicircle-mediated factor VIII gene transfer
Session: Immunity, tolerance, vaccines
A major problem of hemophilia A gene therapy is the development of inhibitory antibodies against the factor VIII (FVIII) transgene. We therefore explore a non-viral gene transfer approach based on the generation of minicircle (MC) vectors for gene therapy and immune tolerance induction. Initially, we applied 0.05 mg (high dose) or 0.01 mg (low dose) of MC-FVIII by hydrodynamic injection technique into the tail veins of C57Bl/6 mice and compared FVIII expression with levels in mice that received an additional dose of 0.05 mg bacterial pUC19 plasmid DNA as contaminant (n = 8/group). Mice treated with 0.05 mg of MC vector showed higher expression levels compared to the low dose treatment group (0.5 IU vs 0.2 IU; day 3). No effect of the pUC19 co-injection was observed on expression levels. Although the onset of the immune response was faster and stronger in mice receiving 0.05 mg MC, FVIII expression only returned in this treatment group 12 weeks post injection. In another experiment, hemophilia A mice were treated with high and low dose of either MC- or pSL1180-FVIII plasmid vector (n = 8/group). The MC vector led to higher expression levels (2.8 IU vs 1 IU (high dose), day 3), but still a drop in FVIII expression and an anti-FVIII immune response was observed in all mice. 20 weeks post treatment, an immunotolerance characterized by a drop in antibodies and a rise in FVIII levels was observed in 50% or 14% of mice treated with high or low dose minicircle and in 40% or 0 % treated with high or low dose plasmid, respectively. In conclusion, minicircle could improve a FVIII gene transfer strategy by lifting the immunological threshold for antibody responses and by tolerance induction through persistent FVIII expression following initial antibody formation.
Lessons from non-gene therapy studies to predict the environmental risk in immune modulation gene therapy
Session: Immunity, tolerance, vaccines
The host immune response against viral vectors and foreign transgenes is a major hurdle in gene therapy and plays a crucial role as a determinant of long-term gene expression and clinical efficacy. We hypothesized that immune modulation might increase the risk for concomitant infections, resulting in a higher risk for recombination of viral vectors with wild type viruses and/or shedding. Fragmentary in vivo animal data using different vectors and types of immune modulation and the lack of human data provide a limited basis to predict the environmental risk. We therefore focused on available literature regarding closely related human, non-gene therapy studies and substantiated the data by interviewing experts. To assess the risk of specific infections in relation to the type of immune modulation, we compared the type of infections occuring in immune deficient/suppressed patients and (stem cell) transplant recipients; to assess the risk for recombination and shedding, we used data from safety and efficacy studies of live-attenuated vaccines. The risk of specific viral infections depends largely on remaining host immunity as well as duration, intensity and type of immune suppression used. Non-gene therapy vaccination studies show that recombination may even occur between distantly related viral strains and can result in recombinant viruses with distinct tropism. Shedding and transfer of vaccine strains to secondary recipients was shown to occur regularly in persons that come in (close) contact with the injection/inoculation site, but rarely to tertiary recipients. Here, we propose a scoring system, based on host-vector interactions, vector characteristics and characteristics of the gene transfer, to predict environmental risk.
Human Gene Therapy Division of Medical Genetics, Akdeniz University Faculty of Medicine , Turkey
Session: Immunity, tolerance, vaccines
Biochemistry and Medical Biotechnology, CEINGE-Advanced Biotechnology, Italy
Session: Immunity, tolerance, vaccines
Adoptive transfer of autologous EBV-CTLs to hematopoietic stem cell and solid organ transplant recipients is effective for prevention and treatment of EBV + post transplant lymphoproliferative disorders (PTLD). However, CTL persistence and efficacy can be limited by immunosuppressive drugs, like FK506 whose effects are dependent on binding of FKBP12 proteins. We hypothesize that EBV-CTLs can be resistant to FK506 by knocking down FKBP12, using a small interfering RNA (siRNA) stably expressed by a retroviral vector. We identified by extensive screening one siRNA (siRNA4) that knocked down more than 90% of FKBP12 in EBV-CTLs. Retroviral vector encoding for siRNA4/eGFP were used to transduce established EBV-CTLs generated from 7 EBV-seropositive donors (Transduction efficiency was in average 46%). We measured the proliferation of transduced CTLs in the presence of FK506 in a short term cultures by thymidine uptake. We found that proliferation was significantly inhibited in control (74.2%) but not in siRNA4-CTL (41.4%). Furthermore, siRNA4-CTLs were stimulated weekly with autologous LCL, low dose IL-2 and in the presence of FK506, to assess the resistance to the drug in a long-term culture. The results confirmed the effect of knocking-down of FKBP12; indeed the proportion of siRNA4-CTLs increased over time in 4 weeks of culture not only as a percentage of GFP + cells but also as absolute count. Moreover, we confirmed their FK506 resistance and anti-PTLD activity in vivo using a xenogenic mouse model, suggesting that the proposed strategy may be of value to enhance EBV-specific immune surveillance in patients at high-risk of PTLD after transplantation.
Human Gene Therapy Division of Medical Genetics, Akdeniz University Faculty of Medicine , Turkey
Session: Immunity, tolerance, vaccines
Aydin et al. Hum Gene Ther
Sanlioglu et al. Cancer Gene Ther
Sanlioglu et al. Cancer Gene Ther
Sanlioglu et al. J Cell Biochem
Session: Immunity, tolerance, vaccines
C peptides (e.g. T20, C46) are highly efficient inhibitors of HIV entry. In vivo secreted antiviral entry inhibitory (iSAVE) peptides produced by gene-modified cells are expected to mediate a bystander protective effect on neighboring non-modified cells and suppress virus replication even if only a small fraction of cells is genetically modified. However, short peptides are only inefficiently translated and exported by the cellular secretory machinery. To circumvent these limitations we expressed therapeutic iSAVE peptides from retroviral vectors as concatamers which were subsequently processed into monomeric peptides by protease cleavage within the secretory pathway. Transfection or transduction of cell lines with these vectors resulted in high-level expression and secretion of iSAVE peptides, which exerted a high antiviral activity in single-round infection assays with replication incompetent lentiviral particles pseudotyped with a variety of different HIV envelope glycoproteins. Moreover, genetic modification of primary human T cells resulted in the secretion of therapeutic levels of iSAVE peptides. In mixed cell cultures peptides secreted from transduced cells produced a bystander effect and prevented HIV-1 infection of non-modified cells.The in vivo secretion of therapeutic iSAVE peptides from gene-modified T cells holds great promise as this is likely to lead to high and stable local concentrations of iSAVE and thus confer a substantial antiviral effect.
Development of therapeutic genome-manipulation tools and its application for AIDS therapy
Session: Immunity, tolerance, vaccines
Current gene therapy approaches concentrate on the introduction into diseased cells a gene providing a missing or defective activity. Such approaches cover only a part of the therapeutic needs and a variety of diseases require therapeutic tools that can perform specific changes in the genome. Moreover, the current replacement therapy approaches are faced with the significant problem of insertional mutagenesis. Recogene is developing site-specific genome manipulation tools for gene therapy that can be used for removal, insertion or inversion of specific DNA sequences. Recogene's Asymmetric-Specific-Recombination (ASR) technology is based on the Cre/loxP system and enables the recognition of any desired site in the genome and opens up the possibility of harnessing the unique properties of Cre recombinase for therapeutic purposes. We are developing unique Cre recombinase variants recognizing the HIV genome. Following the establishment of an in-vitro proof of activity of the Cre variants we have shown that they are able to perform specific removal of sequences flanked by the HIV target sequences in human cells. These experiments will be followed by a direct analysis of HIV infected cells. Current gene therapy approaches concentrate on the introduction into diseased cells a gene providing a missing or defective activity. Such approaches cover only a part of the therapeutic needs and a variety of diseases require therapeutic tools that can perform specific changes in the genome. Moreover, the current replacement therapy approaches are faced with the significant problem of insertional mutagenesis. The first application of the technology is for AIDS therapy. Tools are being developed for the removal of the HIV genome from the genome of the host cell, providing a potential cure. The technology will then be used for the development of tools enabling the specific integration of therapeutic genes into safe locations in the genome. These experiments will be followed by a direct analysis of the Cre variants in HIV infected cells and examination of their ability to reduce viral infection.
Institute for Transfusion Medicine, Hannover Medical School , Germany
Session: Immunity, tolerance, vaccines
Exposure to allogeneic blood products can lead to the development of anti-human leukocyte antigen (HLA) antibodies in multitransfused patients. Refractoriness to platelet transfusion caused by alloimmunization against HLA class I antigens constitutes a significant clinical problem. We developed a RNAi-based approach to permanently silence the expression of HLA class I molecules on platelets derived from CD34+ progenitor cells. A lentiviral-based system was used to express short hairpin RNA (shRNA) targeting β2-microglobulin (β2 m) on CD34 + cells. Silencing of β2 m and HLA class I expression was evaluated by real time RT-PCR and by flow cytometry. Differentiation to platelets was performed by incubating 1 × 105 HLA-silenced progenitor cells in the presence of 50ng/ml thrombopoietin and 50 ng/ml interleukin-3 for 20 days. In CD34 + progenitor cells, the transduction of RNAi cassettes containing the sequences for shRNAs targeting β2 m caused a reduction of the β2 m mRNA level by up to 95%. A reduction of HLA class I molecules by up to 80% was observed on the cell surface of the hematopoietic progenitor cells. After 6 days, 75% of the progenitor cells had differentiated to HLA-silenced CD41 + megakaryocytes. RNAi-mediated knockdown of HLA expression was maintained and detected beyond 15 days after beginning of differentiation. A mean of 4 × 105/ml of HLA-deficient platelets was obtained upon 20 days of differentiation. Our data show that generation of HLA class I-deficient platelets from hematopoietic progenitor cells is feasible. Provision of HLA-deficient platelet units from large-scale production of cultured thrombocytes may become an important component in the management of patients with platelet transfusion refractoriness.
Targeting the immunological synapse by silencing PECAM-1 expression for organ transplantation
Session: Immunity, tolerance, vaccines
Immune recognition and rejection of allogeneic transplants is the main obstacle for long term allograft survival. Platelet endothelial cell adhesion molecule (PECAM-1) is an immunoglobulin-like glycoprotein, involved in leukocyte migration, cellular adhesion, immunological synapse stabilization, and signal transduction. We specifically silenced PECAM-1 expression by RNA interference (RNAi) to circumvent allorecognition. We designed lentiviral vectors expressing short hairpin RNA (shRNA) to target PECAM-1. Silencing of PECAM-1 on monocytes and endothelial cells was measured at protein level by flow cytometry and at RNA level by Real Time RT-PCR. PECAM-1 expression was quantified in an antigen binding capacity assay. The cytotoxic potential of T cells directed against PECAM-1 silenced cells was evaluated by measuring granzyme B mRNA levels using real time PCR. The expression of PECAM-1 was decreased by up to 80% at the protein as well as the mRNA level on monocytes and endothelial cells. The antigen capacity assay showed that PECAM-1 silencing induced a reduction in cell surface expression by up to 80%. Granzyme B production decreased by up to 85% on the T cells stimulated with the PECAM-1 silenced monocytes in comparison with the non-specific shRNA expressing monocytes. We showed that silencing PECAM-1 expression is feasible in a permanent way. Silencing of PECAM-1 expression demonstrated an efficient destabilization of the immunological synapse, and may significantly decline the allogeneic inflammatory process by limiting leukocyte extravasation and graft allorecognition. Targeting the immunological synapse and lymphocyte migration may provide a new approach to overcome rejection in allogeneic transplantation.
Clinic of Immunology and Rheumatology, Hanover Medical School , Germany
Session: Immunity, tolerance, vaccines
Halting the spread of Hepatitis C virus (HCV) and also eradicating HCV in subjects with chronic infection are major goals for global health. Several years of HCV vaccine development were undertaken with the concluding remark that potent multi-antigenic and multi-functional vaccine types will be warranted for effectiveness. In this study, we evaluated lentiviral vectors (LV) expressing clusters of HCV structural (LV-HCV-S: co-expressing Core, E1, E2, p7) and non-structural (LV-HCV-NS: co-expressing NS2, NS3, NS4A, NS4B, NS5A, NS5B) genes for future vaccine development. Batches of high titer (10 mcg p24/ml, equivalent to > 10e8 particles/ml) LV were used to transduce differentiated human Dendritic Cells (DC) or primary monocytes. We report successful delivery of HCV gene clusters to monocyte derived DCs (grown with rGM-CSF and rIL-4), but particularly into monocytes prior to their differentiation into DCs, leading to > 95% LV-HCV-S and LV-HCV-NS transduced cells (assessed by intracellular staining of HCV proteins). Intracellular expression of HCV proteins in monocyte-derived DC resulted in immunophenotypic changes, such as downregulation of CD83 and CD86. Nevertheless, monocytes expressing NS proteins and differentiated into DC stimulated effectively allogeneic and autologous CD8 + and CD4 + T cells in vitro and resulted in antigen-specific CD8 + T cell responses against NS3, NS4A and NS5B. Hence, lentivirus-mediated expression of the multi-antigenic HCV-NS cluster in monocytes subsequently differentiated into DC is a novel potential anti-HCV vaccine modality.
Clinical Immunology Division, Uppsala University , Sweden
Session: Immunity, tolerance, vaccines
Chemotherapy is standard cancer treatment but it is rarely curative and adjuvant therapies are warranted. We have shown that adenoviral CD40L gene therapy is successful in bladder cancer. CD40L is a strong inducer of anti-tumor immune responses. It is also inducing growth inhibition and apoptosis in CD40 positive tumors. We have shown that the combination of 5-Fluorouracil (5-FU) and AdCD40L enhances the apoptotic effect in human bladder cancer cells. In this study we combined AdCD40L and 5-FU to treat murine orthotopic bladder cancer. AdCD40L and 5-FU synergized to inhibit the proliferation of MB49 cells in vitro and up-regulated cell surface molecules and immunoproteasome components associated with tumor antigen processing and presentation, such as MHCI, Fas, B7.1 and TAP1/2. MB49 cells were implanted into C57BL/6 mice. Mice received 3 local treatments of AdCD40L (1 × 10e8 ffu), 5-FU (50 mg/kg), or the combination. Survival of mice, systemic immunity and the presence of activated tumor infiltrating T cells were monitored. The combination of AdCD40L and 5-FU was superior to either agent alone. Thus, the combination therapy cured 68% of large orthotopic tumors while AdCD40L alone cured 32%. Cured animals were re-challenged with a s.c. injection of MB49. All cured mice treated with the combination showed systemic MB49-specific immunity. To investigate the role of the immune system, the treatment regiments were tested on MB49 cells growing in NUDE mice. Although the combination treatment could enhance survival, no mice were cured. In conclusion, AdCD40L gene therapy is promising as adjuvant treatment to conventional chemotherapy such as 5-FU.
Factor IX gene therapy for the treatment of hemophilia a complicated with inhibitory antibodies
Session: Immunity, tolerance, vaccines
The treatment of patients with inhibitory antibodies remains a major challenge in hemophilia therapy. Here, we pursue a gene transfer strategy using FIX variants which do not require FVIII. In initial variant screening experiments, we combined three to five amino acid substitutions which led to the generation of FIX variants with 17% clotting activity (V181I/K265T/I383V) in absence of FVIII (ca. 100-fold increase compared to wild type FIX). FVIII inhibitor bypassing activity was confirmed in plasma of patients with high titers of inhibitory antibodies. Further, three different variants were expressed in FVIII knockout mice using non-viral gene transfer. At FIX expression levels ranging from 7500 to 19000 ng/ml partial normalization of the aPTT (from 70 to < 50 sec.) and of blood loss following tail clip assay (1.5 and 3 mm) were observed in all three variant groups (n = 5–9 mice/group, p < 0.05–0.005), while wild type FIX expressing mice did not differ from untreated animals. Similar results were obtained in FVIII knockout mice with high titers of anti-FVIII antibodies. Further, the efficacy of the FIX variants with FVIII bypassing activity was confirmed following laser induced injury of a cremaster arteriole by in vivo imaging technology. While no formation of a stable clot could be observed in FVIII knockout mice treated with wild-type FIX, a stable clot formed at 8 out of 8 injury sites using the V181I/K265T/I383V variant. The described FIX variants therefore offer a new FVIII inhibitor bypassing strategy. The use of these not previously activated proteases could presumably allow prophylactic treatment or even gene therapy in inhibitor patients.
Department of Virology, University of Turku , Finland
Session: Immunity, tolerance, vaccines
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the CNS and the primary disease model for multiple sclerosis (MS). T-helper 1(Th) cells and Th17 cells are believed to be critical during disease onset, whereas Th2 cells are connected with the recovery phase. The Th1/Th2 paradigm has been targeted for potent treatments of EAE. We and others have previously shown that HSV1 vectors expressing interleukin- (IL-) 4 have beneficial effects in EAE recovery (Broberg et al 2001). IL-10 also has a regulatory role in EAE. The role of IL-5 has not been widely studied. In the present study we treated female SJL mice, induced for EAE, with intranasally administered HSV1 vectors encoding IL-5 or IL-10 (the viruses R8316 and R8308) on day 6 post EAE induction. We observed the clinical EAE scores of the mice daily and obtained samples from the brains and trigeminal ganglia (TG) on days 10, 14 and 21 p.i. for study of the cytokine and TLR gene expression. The treatment with both the IL-5 and IL-10-coding HSV alleviated the clinical EAE. The IL-5 HSV was most effective showing a significant decrease in clinical grades starting day 15 p.i. Mice treated with the IL-5 HSV also showed a significant reduction in infiltrating lymphocytes in the brain at day 21 p.i. From the cytokine PCR-panel we found that both HSV1 vectors downregulated IL-17 during the onset of disease (day 10 p.i) and upregulated IFN-β. We also observed a significant decrease in TLR2, TLR3, and TLR9 gene expression in brains in the HSV vector-treated groups in comparison with the untreated EAE on day 10 p.i. The TLR changes, induced by HSV vectors, may interfere with the establishment of EAE. We thank Dr. B.Roizman for the viruses.
Department of Gene Regulation and Differentiation, Helmholtz-Centre for Infection Research , Germany
Session: Immunity, tolerance, vaccines
Tumour associated antigens (TAA) are usually mutated auto-antigens or auto-antigens with an altered expression pattern which are hardly recognized by the immune system due to tolerance mechanisms. For immunotherapy of tumours it is necessary to break this tolerance and minimize side effects induced by autoimmunity. Previous studies showed that immunization with dendritic cells (DC), which play a decisive role in inducing and regulating immune responses, could induce an effective anti-tumour response. To investigate the effect of adenoviral transduced DC on immunity and auto-immunity we developed a mouse model based on InsHA mice which have the influenza hemagglutinin (HA) as self-antigen expressed in the prancreatic beta cells of the islets and therefore would develop diabetes due to an auto immune response. Bone marrow derived DC from BALB/c mice were transduced with adenoviral vectors encoding HA-variant model antigens. After application of these adenovirally modified DC we obtain an HA specific immune response in BALB/c mice but not in InsHA mice. This emphasizes the strong tolerance of the InsHa mice against the hemagglutinin-antigen. To boost the immune response an adoptive transfer of CD8 + or CD4 + T cells with HA-specific transgenic T cell receptors was done. In BALB/c mice the immune response was enhanced by this transfer and in InsHA mice we observed HA-specific immune response in spleen and insulitis in the pancreas indicating breaking of HA-tolerance by the adenovirally modified DC vaccine.
Department of Hematology Hemostasis Oncology, Medizinische Hochschule Hannover , Germany
Session: Immunity, tolerance, vaccines
Wilms tumor protein 1 (WT1) is a prognostic marker and a leukemia antigen. Dendritic cells pulsed with WT1 epitopes or transfected with WT1 mRNA are currently being tested in cancer immunotherapy trials. The effects of expression of a truncated form of WT1 (tWT1, lacking the zinc finger domain) in the differentiation of dendritic cells (DCs) were unknown. We have previously demonstrated that DC precursors transduced with lentiviral vectors (LV) expressing GM-CSF and IL-4 lead to development of potent and long-lived Self-differentiating Myeloid-derived Antigen-presenting-cells reactive against Tumors (SMART-DCs). SMART-DCs co-expressing tWT1could thus enhance the graft-versus-leukemia effect. A tricistronic self-inactivating third generation LV co-expressing GM-CSF, IL-4 and tWT1 with 2A elements and non-coding WPRE was produced (>3g/mL of p24 equivalent, equivalent to approximately 3 × 107 particles/ml). CD14 + monocytes transduced with the tricistronic LV and maintained in the culture without exogenous addition of cytokines resulted in the generation of SMART-DCs (CD209 + , CD86 + , CD80 + and HLA-DR + ; purity 50% at day 7 and 40% at day 14), expressed cytokines autonomously (GM-CSF 2 ng/ml; IL-4: 0.3 ng/ml), co-expressed tWT1 mRNA (at day 14: 200 fold higher WT1 mRNA copies compared to baseline endogenous control ABL mRNA) and were viable for 3 weeks. Thus, tWT1 co-expression did not hinder DC differentiation of long-lived SMART-DCs. Preclinical evaluation of autologous and allogeneic T cell stimulation/expansion with SMART-DCs/tWT1 is underway.
Graduate School of Pharmaceutical Sciences, Osaka University ., Japan
Session: Immunity, tolerance, vaccines
Infection with replication-incompetent adenovirus (Ad) vectors results in rapid activation of innate immune responses, such as inflammatory cytokine production and tissue damage. The precise mechanism of the innate immune response against Ad vectors remains to be clarified. We and others have demonstrated that activation of innate immune responses against Ad vectors is partially dependent on TLR9/MyD88 signaling. As (a) sensor molecule(s) to detect DNA or RNA in the cytosol, DNA-dependent activator of IFN-regulatory factors (DAI), retinoic acid-induced gene I (RIG-I), and melanoma differentiation-associated gene 5 (Mda5) are well known. The adaptor protein, IFN-b promoter stimulator (IPS)-1, is recruited to such sensors after stimulation with DNA or RNA, leading to innate immune responses including type I IFN production. To examine the role of RIG-I, Mda5, and IPS-1 in the Ad vector-induced innate immune response, we prepared MEFs from wild-type, RIG-I-deficient, Mda5-deficient, and IPS-1-deficient mice, and examined IFN-b production after infection with Ad vectors. Following stimulation with Ad vectors, the amount of IFN-b production was significantly decreased in IPS-1-deficient MEFs, when compared with wild-type cells. On the other hand, both RIG-I- and Mda5-deficient MEFs produced the same level of IFN-b as wild-type cells after infection with Ad vectors. These results suggest that TLR-independent recognition is involved in innate immune activation by Ad vectors. This study provided important insights into mechanisms of Ad vector-triggered innate immune responses.
Center of Animal Biotechnology and Gene Therapy, Universitat Autonoma de Barcelona , Spain
Session: Liver, intestine, pancreas, lung
Type 1 diabetes is a severe metabolic disorder which results from the autoimmune destruction of pancreatic β cells leading to insulin deficiency. In addition to developing more efficient insulin replacement therapies, new strategies able to prevent the development of the disease by restoration of tolerance to self antigens and/or control of autoreactive T lymphocytes are also needed. Exploiting the unique tolerogenic properties of the liver by using an appropriate transgene who's expression is targeted to specific immunomodulatory cells is one approach to achieve this end. We have previously reported that expression of IGF-I in β cells of transgenic mice counteracts diabetes development. In addition, subcutaneous administration of recombinant IGF-I to NOD mice reduces the incidence of T1D. Therefore, the aim of this study was to combine the immunomodulatory properties of the liver with the protective effects of IGF-I to develop a non-viral, liver-directed gene therapy approach for type 1 diabetes. We found that hydrodynamic tail vein (HTV) injection of a plasmid expressing IGF-I under the control of a ubiquitous promoter decreased the incidence of diabetes in a mouse model of the disease, reaching permanent protection after ten administrations of IGF-I plasmid. Our results suggested that IGF-I expression in liver non-parenchymal cells may be a key process in mediating protection from T1D. We also show that Treg cells may play a central role in disease prevention. Overall, this study indicates that non-viral, liver-targeted expression of IGF-I may represent a new gene therapy approach to prevent T1D. Supported by grants from Plan Nacional I + D + I (SAF2008–00962) and the European Community (FP6 CLINIGENE, LSHB-CT-2006–018933).
Department of Internal Medicine I, ZAMED, Martin Luther University Halle-Wittenberg , Germany
Session: Liver, intestine, pancreas, lung
Recent studies indicated an important role of stem cells during tumor formation. Mesenchymal stem cells (MSC) are multipotent cells participating in repair of damaged tissues and organs. According to several analyses MSC can be a source of neoplasia (“tumor stem cell”) and are able to affect their growth potential and phenotypic properties. In the present study we investigated the interaction between adipose derived MSC (adMSC) and liver tumor cells (HUH7) in vitro and in vivo. Coculture of adMSC and HUH7 cells resulted in nucleus aberrations in tumor cells and an increased expression of the oncofetal RNA binding protein IGF2BP1 in stem cells depending on the ratio of cultivated cells. Secondly, we injected a mixture of adMSC and HUH7 cells in immundeficient mice subcutaneously to see whether the ratio of adMSC influences the formation of xenotumors. As compared to control tumors composed of HUH7 cells mixed xenotumors with an increased ratio of adMSC were smaller in shape or even the tumors were not formed. Histochemical analyses of mixed xenotumors demonstrated distinct areas of adMSC and HUH7 cells. When adMSC were injected into preformed small HUH7-xenotumors the tumors stopped to grow or disappeared. This results might be open the perspective using MSC isolated from adipose tissue in the treatment of liver cell tumors.
Molecular Virology and Gene Therapy, Catholic University of Leuven , Belgium
Session: Liver, intestine, pancreas, lung
JRG Stem Cell Biology, Hannover Medical School , Germany
Session: Liver, intestine, pancreas, lung
Alpha1-antitrypsin is secreted by the adult liver into the bloodstream where it inhibits a wide variety of proteases by covalent binding. In humans, the PiZ mutation in the SERPINA1 gene encoding alpha1-antitrypsin was described. Individuals homozygous for the PiZ-isoform (PiZZ) suffer from severe alpha1-antitrypsin deficiency as the protein aggregates and is retained in the endoplasmic reticulum of hepatocytes. The accumulation of this misfolded protein severely deteriorates the metabolic liver function and leads to chronic liver damage. Here, we investigate a combined stem cell-based and gene therapeutic approach in a transgenic mouse model overexpressing the human PiZ form of alpha 1-antitrypsin. We generated induced pluripotent stem cells (iPS) from this mouse strain using lentiviral vectors encoding the three transcription-factors Oct4, Sox2 and Klf4 (MOI = 4 per vector). Two independently established PiZ-iPS lines were transduced using another lentiviral vector encoding a dsRedEx-coupled shRNA directed specifically against the point-mutation (E342K) of the human PiZ transgene (MOI = 126). These gene-corrected and pluripotent cells are currently being subjected to a hepatic differentiation protocol and down-regulation of the PiZ-transgene expression will be analyzed in vitro. In conclusion, we provide evidence for a stem cell-based gene therapy approach in liver disorders by transplanting these cells back into PiZ-mice given the regenerative potential of such gene-corrected cells.
Institute of Cell and Molecular Pathology, Hannover Medical School , Germany
Session: Liver, intestine, pancreas, lung
Airway regeneration after lung transplantation depends largely on the epithelial-mesenchymal interactions, whose failure can result in a fatal disease like bronchiolitis obliterans. Crucial initiating events of this disease seem to originate in the subepithelial mesenchymal tissues rendered incapable of normal interaction with epithelial stem or progenitor cells, from which the regeneration apparently starts. To characterize such events we should first understand more precisely the mechanisms of the tissue interactions. In pursuance of the in vitro 3D approach, a membrane insert system enabled us to combine a fetal hamster stem-equivalent cell line M3E3/C3 and human airway wall-derived mesenchymal cells (MCs). Seven different combinations were tested including M3E3/C3 plus MCs, M3E3/C3 or MCs alone. The most important was a complete combination consisting of M3E3/C3 plated on MCs embedded in a collagen gel in which the medium kept flowing cyclically using a regulated pump, while the air-liquid interface level was predetermined by a photoelectric sensor. This combination yielded the best 3D tissue architecture, in which clearly pseudostratified epithelia and long cilia were observed. Immunohistochemical and -cytochemical detection of an indicator of polarized apical membrane, ezrin, an initiating transcription factor for ciliogenesis, Foxj1, and cilia was practically comparable to the in vivo observations. A sheet of fibrillar collagen I, collagen IV or Matrigel instead of the living MCs could not support pseudostratification or columnar architecture in the epithelium, although beating short cilia were induced. Medium circulation inside the MCs compartment was essential for the building of columnar epithelium resembling the in vivo situation.
Selective removal of residual embryonic stem cells during differentiation towards insulin-producing cells
Session: Liver, intestine, pancreas, lung
Embryonic stem cells (ESCs) are attractive candidates for generation of surrogate insulin-producing cells for the therapy of diabetes. However, their transplantation may result in teratoma formation. Therefore, a selection process must be applied for removal of undifferentiated cells. We have established an ESC line expressing a suicide gene HSV-1 thymidine kinase (HSVtk) that renders cells sensitive to Ganciclovir (GCV). HSVtk cloned under the ES-specific Oct4 promoter allows elimination of undifferentiated cells. Mouse ES-D3 cells were transfected with a pOct4-HSVtk-eGFP construct. The clonal cell line eHSVtk was subjected to a 4-stage differentiation protocol comprising 7 or 14 days of incubation with 1 μM GCV. Cells were analyzed by qPCR, flow cytometry and Western blot. The eHSVtk cell line remained similar to wild type cells in morphology and ESC marker expression. 19 days of differentiation with 7 days of GCV incubation showed a loss of eGFP fluorescence and small population of SSEA-1-positive cells. Untreated cultures were composed of up to 40 % of eGFP- and SSEA-1-positive cells. GCV treatment showed reduction in mRNA and protein level of most embryonic markers. 14 days of GCV incubation further decreased the percentage of eGFP- and SSEA-1-positive cells and a significant drop in embryonic marker expression was observed. GCV-treated cells showed higher insulin content than untreated cells. The results indicate that the suicide gene HSVtk driven by the stem cell-specific Oct4 promoter can be used to specifically target and effectively remove undifferentiated stem cells. This technique, together with a differentiation protocol into insulin-producing cells, provides the basis for a safer application of stem cells in diabetes treatment.
Pharmacology, University of Valencia-Hospital La Fe , Spain
Session: Liver, intestine, pancreas, lung
This work presents our first approach to human liver hydrocatheterism, emulating the pigs in vivo experiments. Human hepatic segments from chirurgically fresh resected livers were weighed and an injection volume of aprox. 1/5 weight, of saline solution with 20μg/ml of a plasmid (p3c) containing the green fluorescent protein (EGFP) gene was prepared. A catheter with large flow capacity was then introduced through the suprahepatic vein in order to reach an area as large as possible, whose vein access was completely closed by the catheter to avoid the flow back. This correct positioning was confirmed by fluoroscopy and then the plasmid solution was injected at 20 ml/s flow rate. After 3 minutes, thin slices of different areas of the segment were cut to be kept in culture, with DMEM medium at 37°C, 5% CO2. Gene expression of transfected egfp gene was confirmed by fluorescent microscopy. RT-PCRq also demonstrates transfection, more efficient 48 h after injection, reaching up to 425 copies of EGFP mRNA/copy of endogenous GAPDH mRNA, with 2^-DCt = 4 and a transcription index of more than 500.000 copies of EGFP mRNA/copy of EGFP plasmid. Emulation of the in vivo hydrocatheterism process results in good transfection in ex vivo human liver segments which represents another step towards the clinical application of the method. However more experiments with different injection volumes, flow rates and perfussed area sizes must be performed. Work supported by SAF 2007–64492 and AP-095/09.
Human Gene Therapy Division of Medical Genetics, Akdeniz University Faculty of Medicine , Turkey
Session: Liver, intestine, pancreas, lung
Kahraman S Molecular Imaging and Biology in press
Dirice E Human Gene Therapy in press
Session: Liver, intestine, pancreas, lung
Kahraman S. Molecular Imaging and Biology in press
Dirice E. Human Gene Therapy in press
Session: Liver, intestine, pancreas, lung
A number of different epithelial cell types form the inner surface of the conducting airways. Goblet cells produce mucins for the protection of the respiratory epithelium and ciliated cells are responsible for the mucociliary clearance of the airways. Clara cells (CC) are non-mucous and non-ciliated secretory cells. One of their major function is to protect the bronchiolar epithelium of mammals and the upper airways of some species such as mice by producing several products like the Clara cell secretory protein. Furthermore, they may serve as progenitor/stem cells. At present, allogeneic lung transplantation seems to be the only approach to treat patients with terminal pulmonary failure. However, donor organs are considered to be a limiting factor. Therefore, the ability to produce CC in vitro would offer new therapeutic options to treat pulmonary injuries and diseases, including genetic disorders like cystic fibrosis. ESCs may represent a suitable source for the production of CC. Our aim is the sufficient differentiation of CC from mESCs via enhanced definitive endoderm formation. In this context, we are testing the influence of different substrates and growth factors on the serum-free monolayer-based mESC differentiation. The establishment of air-liquid interphase cultures might further increase CC differentiation. By means of RT-PCR, immunocytological and electronmicroscopical analyses we are able to examine the efficiency of our protocol.
Division of Transplantation Surgery, Medical University of Graz , Austria
Session: Liver, intestine, pancreas, lung
Genetic Therapeutics, Jichi Medical University , Japan
Session: Liver, intestine, pancreas, lung
Phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase (PAH) which converts phenylalanine (Phe) to tyrosine (Tyr). In addition to neurotoxicity in infancy and childhood, excessive Phe leads to neuropsychological disturbances in adults, associated with aberrant metabolism of monoamine neurotransmitters. We showed that a self-complementary adeno-associated virus (scAAV) vector with type 8 capsid (scAAV8/LP1-mPAH) completely corrected hyperphenylalaninemia in a mouse model of PKU (Pah[sup]enu2[/sup]). Here we addressed whether the liver-targeted gene therapy also improves the brain monoamine metabolism. Female Pah[sup]enu2[/sup] mice were given 1 × 10[sup]11[/sup] or 1 × 10[sup]12[/sup] vector genomes of vector and sacrificed at 8 weeks postinjection, when the blood Phe was stabilized at normal to near-normal level. The brain amine content in the untreated Pah[sup]enu2[/sup] was dramatically decreased from the heterozygous littermates. In the scAAV-treated Pah[sup]enu2[/sup], the brain amine content was significantly recovered and the catecholamine levels were comparable with the controls. The aminergic deficit in Pah[sup]enu2[/sup] mouse may arise from an inhibitory effect of excessive Phe on precursor uptake such as Tyr and tryptophan. Indeed, an imbalance of brain Phe and Tyr was corrected in the AAV-treated Pah[sup]enu2[/sup]. The result showed that the liver-targeted gene therapy ameliorated the brain amine metabolism in PKU.
Interaction of the enteric nervous system and enterocytes in vitro is increased by growth hotmone treatment
Session: Liver, intestine, pancreas, lung
Department of Pharmacy, Ludwig-Maximilian University , Germany
Session: Liver, intestine, pancreas, lung
Plasmid based gene therapy approaches often lack long term transgene expression in vivo due to silencing or loss of the vector. One way to overcome these limitations is to combine non-silenced promoters with strong viral enhancers. Here we combine cytomegalovirus (CMV) derived enhancer elements with the strong, human elongation factor 1 alpha (EF1a) promoter in a plasmid backbone devoid of potentially immunostimulating CpG sequences. Plasmids containing either the murine or human immediate early enhancer were injected into mice by hydrodynamic delivery and the luciferase transgene signal in the liver was monitored by in vivo imaging. The human CMV enhancer led to an enhanced and prolonged transgene signal compared to the murine enhancer. The elevated expression in the case of the human enhancer correlated with a higher plasmid copy number found in the liver two months after gene delivery. Furthermore, a plasmid with two human CMV enhancer elements led to even higher long term transgene expression. As the human CMV enhancer contains more binding sites for transcription factors than the murine version, we conclude that the number of transcription factor binding sites in the enhancer region is decisive for the expression strength and subsequently the retention of the plasmid in the host cell.
Division of Genetic Therapeutics, Jichi Medical University , Japan
Session: Liver, intestine, pancreas, lung
Adeno-associated virus (AAV) 1-, 8- and 9-based vectors are promising for muscle and liver-directed gene therapy approaches. To estimate the efficacy in humans, we utilized cynomolgus monkey (Macaca fascicularis) for in vivo expression. For intramuscular (IM) delivery, an AAV1-based vector driven by CMV promoter was used; for portal vein (PV) administration, AAV8 or 9 vector driven by a liver specific promoter was prepared. After screening of pre-existing neutralizing antibody (NAb) against corresponding AAV serotype capsid, each vector was injected into young adult male macaques at a dose of 1 × 1012 vg/kg. In IM group, all 3 animals exhibited significant and prolonged transgene expression with various plasma levels (3.9–45.2 % of normal at peak). The difference in expression levels has not been explained by any of the known factors, including NAb status. In PV group, AAV8 was used in 4 macaques, and only 1 animal showed significant expression level (28.8% of normal at peak) throughout the observation period. The rest of the animals turned out to be low-positive for NAb against AAV8 capsid, according to the re-examination using improved analysis. AAV9-based vector was injected into 2 macaques, and one animal showed positive results (5.9% of normal at peak) for about 3 years. Both animals in this group were negative for NAb against AAV9 capsid even in the improved assay. In all PV animals, vector DNA copy number within the liver tissue corresponded well with the plasma level of transgene product. These results indicate the significance of NAb in liver-mediated expression, along with the necessity of improved detection system for NAb especially against AAV8 and 9 capsids.
Department of Veterinary Clinical Sciences, Royal Veterinary College University of London , UnitedKingdom
Session: Liver, intestine, pancreas, lung
Optimal glycaemic control required to prevent diabetes mellitus complications is difficult to attain with current insulin therapy. Muscle targeted gene therapy (MTGT) employing (pro)insulin plasmids proves promising as an alternative. However, decreased hormone levels are encountered with mutant plasmids compared to wild-type plasmids. This study aimed to assess the influence of proteasomal decay inhibition on the efficacy of MTGT with wild-type (hppI1, producing proinsulin) and mutant (hppI4, furin-cleavable, producing insulin) proinsulin-plasmids. pVR1012-hppI1 and -hppI4 transfections were performed on C2C12 murine muscle cells. Medium and lysate samples were harvested 48 hours later. Proteasomal inhibitor MG132 was added to medium for various time periods and at various concentrations (5–24 hours prior to study end; 0–20μmol/l). (Pro)insulin concentrations were determined using ELISA. MG132-free studies confirmed the decreased efficiency of hppI4 (insulin: 17.1 ± 5.7pmol/l) compared to hppI1 (proinsulin: 134 ± 3.5pmol/l). MG132 addition resulted in cell death when exceeding 10μmol/l or 5 hours. HppI1-induced proinsulin levels were unaffected by MG132 addition. HppI4-induced insulin expression was not significantly affected by various MG132 concentrations (5 hours of 2.5μmol/l MG132: 15.6 ± 3.1 pmol/l insulin; 5.0μmol/l: 13.5 ± 2.7 pmol/l; 10μmol/l: 14.1 ± 1.9 pmol/l). The current study could not expose proteasomal decay as a likely cause for inefficiency of mutant constructs.
Ratanamart Current Gene Therapy
Shaw Journal of Endocrinology
Session: Liver, intestine, pancreas, lung
Prospective cell and gene therapy approaches designed to extend islet graft survival in patients with type 1 diabetes
Session: Liver, intestine, pancreas, lung
Diabetes is the fourth leading cause of death in North America. Currently, the main treatment modality for patients with type 1 diabetes (T1D) is insulin injection. Unfortunately, this approach sometimes fails to protect patients from the recurrence of hypoglycemic events, seizures, and coma. The best option of a cure is pancreas transplantation, which can dramatically prolong and improve quality of life. However, this means of treatment remains controversial because of the less favorable outcome due to major surgery and need for long-term immunosuppression. As an alternative, pancreatic islet transplantation may be a better therapeutic option because it avoids the complications associated with whole-organ transplant surgery or the need for lifelong enzyme injections. Even though intra-hepatic islet transplantation has been a promising approach for the treatment of patients with T1D, the success of this approach is limited by the high frequency of non-functioning grafts and secondary graft failure leading to the majority of patients needing to resume the administration of insulin within five years. Thus, the use of gene-altered islets has been suggested as a viable option to increase the success rate in islet transplantation. The envisioned scenario relies on the premise that pancreatic islet grafts protected through therapeutic gene transfer (FasL, TNF and TRAIL) from the immune-mediated cytotoxic T cell attack are expected to function longer after transplantation.1,2
Dirice E et al Hum Gene Ther 10.1089. (2009)
Sanlioglu AD et al J Cell Biochem
Session: Liver, intestine, pancreas, lung
Medical Biology and Genetics and Gene Therapy Unit, Akdeniz University Faculty of Medicine , Turkey
Session: Liver, intestine, pancreas, lung
Influence of keratinocyte growth factor (KGF) on the differentiation of murine embryonic stem cells into alveolar type II epithelial cells
Session: Liver, intestine, pancreas, lung
Type II alveolar epithelial cells (AT2 cells) have important functions, including the production of surfactant and regeneration of lost type I pneumocytes. Therefore, the ability to produce AT2 cells would offer a variety of new therapeutic options to treat pulmonary injuries and diseases. Recently we demonstrated that murine embryonic stem cells (mESCs) are able to differentiate into AT2 cells under partially and completely serum-free conditions. Since current differentiation protocols are not efficient and specific differentiation is so far impossible, identification of key factors of differentiation is mandatory. Potential candidates are especially members of the fibroblast growth factor family (FGF). Aim of this ongoing project is to analyze whether keratinocyte growth factor (KGF, FGF-7), which is known as a strong selective growth factor for primary AT2 cells, is able to support the differentiation of mESCs into AT2-like cells. For this aim, mESCs are differentiated via embryoid bodies. KGF is applied in different concentrations and at different time points during differentiation. Quantitative evaluation is performed in particular by qPCR for surfactant protein C, the only known specific AT2 marker. Preliminary data show that recombinant human KGF supplementation starting early during differentiation has a positive impact on the formation of mESC-derived AT2-like cells. At present, studies are ongoing to verify these initial data and to gain mechanistic insights.
JRG Stem Cell Biology, Hannover Medical School , Germany
Session: Liver, intestine, pancreas, lung
Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs) are thought to hold a promise in regenerative medicine because of their strong self-renewal capacity and their ability to differentiate into derivatives of all three germ layers and to generate functional cell types. In this study, we are aiming for an efficient hepatic differentiation protocols that is applicable for both, hESCs and hiPSCs. Therefore we compared two different protocols for their efficiency in terms of hepatic cell number yield and hepatic maturation. The status of the hepatogenic differentiation was determined by qPCR comparing the expression of hepatic markers as AFP, ALB, and HNF4 and the loss of pluripotency markers (Oct4, Sox2 and Nanog). Functional evidence of the hepatic differentiation was obtained by intracellular glycogen detection, by secretion of albumin into the supernatant, and by analyses of cytochrome P450 type 1A1 activity. The expression of GFP under the hepatocyte-specific albumin promoter after lentiviral transduction was utilized for quantification of the number of hepatogenic differentiated cells by FACS. In conclusion, our study indicates that both protocols lead to differentiated hepatic cells that show an ambiguous gene expression pattern and differences in hepatic functions. For further studies a modified protocol taking advantages of parts from both protocols may result in hepatic cell populations with a higher number of cells with a more mature phenotype that might be validated in xeno-transplantation experiments.
Generation of hepatic-like cells from multipotent adult mouse germline stem cells
Session: Liver, intestine, pancreas, lung
Previously, we reported the establishment of multipotent adult germline stem cells (maGSCs) from mouse testis. Similar to embryonic stem cells, these cells are able to self-renew and differentiate into derivatives of all three germ layers. These properties make maGSCs a potential cell source for the treatment of disease such as diabetes or liver cirrhosis. In this study, we describe the generation of maGSC-derived hepatic-like cells using different differentiation systems. For monitoring and selection of functional hepatocytes maGSCs were stable transfected with a plasmid containing the gene encoding green fluorescent protein (GFP) under the control of the mouse albumin (ALB) promoter-enhancer. Three different pALB-GFP+ cell lines (ALB5, 16, 39) were established and analyzed concerning their hepatic differentiation potential. The amount of undifferentiated Oct3/4+ cells decreased gradually during differentiation in contrast to an increase of endoderm- and hepatocyte-specific markers. 5% ALB-GFP+ cells were found at late stages using cell line ALB16. To receive a higher amount of ALB+ hepatocytes at late differentiation stages we established lineage-selective differentiation protocols. We demonstrated that Activin A increased and accelerated the expression of liver-specific genes and proteins such as AAT, AFP, TTR and ALB. Approximately 50% of the cells expressed AFP and 30% expressed ALB at late stages. Furthermore, these cells exhibited functions associated with mature hepatocytes including albumin secretion. Taken together, these data show that maGSCs can differentiate into hepatic-like cells and make them to a potential autologous source for further developmental studies and biomedical applications.
Overexpression of human ATPase 7B confers high copper resistance to mesenchymal stem cells for therapy of wilson disease
Session: Liver, intestine, pancreas, lung
Mesenchymal stem cells (MSCs) are promising vehicles for cell and gene-based treatment of disease. MSC-based therapy has been applied with great success to various diseases including liver. ATPase 7B, predominately expressed in liver, is a human copper transporter protein. Malfunction of ATPase 7B in Wilson disease (WD) is found in human and in several animals, e.g. LEC (Long Evans Cinnamon) rat. In WD up to 3 mg/g of copper accumulates primarily in liver leading to significant cell death. The study describes for the first time the investigation of ATP7B expression in MSCs and the genetic adaptation of these cells to toxic environments. To overexpress ATP7B we transduced MSCs as well as CHO and HepG2 with vector pMYATP. Transduced cells were incubated with high concentrations of copper (1 mM), and the expression of reporter gene GFP was monitored by flow cytometry. A high expression of GFP was observed in cell lines (>80 %) after copper treatment. The extent of viable cells after prolonged incubation of cells with copper was determined by MTT assays. The percentage of viable cells was compared between untransduced cells or after transduction with vector pMYATP. ATP7B transduced MSCs showed a unique high viability (>65%) at higher copper concentrations (0.5–1.0 mM) that outperformed the viability of HepG2. Furthermore, ATP7B transgene expression is stable over a time period (>60 days) that is sufficient for regeneration of liver. Our in vitro results using long-term cultivated transduced MSC suggest that ATP7B expression may result in a positive selection of such cells in a toxic copper microenvironment. Thus, ATP7B expressing MSCs may represent superior cell transplants for therapy of WD.
Effect of fluid flow on endothelial cells cultivated on poly 4-methyl-1-pentene (PMP) gas exchange membranes
Session: Liver, intestine, pancreas, lung
We could recently show the endothelialization of polymethylpenthene (PMP) gas exchange membranes used in membrane oxygenators and artificial lung systems using human cord blood derived endothelial cells (hCBEC). This endothelialization resulted in significantly reduced thrombogenicity of the membranes without having a major impact on their gas permeability. This is a first promising step towards the development of a biohybrid lung system suitable for the replacement of a deceased organ. In our previous study all experiments were conducted under static conditions, which does not reflect the in vivo situation where cells are exposed to the dynamic environment of the flowing blood. In order to obtain results with more informative value with respect to in vivo performance of the endothelialized membranes, the behavior of cells under flow conditions was investigated. For the desired contribution to the performance of the endothelialized surface the maintainance of an intact monolayer is a critical requirement. We observed good adherence and only minimal loss of cells under physiologic shear stress. There are several publications reporting the impact of shear stress on endothelial cell thrombogenicity and expression levels of leukocyte adhesion molecules. Exposure of endothelialized membranes to shear stress resulted in a slight activation of the endothelial cells but had no adverse impact on the thrombogenicity of the cell layer. This study shows the applicability of endothelialized gas exchange membranes even under dynamic conditions.
Gastroenterology Hepatology and Endocrinology, Hannover Medical School and Twincore Cente r, Germany
Session: Liver, intestine, pancreas, lung
Fetal liver progenitor cell suspensions (FLPC) and hepatic precursor cells derived from embryonic stem cells (ES-HPC) represent a potential source for liver cell therapy. However, the relative capacity of these cell types to engraft and repopulate a recipient liver compared to adult hepatocytes (HC) has not been comprehensively assessed. We transplanted mouse and human HC, FLPC and ES-HPC into a new immunodeficient mouse strain (Alb-uPAtg(+/−)Rag2(-/-)γc(-/-) mice) and estimated the percentages of HC after 3 months. Adult mouse HC repopulated approximately half of the liver mass (46.6 ± 8.0%, 1 million transplanted cells), whereas mouse FLPC derived from day 13.5 and 11.5 post conception (p.c.) embryos generated only 12.1 ± 3.0% and 5.1 ± 1.1%, respectively, of the recipient liver and smaller cell clusters. Adult human HC and FLPC generated overall less liver tissue than mouse cells and repopulated 10.0 ± 3.9% and 2.7 ± 1.1% of the recipient livers, respectively. Mouse and human ES-HPC did not generate HC clusters in our animal model. In contrast to what was expected, adult HC of human and mouse origin generate liver tissue more efficiently than cells derived from fetal tissue or embryonic stem cells in a highly immunodeficient Alb-uPA transgenic mouse model system. These results have important implications in the context of selecting the optimal strategy for human liver cell therapies.
Biochemistry, University of Medicine and Pharmacy Victor Babes , Romania
Session: Mesenchymal and tissue-resident stem cells
The aim of the study is to evaluate the number of cells from different subpopulations of circulating EPC in two groups of metabolic syndrome patients with different ages (30–40 vs. 50–70) before they follow up therapeutic guidelines in order to identify homogenous population groups for a larger study investigating the possibility to use the number of EPC in evaluation of therapy efficiency. The number of cells from CD34 + /VEGFR2 + , CD34 + /CD133 + , CD34 + /CD133 + /VEGFR2 + subpopulations in peripheral blood samples from 20 metabolic syndrome patients (male) divided in two groups (n = 10, age 35 ± 2 vs. n = 10, age 60 ± 3) were measured. The results have shown much higher number of cells for all considered subpopulations in the young vs. aged patients (CD34 + : 124 vs. 25; CD34 + /VEGFR2 + : 43 vs. 9, CD34 + /CD133 + : 86 vs. 16, CD34 + /CD133 + /VEGFR2 + : 27 vs.8). In the younger subjects, each of these subpopulations expresses different surface levels of CD34, VEGFR2 respectively CD133. These subdivisions were not observed in the group of 50–70 years age, suggesting that distinct subgroups with different degree of differentiation exist in the group of 30–40 years age. Age could be used as a parameter to differentiate EPCs endothelial repair capacity based on EPC numbers and the differentiation stages. Consequently, the number and the differentiation stage of EPCs corresponding to a specific range of age could be criteria to appreciate the therapy efficiency.
Enhancement of adenoviral gene transfer to bone-marrow derived mesenchymal stem cells of the rat by magnetic force
Session: Mesenchymal and tissue-resident stem cells
Bone marrow derived mesenchymal stem cells (MSC) have gained considerable attention in view of their potential use in regenerative medicine and tissue engineering. The combination with gene therapy allows to provide MSC with new traits for potentially improved tissue repair. We sought to enhance adenoviral (adv) gene transfer to rat MSC without increasing toxicity. Ad expressing reporter eGFPLuc or bone morphogenetic protein 2 (BMP2) were associated with PEI-stabilized iron oxide magnetic nanoparticles (MNP) and used to infect rat MSC under influence of a gradient magnetic field (MF) (magnetofection). Magnetofection of MSC with an MOI of 2 pfu/cell resulted in ca. 90% eGFP positive cells, corresponding to 5 ng luciferase/μg protein in presence of MNPs and MF; this corresponded to an 80fold increase of luciferase expression as compared to conventional infection. Magnetofection of MSC with AdBMP2 resulted in a more than 100 fold increase in BMP2 secretion as compared to conventional infection. Differentiation potential was not altered when using AdeGFPLuc, but AdBMP2 infection of MSC resulted in an increase in calcium deposition and GAG formation as indicators of osteo- and chondrogenic differentiation. Magnetofection is able to enhance adv gene transfer of rat bone marrow derived mesenchymal stem cells at low MOI without loss of differentiation capacity. Enhanced adv mediated BMP2 expression leads to increases in osteo- and chondrogenic potential without major toxicity.
University of Veterinary Medicine , Small Animal Clinic, Germany
Session: Mesenchymal and tissue-resident stem cells
A decreasing expression of the DNA-binding protein high mobility AT-hook 2 (HMGA2) has been observed during aging of hematopoietic as well as neural stem cells. Mechanistically, HMGA2 has been shown to reduce the expression of the INK4a gene which in turn can induce cellular senescence. Accordingly, transfection of aging stem cells with a vector expressing HMGA2 can rescue their self-renewing potential. On the other hand, gene transfer of HMGA2 which when over expressed may induce genomic instability raises several safety issues. Thus, we have made a fully synthetic fragment of HMGA2 still containing its three DNA-binding domains. We were able to show that this fragment - is able to penetrate spontaneously hematopoietic as well as mesenchymale stem cells of bone marrow and adipose origin - increases the proliferation of mesenchymal stem cells up to 4.28 fold (bone marrow derived) and 2.23 fold (adipose tissue-derived), respectively, at a concentration of 50 mM/ml culture medium. In summary, synthetic fragments of HMGA2 offer promising tools for substitution of this protein in somatic stem cells aimed at increasing their growth potential.
Lab. Exp. Orthopaedics, Saarland University Medical Center , Germany
Session: Mesenchymal and tissue-resident stem cells
Articular cartilage lesions are an important problem in orthopaedic surgery. Bone-marrow-derived mesenchymal stem cells (MSCs) may provide suited platforms to deliver therapeutic genes in sites of cartilage injury, allowing for tissue healing and regeneration. Here, we tested the hypothesis that overexpression of a human FGF-2 coding sequence via rAAV transduction allows for the proper chondrogenic differentiation and expansion of human MSCs in an environment relevant for application in vivo. Efficient, sustained FGF-2 overexpression was noted in three-dimensional (pellet) hMSC cultures (up to 375.1 ± 1.6 pg FGF-2/ml/24 h in FGF-2 pellets versus 1.51 ± 0.4 pg/ml/24 h in lacZ pellets). Chondrogenic differentiation was observed in all types of pellets, as evidenced by staining for toluidine blue, type-II collagen, and SOX9, whereas reactivity to alizarin red, type-X collagen, and Runx-2 (as markers of terminal differentiation) was less intense. Remarkably, the diameters (856 ± 30 versus 538 ± 36 μm), cell numbers (862 ± 14 versus 435 ± 12 cells/mm2), and DNA contents (1.039 ± 0.005 versus 0.942 ± 0.009 ng/mg total proteins) were higher in the FGF-2 pellets compared with the lacZ pellets (P < 0.001), although there was no difference in the proteoglycan contents (0.80 ± 0.10 versus 0.77 ± 0.05 ng/mg total proteins) and type-II collagen contents (0.023 ± 0.005 versus 0.020 ± 0.010 ng/mg total proteins) between the pellets (P = 1). The data show that FGF-2 can be overexpressed in hMSCs via rAAV, allowing for the chondrogenesis and proliferation of cells in a three-dimensional system. These results suggest that genetic modification of hMSCs via rAAV has value in developing treatments for articular cartilage lesions.
Clinical Medicine, Faculty of Medicine of Ribeirao Preto , Brazil
Session: Mesenchymal and tissue-resident stem cells
The most widely used source of MSC for cell therapy is the bone marrow (BM). Other tissues such as the umbilical cord also contain MSC and represent an alternative to BM as a source of cells for therapeutic applications. However, verifying if MSC from the umbilical cord vein (UCV-MSC) are equivalent to BM-derived MSC (BM-MSC) is necessary to validate their use in stem cell therapy. Thus, this present work focused on a direct comparison between the biological characteristics of BM-MSC and UCV-MSC. The parameters studied included morphology, immunophenotypic profile, in vitro differentiation potential, proliferative potential, cell cycle, cytogenetic, telomere length, relative expression of selected genes assessed by real-time PCR, angiogenic potential and immunomodulatory activity in vitro. Not surprisingly, UCV-MSC shared most of the characteristics with BM-MSC, including fibroblastic-like morphology, multilineage differentiation capacity, surface molecule profile, doubling time (approximately 67 hours), cell cycle status, absence of chromosome abnormalities, expression of CD140b, Anexin V, NG2, CD271, and expression of immunomodulation-, migration- and osteogenesis-related genes. UCV-MSC exhibited high proliferative potential (17.86 ± 9.98 PD), longer telomeres (13.48 ± 2.55 Kb), marked pro-angiogenic activity in vivo (6,83 ± 4,44 vessels/area), higher levels of some surface proteins (CD146, CD73, CD54, CD105, CD106) as well as some genes (CXCR4 and ITGA4), and an important immunossupressive effect on CD3 + cells in vitro. Taken together, these results indicate that UCV-MSC can be used as an alternative to BM-MSC in different types of therapy, with the added advantage of greater accessibility and painless procedures to donors.
Integrin alpha 2: loss-of-function analysis in human mesenchymal stem cells
Session: Mesenchymal and tissue-resident stem cells
Human mesenchymal stem cells (hMSCs), as a source for cell-based regeneration and tissue engineering, are extensively studied on different levels of complexity. Although there is a considerable amount of data collected, a further exploration of the mechanisms that control hMSC adhesion and migration will be beneficial for their in vivo application. Therefore, in this study we aimed to investigate the role of integrin alpha 2, a major collagen I-binding receptor, in hMSCs. First, HMSCs were lentiviraly transduced with integrin alpha 2-specific-shRNA. We achieved 80–95% knockdown of the integrin mRNA, a finding which was also confirmed on protein level. Subsequently to the transduction, we did not notice pronounced morphological changes, however, a clear decrease of alpha 2-shRNA-hMSC numbers was observed during cultivation. Using a quantitative adhesion assay, we estimated that 30 min after platting only 30% of alpha 2-shRNA-hMSCs were attached while control hMSCs have already reached 65% adhesion. Furthermore, a time lapse-based investigation showed that control hMSCs needed approx. 35 min to fully spread out on collagen I whereas alpha 2-shRNA-hMSCs took twice the time for spreading. In addition, hMSCs with alpha 2 knockdown demonstrated significantly reduced migration capacity (50%) which was measured by migration distance and cell velocity. Since a loss of alpha 2-shRNA-hMSCs was observed, we next performed JC-1 staining that visualizes mitochondrial depolymerisation, a hallmark of apoptosis. The majority of alpha 2-shRNA-hMSCs exhibited mitochondrial leakage whereas control hMSCs showed intact mitochondrial staining. Our results strongly suggest that integrin alpha 2 mediates an important signalling for hMSC adhesion, migration and survival.
Institut für Technische Chemie, Leibniz Universität Hannover , Germany
Session: Mesenchymal and tissue-resident stem cells
III. Medizinische Klinik, Klinikum rechts der Isar München , Germany
Session: Mesenchymal and tissue-resident stem cells
Mesenchymal stem cells (MSC) are a mixture of different cell types, of which only a some are therapeutically relevant. Surface markers specifically identifying non-differentiated MSC from their differentiated progeny have not been described in sufficient detail. We here compare the gene expression profile of the in vivo bone-forming bone marrow-derived MSC (BM-MSC) with non-bone-forming umbilical vein stromal cells (UVSC) and other non-MSC. Clustering analysis shows that UVSC are a lineage homogeneous cell population, clearly distinct from MSC, other mesenchymal lineages and hematopoietic cells. We find that 89 transcripts of membrane-associated proteins are represented more in cultured BM-MSC than in UVSC. These include previously identified molecules, but also markers like NOTCH3, JAG1, and ITGA11. We show that the latter three molecules are also expressed on fibroblast colony-forming units (CFU-F). Both NOTCH3 and ITGA11, but not JAG1, further enrich for CFU-F when combined with CD146, a known marker of cells with MSC activity in vivo. Differentiation studies show that NOTCH3 + and CD146 + NOTCH3 + cells sorted from cultured BM-MSC are capable of adipogenic and osteogenic progeny, while ITGA11-expressing cells mainly show an osteogenic differentiation profile with limited adipogenic differentiation. Our observations may facilitate the study of lineage relationships in MSC as well as facilitate the development of more homogeneous cell populations for mesenchymal cell therapy.
Institute of Cell Biology Histology and Embryology, Medical University of Graz , Austria
Session: Mesenchymal and tissue-resident stem cells
Recent research in stem cell biology identified the fetal membranes of the human placenta as a new source of cells which show phenotypical similarities to bone marrow-derived mesenchymal progenitor cells regarding their multipotent differentiation potential and immunomodulatory properties [1]. Even though amnionic membrane - human mesenchymal stromal cells (AM-hMSC) currently play an important role in stem cell research, their characterization is mostly limited to immunodetection by flow cytometry. Therefore, we performed immunohistochemical studies on cultured cells and term placental cryosections to compare the in situ with the in vitro situation. Furthermore, we cultivated the cells under angiogenic conditions in order to investigate their endothelial differentiation potential. AM-hMSC display a hybrid epithelial-mesenchymal phenotype. They are concordantly positive for the mesenchymal marker vimentin, but some cells also express the epithelial marker cytokeratin. The in situ and in vitro expression of common mesenchymal stem cell markers (CD73, CD105, CD90) and the embryonic stem cell markers Oct-4 and SSEA-4 indicates a multipotent differentiation potential. In the presence of vascular endothelial growth factor (VEGF), the cells change their morphology towards an endothelial, cobblestone-like phenotype and upregulate VEGF receptor-2. This receptor can also be found on vasculogenic and angiogenic precursor cells [2]. In contrast to undifferentiated control cells, they take up DiI-AcLDL and form distinct vascular networks in the matrigel assay. These results suggest that AM-hMSC have the potential to differentiate along the endothelial lineage and might be useful tools for the development of vascular grafts.
O. Parolini et al. Stem Cells
R. Demir et al. Placenta
Session: Mesenchymal and tissue-resident stem cells
Endothelial progenitor cells (EPCs) have moved in the focus of interest due to their ability of postnatal vasculogenesis [1]. We have recently demonstrated that fetal venous endothelial cells show a juvenile phenotype with progenitor cell character [2]. Here, we investigated the progenitor and differentiation potential of adult arterial (a-AEC) and venous endothelial cells (a-VEC) from different human vascular beds (ilia, mesenteries) and compared it to the pheno- and genotype of fetal arterial (f-AEC) and venous endothelial cells (f-VEC) from human term placenta. f-AEC and a-AEC are polygonal shaped cells which grow in loose arrangements and form classical cobblestone monolayers, whereas f-VEC and a-VEC are more spindle-shaped cells growing closely apposed to each other. Both f-VEC and a-VEC show a higher proliferation potential and a stronger gene expression of Oct-4, Sox-2, and Nanog (transcription factors in undifferentiated cells) and the EPC markers CD34 and CD14 than their arterial counterparts. Furthermore, these genes are higher expressed in fetal than in adult endothelial cells. More than 50% of the f-VEC show lipid droplets and calcium deposits after adipogenic and osteogenic conditions, respectively. In contrast, only limited or no induction could be observed in the other cell types.We suggest that endothelial cell cultures include several subpopulations of cells at different stages of maturation. The higher expression of progenitor cell markers on VEC and in particular the adipogenic and osteogenic differentiation potential of f-VEC may indicate the role of VEC as tissue-resident endothelial progenitors. Thus, they may represent a potential tool for regenerative cell therapy.
A. Schmidt et al. Circ Res
I. Lang et al. Differentiation
Session: Mesenchymal and tissue-resident stem cells
Calciphylaxis is a rare disorder characterised by vascular calcifications which most frequently occur in patients with end-stage renal disease (ESRD). Calciphylaxis leads to ischemia and skin and soft tissue necrosis with mortality rates up to 80%. The underlying pathogenetic events are poorly understood. Both elevated serum parathyroid hormone and elevated calcium-phosphate product have been discussed as single causative agents. We hypothesize that mesenchymal stromal cells (MSC) are the cellular origin of the vascular calcifications and that differentiation into the osteogenic lineage occurs after stimulation by serum-derived factors. Since calciphylaxis is often located at the gluteal region, MSC were isolated from adipose tissue of this origin. According to their surface CD marker profile these cells were classified as MSC. These fibroblastoid plastic adherent cells were further cultivated and successfully differentiated into the osteogenic lineage using an established osteogenic induction medium. Moreover, these cells showed the capacity to differentiate into the adipogenic and chondrogenic lineage after application of classical induction media. To elucidate the role of parathyroid hormone and calcium phosphate, MSC were treated independently with one of the agents at concentrations found in patients with ESRD/calciphylaxis and osteogenic differentiation was analyzed. Both substances were sufficient to induce osteogenic differentiation of gluteal adipose tissue-derived MSC in the absence of established osteogenic factors. These results point to a joint role of parathyroid hormone and calcium-phosphate product in the pathogenesis of calciphylaxis and suggest that MSC are involved in the development of vascular calcifications in ESRD.
Neurology, Buddhist Tzu Chi General Hospital , Taiwan
Session: Mesenchymal and tissue-resident stem cells
It has been found that bone marrow mesenchymal stem cells (BMMSCs) have the ability to differentiate into many kinds of tissue-specific cells. Because of ease to isolate and expand rapidly in vitro, bone marrow BMMSCs have great potential for treatment of many acquired and hereditary diseases. We have successfully isolated and expanded human BMMSCs. Ten adult male C57BL/6 mice were subjected to intramuscular injection of 30 μL of 1.2% barium chloride in the left anterior tibialis. One week after the chemical injury, 5 mice in the study group received intramuscular injection of 1 × 106 human BMMSCs in the left anterior tibialis. The mice in the control group received intramuscular injection of the same volume of normal saline. The BMMSCs were labeled with 4′, 6-diamidino-2-phenylindole before implantation. All the animals were sacrificed three weeks after the last injection. The left anterior tibial muscles were isolated and prepared for histochemical and immunofluorescent analysis. We found that the mice receiving BMMSCs had less destructive changes, in terms of variation in fiber size, central nuclearization and fibrosis, as compared with the mice in the control group. Few centrally localized nuclei were noted in the study group. Most of them were transplanted stem cells as evidenced by fluorescent microscopy. Surprisingly, numerous muscle fibers expressed human dystrophin in the mice receiving BMMSCs.Our study shows that BMMSCs can fuse with skeletal muscle cells and rescue the damaged muscle caused by barium chloride. This study indicates a potential autologous cell therapy for acquired muscle diseases.
The role of the endosomal machinery on the maintenance of somatic stem and progenitor cells
Session: Mesenchymal and tissue-resident stem cells
Somatic stem cells have the ability to balance the production of self-renewing and differentiating daughter cells. To maintain this capacity over a long period of time it is required to keep the stem cell pool relatively constant. There is good evidence that the delicate equilibrium of self-renewal versus differentiation is orchestrated by intrinsic as well as by extrinsic factors. Even though different groups observed that daughter cells of hematopoietic stem and progenitor cells (HPSCs) often adopt different cell fates, it remained an open question whether these differences were established by asymmetric cell divisions or by post mitotic decision processes. By the identification of four different proteins which frequently segregate differently into the arising daughter cells, we could show for the first time that human HPSCs indeed can divide asymmetrically (Beckmann, 2007). Remarkably, three of the four proteins are associated with the endosomal compartment. Coupled with findings in model organisms, it seems that asymmetric segregation of endosomes is a general feature of asymmetrically dividing cells (Giebel & Beckmann, 2007). In our ongoing studies we are investigating the role of the endosomal machinery on cell fate specification of somatic stem and progenitor cells. To evaluate changes of the endosomal compartments after interfering with its machinery, we qualified markers allowing us to distinguish different subcellular compartments in HPSCs, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs). Preliminary results will be presented.
Microporation of human mesenchymal stem cells promotes high cellular recoveries and efficient plasmid gene delivery
Session: Mesenchymal and tissue-resident stem cells
Human mesenchymal stem cells (hMSC) constitute an attractive source of transplantable cells, being also considered potential vehicles for cell and gene specific therapy. In this context, non-viral vectors, including plasmid DNA, represent a safe alternative to viral vectors. In recent years, electroporation of hMSC has been attempted and despite promising transfection efficiencies and high cell viabilities (CV), cell recoveries (CR) around 40% were reported. Herein we developed a protocol for hMSC microporation using a Central Composite Face-Centred design (CCF) approach. We were able to evaluate the effect of multiple independent variables, namely electric pulse (EP), pulse width (PW) and DNA amount, and their degree of interaction on microporation efficiency. The response variables were: number of GFP + (green fluorescent protein) cells; CV; CR; and transfection yield. Using 1400 V, 40 ms and 4.5 ug of DNA we have obtained 75% of GFP + cells and a CR of 30%. Using the same EP, higher PW and lower DNA amount, the number of GFP + cells decreased to 58% and CR increased up to 90%. Transfection yield ranged 16–55% and CV higher than 80% were obtained. Cells maintained their differentiation potential. These findings are extremelly useful to delineate efficient non-viral strategies to exploit gene delivery to hMSC, without compromising cell recovery and functionality, in order to harness its potential as cell-based delivery agents in multiple clinical settings.
Optimization of novel nonviral vectors for gene transfer in human bone marrow-derived mesenchymal stem cells
Session: Mesenchymal and tissue-resident stem cells
Bone marrow-derived mesenchymal stem cells (MSCs) might be suitable platforms to deliver therapeutic genes to articular cartilage lesions. Here, we evaluated the efficacy of 15 nonviral vectors to deliver candidate genes (Firefly luciferase versus human IGF-I) in human MSCs, i.e. FuGENE 6 (Fg; Roche Applied Science), Metafectene (Biontex), Lipofectamine 2000/Lipofectamine with PLUS /DMRIE-C (DC) (Invitrogen), Dreamfect/Dreamfect Gold/Ecotransfect (Ec) (Oz Bioscience), Gene Jammer (GJ; Stratagene), Effectene (Qiagen), Turbofectin (OriGene), Mirus (Ms; Mirus Bio Corporation), Gene Juice (Novagen), Transpass D2 (TP; New England Biolabs), and Jet Pei (Polyplus Transfection). Optimal luc activity was noted with Gene Jammer, an effect even more marked in the presence of hyaluronidase. In (GJ)-Luc cells, activity was of 11,053.5 ±7,872.3 (no hyaluronidase) and 15,356.9 ± 11,001.4 RLU/mg total protein (with hyaluronidase, 1.4-fold increase), with control values of 1,206.8 ± 710.2 to 1,373.1 ± 1,021.7 RLU/mg total protein. With the other compounds, activity was of 1,382.1 ± 719.2 (DC) to 6,798.4 ± 2,315.9 (Ec) RLU/mg total protein (no hyaluronidase) and of 1,080.6 ± 625.9 (Fg) to 7,437.3 ± 3,307.4 (Ms) RLU/mg total protein (with hyaluronidase), with control values of 260.5 ± 348.2 (Fg) to 5,768.4 ±4,533.4 (TP) RLU/mg total protein. Gene Jammer was among the less toxic methods tested (13.8 ± 3.4%; Cytotoxicity Detection Kit LDH, Roche Applied Science), with values reaching up to 47.4 ± 2.2%. Successful transgene expression can thus be achieved in hMSCs using the nonviral Gene Jammer compound, suggesting that this method may have value in developing safe therapeutic regimens to treat articular cartilage lesions.
Institute of Technical Chemistry, University of Hannover , Germany
Session: Mesenchymal and tissue-resident stem cells
Searching for new and easily accessible sources of mesenchymal stem cells (MSC), other than bone marrow, is a timely subject that is attracting more and more attention. Research over the last decade shows, that MSC reside in virtually all organs, including extra-embryonic tissues available after child-birth. Placenta, amnion and umbilical cord (UC) represent a rich and non-controversial source of MSC-like cells. The main objective of the present study was the detailed characterization of stem cell cultures derived from whole UC tissue. To obtain cells from UC an explant culture was used. Collected cells were seeded at a density of 4,000 or 500 cells/cm2, respectively, and expanded in αMEM containing 10% human serum. Cell surface epitopes, cell growth and expression of tissue-specific markers following the addition of adipogenic, chondrogenic and osteogenic agents were analyzed by microscopic, spectroscopic, immunological and flow cytometry methods. Cells showed a pronounced presence of CD73, CD90, CD105, and CD44, whereas MSCA-1, CD271, CD31 and CD34 remained undetectable. Moreover, they exhibited the expression of alkaline phosphatase, remarkable and unchanged proliferative activity over approx. 20 population doublings and distinct adipogenic and chondrogenic potential. Cell differentiation into bone-forming cells was, however, less efficient even after addition of potent osteoinductive factors such as BMP-2/7 and 1α,25-dihydroxyvitamin D3. Despite the homogenous immunophenotype we identified in UC-derived cultures two clearly distinct subpopulations displaying differences in cell size, morphology, growth properties and the level of MSC markers expression. Thus, UC represents a valuable alternative source of MSC for regenerative medicine.
Department of Experimental Medicine I, University of Erlangen-Nuernberg , Germany
Session: Mesenchymal and tissue-resident stem cells
Umbilical cord blood (UCB) can be collected without any harm and is used successfully for cell therapies. CD133 is one of the markers employed to isolate stem cells from different sources including UCB. A subpopulation of CD133 (+) cells has been reported to be capable of differentiating into mesenchymal lineages. Here we investigated conditions, under which such differentiation can be induced. CD133 (+) human UCB cells, expanded by stimulation with PDGF and EGF, were labelled with a fluorescent marker and co-cultivated with rat osteoblasts, chondrocytes, myoblasts or cardiomyocytes, respectively. In control experiments, the two cell types were separated by microporous membranes to avoid cell-cell contacts. Differentiation was analysed by immunochemistry, flow cytometry and RT-PCR. Direct co-cultivation of labelled UCB cells with the inducer cells resulted in significant morphological changes and up-regulation of lineage-specific markers. Detection of osteocalcin, collagen type II, myosin heavy chain or alpha-actinin confirmed differentiation of UCB cells to an osteoblastic, chondroblastic, myoblastic or cardiomyocytic phenotype, respectively. In contrast, co-cultures of UCB cells with inducer cells under conditions preventing cell-cell contacts yielded minor if any evidence for such cellular re-programming. Thus, UCB cell expansion and subsequent direct contact with mesenchymal cells can produce distinct mesenchymal cell types, specific to the cells they are in contact with.
Park et al. Tissue Eng
Session: Mesenchymal and tissue-resident stem cells
Adipose tissue-derived stem cells (ASC) may have the potential to be used for various clinical applications, including bone and cartilage regeneration. Recent studies revealed a cell population within freshly isolated ASC that showed a distinct expression of CD34 and exhibited pericyte character. Since ASC are still poorly characterized, we have studied the expression of stem cell markers and cadherins, i.e. transmembrane proteins involved in morphogenesis, in CD34-positive ASC in dependency on osteogenic and adipogenic differentiation. Mesenchymal stem cell markers CD29, CD44, CD105 and CD166 were almost completely expressed (>95% positive cells, analysed by FACS). Whereas expression of CD29 and CD44 remained unaffected by differentiation, CD166 was upregulated in osteogenic differentiation (210% compared to the untreated control set as 100%). CD105 was downregulated with cultivation time (30% reduction after 14 d); differentiation did not induce further changes in CD105 expression. Furthermore, subpopulations of CD34-positive ASC were positive for E- and OB-cadherin (negative for N-, P-, VE-cadherin, analysed by FACS). OB-cadherin positive subpopulation was enriched with cultivation time and by osteogenic differentiation, adipogenic differentiation did not increase E- and OB-cadherin expression. These changes in stem cell marker and tissue-specific cadherin expression could reflect the progression of differentiation. However, knowledge about differentiation characteristics of ASC-subpopulations remains fragmentary.This work is supported by the European Union and the Federal State of Mecklenburg-Vorpommern.
CD14+CD34+ murine bone marrow-derived stem cells show an endothelial and neuronal potential and form spheres in-vitro
Session: Mesenchymal and tissue-resident stem cells
Bone marrow-derived primitive cells can give rise to various cell lineages, however, little is known about the potential of particular defined subpopulations. Here we investigated differentiation capacity of bone marrow-derived cells in long-term in vitro culture. Mononuclear cells were cultured for over 10 passages in DMEM with medium containing 5.5 mM glucose. Such cells expressed mesenchymal markers CD29, CD44, CD105, but they also sustained an expression of non-mesenchymal markers CD14 and CD34 along with neuronal markers βIII-tubulin, nestin and GFAP. The one-week culture in medium with angiogenic mediators induced endothelial differentiation: upregulation of VEGFR-2, CD31 and von Willebrand factor was accompanied by ability to bind B. simplicifolia lectin, incorporation of acLDL, upregulation of V-CAM expression after TNF stimulation and formation of tubes on Matrigel. In contrast, one week treatment by B27 supplement, bFGF, EGF and retinoic acid upregulated neurofilament heavy, tau, tenascin R and neuroglycan C. Moreover, cells were able to generate floating spheres when cultured in B27 without retinoic acid. Interestingly, although sphere formation was proposed to increase multipotency, we found that this protocol abolished differentiation to endothelium and was not critical for induction of neuronal markers. In sum, CD14+CD34+ bone marrow-derived cells showed capacity to differentiate towards endothelial and neuronal cells and could be maintained in long-term culture without losing this potential.
Role of PGE2 in the immunosuppressive properties of adipose tissue-derived mesenchymal stromal cells
Session: Mesenchymal and tissue-resident stem cells
Adipose tissue derived mesenchymal stromal cells (Ad-MSCs) have important immunosuppressive properties over activated T cells proliferation and prevent GVHD in mouse allo-hematopoietic stem cells transplants (Yanez, Stem Cells 2006). Focusing on the mechanisms involved in these effects, we detected that the MSCs inhibited T cell proliferation and production of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6 and IL-12) and increased the immunosuppressive IL-10. These co-cultures produced high levels of PGE2, that when blocked by indomethacin (IDM), preserved activated T lymphocytes proliferation but did not restore the pro-inflammatory cytokine secretion profile. However, we detected an increase in the expression of genes related with proliferation as well as transcription factors and cytokines involved in the TH1/TH2 differentiation pathway of T cells. This shows that PGE2 plays an important role in the immunosuppressive effects of MSCs over activated T cells proliferation, but its blockade does not restore immediately the cytokine secretion profile of activated T lymphocytes. Additionally, we found that MSCs inhibited the maturation of dendritic cells (DCs), demonstrated by low CD83/CD80 expression, decreased secretion of TNF-α by m-DCs, and increased IL-10 by p-DCs. The high levels of PGE2 detected in the culture, blocked by IDM allowed p-DCs but not m-DCs maturation, showing that PGE2 has a key role in the immunosuppressive effects of MSCs over p-DCs, but not m-DCs.
Department of Medical Biotechnology, Jagiellonian University Cracow , Poland
Session: Mesenchymal and tissue-resident stem cells
Recently, we identified in murine adult bone marrow (BM) a population of very small embryonic-like (VSEL) stem cells that are i) smaller than erythrocytes, ii) SSEA-1 + Oct-4 + Sca-1 + CXCR4 + Lin-CD45-, iii) express pluripotent markers. We also found VSEL in other adult organs and in fetal liver. Due to the expression of epiblast antigens and unique methylation status of genes, we hypothesized their embryonic origin. Therefore, we examined if VSEL are present in embryonic tissues and if they develop in embryo or migrate from extra embryonic tissue. We used flow cytometric and ImageStream analysis of VSEL presence in embryo in stages: 8.5 dpc (3–7sp), 9.5 dpc (19–24sp), 10dpc, 11dpc, 12–13dpc and 15–16dpc. We established that very small (4.13 ± 0.32μm) Oct-4 + Sca-1 + Lin-CD45- VSEL are present in embryo in all stages of development including 8.5dpc (before cellular migration from yolk sac). Interestingly, absolute number of VSEL in stages 8.5dpc and 9.5dpc was very low (6.1 ± 1.8 and 1.6 ± 0.1 K cells, respectively) and then increased peaking at 11dpc (312.5 ± 15.9 K). The total number of VSELs per embryo was established on the level observed in adult body at 15–16dpc (163.2 ± 5.9 K). Real-time RT PCR showed high expression of pluripotent genes (Oct-4, Nanog, Fgf5, Lit1 and Eras), confirming primitive nature of these cells. In conclusion, our data suggest that Oct-4 + Sca-1 + Lin-CD45- VSEL detected in adult tissues are originated from embryonic tissues. The number of VSEL increases during development due to proliferation or developmental migration. They may play a potential role in formation of the embryonic body and to establish the future pools of VSEL deposited in different organs which survive until adulthood as embryonic remnants-adult pluripotent stem cells.
Vector Development, Arthrogen BV , Netherlands
Session: Muscle, fat, bone, joints
To support development of our clinical gene therapy treatment of rheumatoid arthritis (RA) a robust assay is needed to measure interferon beta (IFNb) after rAAV5 gene transfer. AAV5 is not efficient in transducing fibroblast-like synoviocytes (FLS) in vitro. Proteosome inhibitors were tested to increase transduction efficiency in FLS, and in a 293 cell line that stably expresses the adenovirus E4orf6 gene product (E4 34 k) under the control of the ecdysone-inducible promoter (2V6.11 cells, ATCC CRL-2784).2V6.11 cells and FLS were infected with rAAV5 expressing IFNβ under control of an NF-kB promoter and treated with various concentrations of the proteosome inhibitors (PI) doxycycline (dox) and etoposide both pre- and post-infection. In 2V6.11 cells ponasterone A (ponA) was added to induce E4orf6 gene expression. To activate the NF-kB promoter, cells were stimulated with TNF with or without IL-1β. 2V6.11 cells with ponA treatment (1 ug/ml) alone gave the highest levels of gene expression when compared to treatment in combination with dox (25 nM to 3.2 uM) and etoposide (5 uM). Both TNF and IL-1b together were required to achieve maximal NF-kB stimulation in 2V6.11 cells. Dox concentrations > 1 uM were toxic with ponA in 2V6.11 cells. FLS were treated with combinations of dox (0.2 to 3.2 uM) and etoposide (5 uM) and dox (0.4 uM) was the most effective PI concentration. FLS only required TNF for maximal stimulation of NF-kB. Dox concentrations > 3.2 uM were toxic to FLS. In all cases Dox treatment was more effective than etoposide treatment at stimulating gene expression. rAAV5 infection of 2V6.11 cells treated with ponasterone A effectively leads to rapid conversion of single stranded rAAV to double stranded DNA and provides robust gene expression.
Department of Biomedical Scienecies, University Teramo , Italy
Session: Muscle, fat, bone, joints
The repairing process occurring in tendon lesions following amniotic epithelial stem cells (AESC) transplantation has been investigated. Experimental lesions were induced in both calcaneal tendons of 5 sheep by injecting 400 I.U. of collagenase. 15 days later AESC were injected under ultrasound guidance into the enzyme-induced lesion of one limb while saline was injected in the controlateral lesion (control). Tendons were harvested from euthanized sheep 7 and 30 days following AESC transplantation. Histological examination of tendons isolated 7 days after the treatment revealed bilateral inflammatory signs with loss of matrix organisation and collagen fibres irregularly oriented. Clear signs of tendon healing were recorded 30 days after stem cell transplantation. The site of injection was in fact characterized by a diffuse presence of repairing connective cells with frequent mitotic pictures and accumulating collagen fibres within the extracellular matrix that started to be organised in parallel arrays oriented along the longitudinal axis of the tendon. Amniotic stem cells, still clearly detectable at this time, did not show any proliferation but they were frequently recorded in close proximity of groups of actively proliferating connective cells. AESC did not cause any detectable inflammatory infiltrations. No clear signs of tendon healing were recorded in the controlateral control tendons were the collagenase-induced lesions were still clearly visible, little cell proliferation was present and collagen fibres were randomly oriented in the extracellular matrix. This research demonstrates that molecular and cellular responses occurring during tendon healing are markedly improved by the presence of allotrasplanted AESC.
Human Gene Therapy Division of Medical Genetics, Akdeniz University Faculty of Medicine , Turkey
Session: Muscle, fat, bone, joints
Terzioglu E. Rheumatology Oxford
Bisgin A. Unpublished
Lub-de Hooge MN Ann Rheum Dis.
Session: Muscle, fat, bone, joints
Therapeutic gene transfer might be a means to regenerate articular cartilage lesions. Here, we tested the hypothesis that simultaneous expression of FGF-2 and tagged (FLAG) sox9 sequences via rAAV improves the healing of osteochondral defects in the femoropatellar joint of rabbits in vivo vis a vis control treatments. 4 months post operation, there were no signs of synovitis, adhesions, or adverse reactions, and no macroscopically differences between the FGF-2/sox9 and lacZ knees. β-gal activity was restricted to lacZ defects, whereas FGF-2 and FLAG expression were seen only in FGF-2/sox9 defects. In contrast, SOX9 was detected in both types of defects, in good agreement with the endogenous expression of this transcription factor. At this time, enhanced healing was observed in FGF-2/sox9 vis a vis lacZ defects. When safranin O-stained histological sections of the defects were graded using a repair scoring system, improved scores were noted for defect filling, matrix staining, cell morphology, architecture, surface, subchondral bone, and tidemark in FGF-2/sox9 defects (P < 0.001), with also improved total score (P < 0.001). Immunoreactivity to type-II collagen was more regular and consistent with the surrounding cartilage in FGF-2/sox9 defects. The data indicate that FGF-2/sox9 can be co-expressed in cartilage defects in vivo via rAAV over extended periods of time, leading to an improved healing capacity. These results suggest that co-application of therapeutic rAAV may have value in enhancing cartilage repair by direct administration to sites of cartilage damage. Further studies are required to evaluate the long-term properties of the repair tissue.
Lab. Exp. Orthopaedics, Saarland University Medical Center , Germany
Session: Muscle, fat, bone, joints
The transfer of therapeutic genes into ligaments may be of use to modulate the healing and remodelling of this tissue. Here, we tested the hypothesis that overexpression of human FGF-2 via rAAV mediates the production of a factor that promotes detectable metabolic changes in human anterior cruciate ligament (ACL) explant cultures in situ vis a vis control treatment. Efficient and sustained transgene expression was observed, as seen by β-gal activity restricted to the rAAV-lacZ-transduced (control) explants or by FGF-2 secretion in the rAAV-hFGF-2-transduced (treated) explants (33.00 ± 0.82 versus 1.25 ± 0.50 pg/ml/24 h in control explants after 19 days), with transduction efficiencies ranging between 80–85%. On immunohistochemical sections, specific transgene reactivity was seen in the entire ligament tissue. The cell densities in the entire ligament and DNA contents of the treated explants were higher than those of the control explants (P ≤ 0.029). In contrast, there was no difference in the type-I and type-II collagen contents between the treated and control explants (P ≥ 0.390), a result confirmed when performing histomophometric analyses (P ≥ 0.228). The data indicate that FGF-2 can be efficiently overexpressed in human ACL explants via direct rAAV transduction in situ over extended periods of time, leading to increased levels of cell proliferation. These results may form a basis for novel treatments in ligament surgery when cell repopulation is the therapeutic goal.
Signalling and Gene Regulation, HZI-Braunschweig , Germany
Session: Muscle, fat, bone, joints
We recently described the BMP2 and SMAD8-dependent formation of tendon-like structures in murine MSCs in vitro and in vivo and their use for regenerative strategies (Hoffmann et al., J. Clin. Invest., 2006). Here, we now assess the capacity of this system to direct the formation of entire ectopic tendon-bone insertions by murine and human MSCs as well. Using this system we were able to control the MSC-dependent generation of ectopic fibrocartilaginous tendon-bone junctions by the adenoviral modification of murine MSCs. Lentiviral modification of human MSCs (hMSCs) exhibited a similar result: hMSCs expressing both the biologically-active Smad8ca and BMP2 generate ectopic tendon-bone insertions, however, without fibrocartilaginous elements. By in situ hybridization and in situ histochemistry we can demonstrate that the modified human or murine MSCs are directly involved in the formation of these ectopic tendinous and bony elements. In addition, we observe a considerable contribution of host-derived stem cells which are recruited into the developing ectopic tendon-bone stuctures. We believe that our finding may allow to control the formation of the type of osteotendinous junction needed for regenerative therapies, eventually. In addition, we were also able to control the formation of tendon-ligament-like structures without bony elements. This may also have considerable importance for the therapeutic replacement of tendon or ligaments as well.
Hoffmann, A. J. Clin. Invest.
Session: Muscle, fat, bone, joints
Notch signaling is involved in many different cell lineage determination processes during embryonic development. Here, we provide evidence that Notch signaling is involved in the regulation of chondrogenic differentiation. We have previously shown that different stages of early chondrogenesis and osteogenesis can be recapitulated during ES cell differentiation in vitro. Condensations of mesenchymal cells expressing markers of early pre-chondrocytic cells such as the transcription factors Sox5, Sox6 and Sox9 developed further into mature chondrocytes which expressed chondrocyte markers such as collagen II. After prolonged cultivation cells showed the phenotype of hypertrophic chondrocytes and later showed signs of ossification. To analyze the function of Notch signaling during this process, we used murine ES cells, carrying a tamoxifen-inducible, activated form of Notch1. The cells were differentiated in vitro as embryoid bodies (EBs). Activation of Notch1 during early stages of EB differentiation (0–2 d and 2–5d) results in induction of chondrogenic differentiation during later stages of EB cultivation. The specific function of chondrogenic regulatory genes for Notch-induced chondrogenic differentiation is further tested by performing knock-down using siRNA. Our data suggest that Notch signaling plays an important role during chondrogenic differentiation.
Department of Nursing, Chung Hwa University of Medical Technology , Taiwan
Session: Muscle, fat, bone, joints
In osteoarthritis (OA), angiogenesis which occurs in osteochondral junction and synovium may accelerate inflammation and contribute to the severity of the disease. Here we investigated the therapeutic effect of an angiogenic inhibitor, thrombospondin-1 (TSP-1), in a rat model of OA by anterior cruciate ligament-transection (ACLT). Adenoviral vector encoding mouse thrombospondin-1 (AdTSP-1) was generated. OA was induced in Wistar rats. The AdTSP-1 was injected intraarticularly in the knee joints after ACLT. The expression of TSP-1, TGF-β, and vessel density were examined in the synovial tissue. The treated joints were assessed morphologically, radiographically, and histologically for disease manifestations. The major sites of adenovirus-mediated gene expression were located in the synovium as well as the transitional and radial zones of cartilage. TSP-1 gene transfer increased TGF-β production, whereas reduced the microvessel density accompanying with reduced infiltrating macrophages and interleukin-1β (IL-1β) levels after treatment. Gross morphological and histopathological examinations revealed that rats treated with AdTSP-1 had reduced severity of OA, as compared with the control rats. Our results show that in vivo adenovirus-mediated TSP-1 gene transfer significantly suppresses the progression of OA, accompanied by reduced microvessel density and inflammation. Furthermore, this study also provides potential applications of TSP-1 gene delivery for the treatment of OA.
Transduction of full-length dystrophin to diaphragm ameliorates respiratory dysfunction in severe dystrophic mouse
Session: Muscle, fat, bone, joints
Myostatin knockdown by exon skipping using anti sense reagents
Session: Muscle, fat, bone, joints
Myostatin (a secreted growth factor) is expressed in skeletal muscle and adipose tissue. Myostatin expression limits the size of muscle during development. Therefore, knocking down myostatin could be a potential strategy to be used in conjunction with dystrophin correction methods for improvement in muscle development in case of muscle wasting conditions. Also, myostatin knockout mice have a dramatic increase in muscle mass, reduction in fat mass and resistance to diet-induced and genetic obesity. Inhibition of myostatin pathway represents a therapy to improve functional outcomes in muscle disorders. Use of anti myostatin antibodies and natural binding partners of myostatin has resulted in increase in muscle mass. These techniques are however accompanied by undesirable immune response and low sustainability over a longer period of time. Other methods involving the use of viral vectors offer long term expression but have the risk of uncontrolled insertion into human genome. Antisense oligonucleotides (AOs) manipulate myostatin pre-mRNA splicing to induce exon skipping as demonstrated in our study. Using different AO chemistries, blockage of motifs (exon splicing enhancers (ESEs) in this case) involved in splicing leads to exclusion of exons and therefore manipulation of gene transcription. We have reported that the use of anti sense oligonucleotides of 2′O-methyl RNA (with a phosphorothioate backbone) and Phosphorodiamidate morpholino chemistries lead to efficient myostatin knockdown and offers a controlled therapy as its dosage can be controlled and monitored as per the need arises. It also has a sustainable effect on the cells as tested in this study.
Full-dsytrophin packaging in lentiviral vector for a gene therapy of duchenne muscular dystrophy
Session: Muscle, fat, bone, joints
A lentiviral vector is one of the efficient gene delivery tools for skeletal muscle fibers as well as myogenic progenitor cells in vivo and ex vivo. The integration ability of lentiviral vector is huge advantage to target DMD muscles, which require a missing gene product, dystrophin, for long life span compare with none integrating gene vectors. As the carrying capacity of lentiviral vector has been considered to be limited within 9 kb, it can package trunciated versions of dystrophin gene, such as micro- or mini-dystrophin, but not 14 Kb of full-length cDNA. Although the over size packaging has been presented, still no useful quantity (titer) of full packed particles could be generated in previous study. Recently, several modifications of lentiviral vector production system successfully achieve to generate relatively higher titer of vector stock. Here we present a cording sequence (CDS) of a full dystrophin cDNA with a 2.5 Kb shorten down in 3′UTR was packed in a VSVG-pseudotyped lentiviral vector. The functional titer was still relatively low, 7.0 × 107 TU/ml, however enough for ex vivo usage, as it could deliver transgene into HEK293T cells, NIH3T3 cells as well as mdx myoblasts. These genetically corrected mdx myoblasts successfully expressed 427 kDa of dystrophin protein, which was provided to formed myotubes by cell fusion in vitro. Our candidate strategy with lentiviral vector carrying full dystrophin CDS might be useful for ex vivo cell therapies.
Technische Universität München , Inst of Experimental Oncology and Therapy Research, Germany
Session: Muscle, fat, bone, joints
TricOs is a resorbable bone substitute already clinically used. It is a biphasic calcium phosphate ceramic composed of a mixture of hydroxyapatite and beta-tricalcium phosphate combining structural stability with osteogenic capacity. For better healing of large bone defects optimization of the biological properties would be an advantage. This could be achieved by localized gene delivery using vectors coding for e.g. growth factors like BMP-2. Based on our knowledge of localized gene delivery using copolymer protected gene vectors (COPROGs) and organic matrices such as collagen sponges or fibrin glue we examined whether a combination of COPROGs and the anorganic calcium ceramic for matrix mediated gene delivery is possible. For in vitro studies we used a novel reporter gene, Metridia luciferase-a protein which is secreted in the tissue culture supernatant. We could demonstrate that our gene vector formulation immobilized on TricOs revealed Metridia luciferase expression over 48 days with a maximum at day 5. Detection of GFP expression exhibited a bright fluorescence of cells which colonized the TricOs granules. For in vivo studies we implanted TricOs granules loaded with COPROGs coding either for BMP-2 or firefly luciferase as control in the musculus lattisimus dorsi in rats. At different time points the bone substitutes were explanted and the formation of newly formed bone was examined by muCT, conventional CT and PETscan. In contrast to the luciferase implants BMP-2 gene vector loaded granules exhibited a better turnover of the bone substitute into newly formed bone and a higher bone density. The finding that ectopic bone formation in the muscle of rats suggests that matrix mediated gene delivery is useful in bone regeneration applications.
Department of Oral and Maxillofacial Surgery, Technische Universität München , Germany
Session: Muscle, fat, bone, joints
Titanium implants with PDLLA coatings comprising copolymer-protected gene vectors were established for localized growth factor gene delivery. Lyophilized vectors were resuspended in PDLLA solutions in organic solvent and applied to titanium foils. Critical size defects were set in rats and covered with titanium coated at different concentrations with PDLLA comprising either BMP-2 COPROGs, or rhBMP-2 or protective copolymer alone, respectively. In vitro, vectors were released in an initial burst within the first day, followed by a phase of sustained release. Gene transfer efficiencies in vitro displayed distinct optima depending on the cell, vector dose and vector-to-PDLLA ratio and correlated with cell viability. Luciferase and BMP-2 gene expression in NIH 3T3 and 293 cells remained detectable for at least one weak after reaching peak expression within 48 hours. In contrast, BMP-2 expression in mesenchymal stem cells became detectable only by day 3, reaching peak levels by day 6. In the in vivo model, BMP-2 COPROGs-comprising PDLLA coated titanium implants gave rise to partial bridging of the bone defects within 14 days and complete bridging within 56 days when the DNA dose per implant did not exceed 12.5 mug. Higher doses led to none-bridging and seroma, possibly due to toxic side effects. Implants coated with rhBMP-2 in PDLLA resulted in a fast defect bridging with a thin, lace-like bone in the defect and a huge amount of heterotopic bone outside the defect. BMP-2 COPROGs induced bone displayed more coarse trabeculae compared with recombinant human BMP-2 induced bone. The delivery of plasmid DNA coding for BMP-2 formulated as COPROG in a biodegradable PDLLA matrix appears to be a potent method for controlled new bone formation.
Studies on expanded c-terminal microdystrophin gene transfer using rAAV vectors in skeletal muscle of dystrophic mdx mice
Session: Muscle, fat, bone, joints
Duchenne Muscular Dystrophy (DMD) is a severe inherited muscle-wasting disease and is caused by mutations in members of a multi-component protein structure called the dystrophin protein complex (DPC). Previous studies have attempted to transfer terminally truncated microdystrophins, however the role of C-terminal domain (CT) is yet to be delineated. CT domain of dystrophin recruits dystrophin associated protein complex (DPC) to form a transmembrane link and perhaps act as mediator of signalling between extracellular matrix and cytoskeleton in muscle fiber. In this study, codon-optimised mouse micro-dystrophin cDNAs have been expanded at the C-terminal region (variants; ΔR4–23/ΔCT, ΔR4–23/Δ75–78, ΔR4–23/Δ76–78, ΔR4–23 microdystrophin) allowing for specific/functional micro-dissection of particular dystrophin regions (eg.dystrophin regional association/localisation studies of syntrophin, dystrobrevin and nNOS binding). These variants have been engineered in AAV plasmid vectors under SPc5/12 muscle-specific promoter, Kozak sequences and domain configurations. We have detected the localization of microdystrophin and part of the DPC at the sarcolemma of the skeletal muscle from mdx mice. Recently reported AAV 9 vector expressing C-terminal extended microdystrophin variants are currently subjected to comparative evaluation in mdx mice by intramuscular injection. Complementation of dystrophin deficiency, transgene stability in muscle tissues and host immune modulation will be monitored with the objective of developing safe and effective muscle gene therapy protocols appropriate to move forward towards clinical trials. The current study will present the role of C-terminal domain of dystrophin protein to restore/assist the function of dystrophin in DMD.
Institute of Virology and Cell Biology, University of Lübeck , Germany
Session: Muscle, fat, bone, joints
Chondrogenesis is a multilevel process with a complex regulatory network that has been shown to be adequatly mimicked by murine embryonic stem cells (ESC) in vitro. Formation of mesenchymal condensations by aggregation of precursor cells is an early key step during this event. Ultrastructural analysis using immunogold-labeling for electron microscopy demonstrated that terminal differentiated stages of cartilage can be investigated in late stages in the ES cell model system. Because we found a high expression of fibronectin (FN) and α5-integrin during early chondrogenic differentiation stages, we hypothesized that FN, a matrix molecule, and its interaction with its main cellular receptor, α5β1-integrin, is of particular importance during early chondrogenic development. FN and α5-integrin expression was analyzed during chondrogenic differentiation of murine ESC using RT-PCR and immunofluorescence staining. Alcian blue staining and immunofluorescence staining for collagen type II were used to monitor chondrogenic differentiation. Early differentiation stages were treated with peptides containing the RGD-tripeptide which is essential for the binding of FN to α5β1 integrin. Integrin downstream signaling was evaluated by analyzation of focal adhesion kinase phosphorylation status. Treatment with RGD-containing peptides altered both the expression levels of FN and α5-integrin. Chondrogenic differentiation was disrupted as seen by a significant decrease of alcian blue stained nodules. These results suggest that the interaction between FN and its main receptor is indeed necessary for an intact chondrogenic development.
Vector Development, Arthrogen BV , Netherlands
Session: Muscle, fat, bone, joints
We are currently developing an AAV vector system to express human interferon beta (IFNb) locally in the joints of rheumatoid arthritis (RA) patients. An additional safety feature is the use of an inflammation inducible nuclear factor (NF)-kB promoter for regulated gene expression during flares of inflammation. Our current vector uses single stranded (ss) AAV packaged into AAV5 capsid. To improve safety by allowing the use of lower doses of vector to achieve the same therapeutic threshold of gene expression we sought to make additional improvements in the vector design. We constructed plasmids with a synthetic intron, WPRE and codon optimized sequence for the IFNb gene, or combinations thereof into self-complementary AAV vectors. The plasmids were transfected into 293T cells and levels of IFNb gene expression were measured by ELISA. During the initial cloning we inadvertently created an open reading frame just upstream of the IFNb gene. The additional ORF reduced NFkB regulated IFNb gene expression by 20% (±2%) (386 vs. 483 ng/ml), and increased non-regulated IFNb gene expression by 284% (±2%) (173 vs. 61 ng/ml), when compared to the double stranded vector with the additional open reading frame removed. The addition of a codon optimized sequence for the IFNb gene gave 352% (±2%) increase in gene expression over the non-codon optimized version (483 vs. 137 ng/ml). Addition of an intron, WPRE or both elements together actually reduced gene expression 29% (±5%), 25% (±9%) and 31% (±14%) (341 and 361 and 332 vs. 483 ng/ml) respectively in the corrected IFNb gene cassette. Codon optimization led to significant improvement in gene expression whereas inclusion of either an intron or WPRE reduced gene expression.
Exploratory Research , Genethon, France
Session: Muscle, fat, bone, joints
Skeletal muscle is an attractive gene therapy target due to easy accessibility, a permissive physiological environment for AAV mediated gene transfer, and an ability to secrete transgenic protein in blood. However, gene therapy in dystrophic muscle may be more difficult than in healthy tissue due to degenerative-regenerative processes as well as to the inflammatory context. In the present work AAV6 vectors encoding chimeric proteins inhibiting the pro-inflammatory TNF-α cytokine (human and murine TNF soluble receptor I fused with the Fc portion of murine IgG1) were injected into either dystrophic mdx mice (a mouse model of DMD) or in healthy C57Bl/10 mice. We conducted an AAV6 dose-response study and determined the expression kinetic of the secreted transgenic proteins by ELISA. Further, we followed the antibody response against the transgene and studied the expression pattern in the injected muscles by immunostaining. When injected at equivalent dose, the transduction efficiency of AAV6 is reduced in dystrophic muscles as compared to the healthy ones; and the humoral response against the transgenic protein is stronger in the mdx mouse. Taken together, our results underscore that the inflammatory context of dystrophic muscles has to be taken into consideration when designing gene therapy approaches. Not only the levels of expression are lower in the dystrophic muscle but the inflammatory context favours immune response against the transgene.
Skeletal muscle repair and clinical improvement in GRMD dogs, after muscle stem (MuStem) cell transplantation
Session: Muscle, fat, bone, joints
Duchenne Muscular Dystrophy (DMD) is a degenerative disorder of skeletal muscle characterized by the lack of dystrophin at the muscle fiber sarcolemma. Today, no effective treatment exists. The demonstration of myogenic potential for several adult stem cells gave a new impetus to the DMD cell-based approach. Here, we investigate the muscle-derived stem cells in the Golden Retriever Muscular Dystrophy (GRMD) dog, the clinically relevant DMD animal model. Using a modified Preplating technique, we isolated from healthy dog muscles stem cells (named MuStem cells) that display extensive proliferation in vitro as adherent or non-adherent cells to form myospheres, asymmetric division and clonogenecity. In specific media, they differentiate into distinct mesoderm cell types (myocytes, adipocytes and osteocytes). Phenotypically and morphologically, they correspond to myogenic cells or uncommitted ones and are distinct from AC133 + cells, mesoangioblasts, SP cells and hematopoietic ones. After intra-muscular injection in GRMD dogs, they can participate to myofiber regeneration, generate satellite cells and restore dystrophin. Also, they could reach skeletal muscle from blood resulting in dystrophin restoration, partial muscle repair and dog locomotion improvement.The MuStem cell use in DMD context displays features in terms of clinical efficiency and persistence that could improve the outcome of DMD cell therapy and positioned these cells as source for future therapeutic strategy.
Department of Microbiology and Immunology, National Cheng Kung University Medical College , Taiwan
Session: Muscle, fat, bone, joints
Physiology Anatomy and Genetics, University of Oxford , UnitedKingdom
Session: Muscle, fat, bone, joints
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that prevent the production of dystrophin protein. Antisense oligonucleotides (AOs) targeted to skip the mutated dystrophin exon can restore functional dystrophin protein in mdx mice. To fully exploit the therapeutic potential of this method requires efficient systemic AO delivery to multiple muscle groups, including heart. Here we report the discovery and application of novel peptides which when conjugated to PMO AOs facilitate highly efficient muscle and heart targeting and enhanced systemic splice correction. Data relating to two novel peptide classes will be presented; chimeric peptide conjugates of the form X-TSP-PMO where X is an arginine rich transduction domain and TSP is a tissue targeting peptide domain (the prototype for which is MSP-a muscle specific heptapeptide), and Pip peptides, a novel series of positively charged cell penetrating peptides, the prototype for which is Pip5e-PMO. Data following systemic intravenous administration of both peptide classes will be presented, showing highly efficient systemic dystrophin splice correction including in heart tissue, with further data showing functional and phenotypic correction in mdx mice. We therefore describe two novel peptide classes with potential for highly efficient targeted AO delivery to muscle and heart which offer promise for the delivery of biotherapeutic agents to these target tissues in clinical studies.
Yin Human Molecular Genetics
Ivanova Nucleic Acids Research
Session: Muscle, fat, bone, joints
Inst. f. Exp. Onkologie u. Therapieforschung , Klinikum rechts der Isar TU München, Germany
Session: Muscle, fat, bone, joints
Therapy of cartilage defects is challenging due to poor self-healing capacity. A combined gene and cell therapeutic approach for in situ production of growth factor Bone morphogenetic protein 2 (BMP-2) by implanted autologous chondrocytes was chosen. Regulation of gene expression was achieved by the Tet on-System, delivered by VSV-G pseudotyped lentiviral vectors.Primary rabbit chondrocytes were coinfected with 2 vectors, expressing the reverse Transactivator (rtTA) and eGFP or BMP 2 under control of the Tet-responsive element (TRE), respectively or a 1-vector system. Transgene expression was induced by doxycycline (dox). The efficacy of the Tet on-System was tested and constructs were optimized using eGFP. Using BMP-2 as therapeutic gene resulted in production of 15–16 ng/ml BMP-2 secreted into the medium by 1- and 2-vector system. After withdrawal of dox and decrease of BMP-2 expression to background levels, the Tet on-System could be reinduced for various cycles without a loss in efficacy. Produced BMP-2 was functional as evidenced by proteoglycan synthesis, in Tet-regulated 1 and 2-vector systems. The lentivirally delivered Tet on-System allows for regulated and repeated expression of BMP-2 in primary rabbit chondrocytes. The amount of BMP-2 produced by chondrocytes after induction in vitro is sufficient for proteoglycan synthesis, a marker for chondrogenesis. The 1-vector system is favourable over the 2-vector system since it allows for reduction of overall virus load.
Experimental Hematology, Hannover Medical School , Germany
Session: Persisting transgenes
Vectors based on adeno-associated virus (AAV) are efficient tools to alter complex genomes by gene targeting. Gene targeting is based on the homologous recombination (HR) pathway, which is mainly available during the G2 phase of the cell cycle. We have shown that insertion of a DNA double strand break (DSB) in the target locus stimulates gene targeting up to 1000-fold, most likely by activating cellular HR repair mechanisms. In a proof-of-concept approach, we generated three human cell lines that carry a copy of a mutated eGFP gene flanked by recognition sites for the meganuclease I-SceI and zinc-finger nucleases (ZFNs). After treatment with different cytostatic drugs, cells were co-transduced with nuclease encoding AAV vectors and HR donor vectors to rescue eGFP expression by gene targeting. The cell cycle profile, the extent of cytotoxicity and the frequency of gene targeting were assessed by flow cytometry. We show that a transient cell cycle arrest before the creation of a DSB in the target locus increased AAV-mediated gene targeting up to 6-fold. Partially, this effect could be attributed to an increase in AAV transduction. Dependent on the cell line and the AAV vector dose, we reached gene editing frequencies of up to 34%. Because the frequency of AAV random integration was not significantly increased, as determined by quantitative real-time PCR, treatment of cells with the chemicals let to a net increase in the targeting ratio. In summary, cytostatic drugs can be used to enhance AAV-mediated gene targeting in different cell lines. The combined beneficial effects of site-specific DSBs and transient cell cycle arrest allowed us to lower the vector dose-and hence random integration-without compromising on the gene targeting efficacy.
Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital , Israel
Session: Persisting transgenes
Lentiviral vector integration into the host cell genome results in long-term transgene expression, however it carries the risk of inducing oncogenesis. In the present study, we transduced fetal mice with a feline immunodeficiency virus (FIV)-based vector carrying the gene encoding EGFP and found that approximately 50% of hepatocytes expressed the transgene at 5 months post-transduction. However, 38% of transduced mice developed hepatocellular carcinomas between the ages of 273 and 484 days. To investigate the link between lentiviral transduction and tumor development, we mapped sites of viral integration and chromosomal aberrations and found them to be unique in each tumor. In contrast, microarray analysis revealed that 5 out of 6 tumors exhibited a common activation pattern of genes associated with the DNA damage response, chromosomal instability, and oncogenesis as well as upregulation of genes controlled by E2F1 transcription factors. Notably, the activated oncogenes and the chromosomal rearrangements were not located in the vicinity of viral integrations. Interestingly, an entirely different gene expression pattern was found in a spontaneous liver tumor from the same mouse species. These findings suggest that FIV-induced oncogenesis in fetal livers may be caused by mechanisms other than a site-specific insertional mutagenic event. We propose that viral transduction induces a global cellular mechanism entailing the DNA repair response that leads to upregulation of E2F1 target genes, resulting in activation of oncogenes, chromosomal instability, and tumor development.
LBPA and CliniGene, Ecole Normale Superieure de Cachan , France
Session: Persisting transgenes (viral and non-viral)
The need for better gene transfer systems towards improved risk/benefit balance for patients remains a major challenge in the clinical translation of gene therapy (GT). We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing genetic integration to heterochromatin. We have designed SIN-insulated retrovectors with two candidate GIEs and could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro (p20) and lentivectors (DCaro4) (see Duros et al, abstract ibid). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, DCaro4 has been further tested with chimeric HIV-1 derived integrases which comprise C-ter chromodomains targeting heterochromatin through either histone H3 (ML6 chimera) or methylated CpG islands (ML10). With DCaro4 only and both chimeras, a homogeneous expression is evidenced in over 20% of the cells which is sustained over time. With control lentivectors, less than 2% of cells express GFP as compared to background using a control double-mutant in both catalytic and ledgf binding-sites; in addition, a two-times increase of expression can be induced with histone deacetylase inhibitors. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent genotoxicity, according to X-SCIDs patients data.Work performed with the support of EC-DG research within the FP6-Network of Excellence, CLINIGENE: LSHB-CT-2006-018933
High performance analysis of genome integrating gene transfer agents
Session: Persisting transgenes (viral and non-viral)
Viral clinical gene therapy trails successfully restored the function of defect genes but also introduced adverse side effects due to insertional mutagenesis. Therefore, integration site (IS) profiling of genome integrating gene transfer agents is inevitable. Viral and non-viral IS can be accessed via LAM- or nrLAM-PCR, which amplify e.g. virus or transposon flanking genomic regions. By incorporating pyrosequencing primers and probe specific tags, hundreds of samples can be pooled and directly sequenced with the 454/Roche Titanium technology. These sequences are then automatically analyzed by Perl scripting together with standardized bioinformatics programs. Pooled samples are sorted by tag and gene transfer vector-specific sequences are removed. The sequences are mapped against the host genome via BLAT and genomic IS are annotated with the next RefSeq gene, CpG island and repeat. Further (semi)automated analysis gives an overview of typical characteristics, such as IS distribution in genes and on chromosomes, IS present at different time points or fractions within one study as well as IS hot spots of certain vector types which are summarized to identify potential harmful clones. With an optimal infrastructure the bioinformatical analysis of more than 400.000 raw sequence reads can be archived in a couple of days. Combination of LAM- or nrLAM-PCR with high-throughput sequencing and an adequate high performance bioinformatics infrastructure gives a time efficient and broad insight into gene transfer studies.
Translational Oncology, German Cancer Research Center , Germany
Session: Persisting transgenes (viral and non-viral)
Retrovirus gene therapy has been successful in inherited monogenetic disorders, but insertion of functional retrovirus LTRs can cause severe side effects. Self-inactivating (SIN) lentiviral vectors have the potential to avoid many of these risks. Here we show that stable and sustained restoration of the genetic defect in cerebral adrenoleukodystrophy (X-ALD) in the first HIV-1 lentiviral SIN-vector gene therapy trial in humans is not accompanied by signs of clonal dominance or even premalignant disproportional distribution of cellular contributions. Integration site (IS) distribution (P1: 2217 IS; P2: 1380 IS) was analyzed in ex vivo transduced cells prior to reinfusion and on engrafted cells (P1: 6–24 months (M); P2: 6 − 20 M) by LAM-PCR and pyrosequencing, revealing gene coding regions as preferred targets for lentiviral vector integration (P1: 72%; P2: 76%). Interestingly, we found common IS (CIS) that affected the same genes in samples of both patients, likely reflecting a preference of lentiviral vector insertion for particular genes. Identical IS were identified in myeloid and lymphoid lineages, indicating successful ex vivo transduction of early hematopoietic progenitors. Our molecular follow-up presents the first molecular data on lentiviral clinical gene therapy in humans. High throughput distribution analysis of the IS repertoire indicates that lentivirus vectors offer great promise for safe and effective correction of human stem and progenitor cells.
Molecular Microbiology and Biotechnology, Tel Aviv University , Israel
Session: Persisting transgenes (viral and non-viral)
Homing endonuclease (HE) genes are selfish elements that reside within introns and inteins and promote their horizontal dissemination. In recent years, HEs have been the focus of extensive research due to their promising potential use in gene targeting procedures. We devised the HomeBase application, a computational assignment of HEs to the targeting of specific human loci, and an experimental methodology for its validation. HomeBase consists of an automated tool for the construction of a comprehensive HE database wherein each HE is listed alongside with the range of sequences it is predicted to cleave. HomeBase also entails a search engine which allows the scanning of the human genome, or any other DNA dataset, for potential HE targets. Using HomeBase we have significantly extended the repertoire of diseases that can potentially be treated by HE-mediated gene-targeting. We validate the capacity of our HEs to induce unique DNA breaks and repair by homologous recombination using a selective yeast system. We then assess the potential of HEs to induce a safe and efficient gene targeting in human tissue cultures and live mice. In particular, we are trying to develop a gene therapy for Hemophilia by inducing the site specific integration of genes encoding coagulation factors into predicted HE target sites that we have found in the human genome.
Departement of Pediatrics, Ludwig-Maximilians-University , Germany
Session: Persisting transgenes (viral and non-viral)
The phiC31 integrase has been used successfully with non-viral gene delivery to mediate recombination between pseudo attP sites in mammalian genomes and plasmid DNA comprising an attB site. Protein delivery of the phiC31 integrase could be a promising alternative to the generally used method of introducing the phiC31 integrase as plasmid DNA together with the target plasmid. The advantages are instant availability of the enzyme resulting in early onset of recombination and transient presence of the phiC31 integrase avoiding unwanted integrations of a phiC31 integrase coding plasmid which may lead to chromosomal aberrations. Our recombinant phiC31 integrase protein comprises a HIV-TAT protein transduction domain for intracellular delivery and nuclear targeting and was expressed in E. coli. We successfully delivered recombinant integrase-TAT fusion protein in various cell lines and confirmed recombination activity by measurement of luciferase expression and FACS analysis. The phiC31 integrase has been previously shown to be of low activity in Jurkat cells if co-transfected as a plasmid DNA. We could confirm recombination activity mediated by recombinant integrase-TAT protein delivered to Jurkat T-cells using a luciferase reporter system. Further, we could observe long-term luciferase expression in Jurkat T-cells transduced with the recombinant integrase-TAT protein and a plasmid encoding the firefly luciferase comprising an attB site for integration. We suggest that transduction of recombinant phiC31 integrase protein is a promising alternative to plasmid DNA codelivery which may improve the safety and efficiency of the phiC31 integrase system in mammalian cells.
Hematology and Immunology, Vrije Universiteit Brussel , Belgium
Session: Persisting transgenes (viral and non-viral)
The EMCV-IRES has been the most popular tool to achieve dual protein expression from one single mRNA molecule. However, a few drawbacks to the system exist: (i) the expression level of the second protein is decreased over the first one and (ii) a well-defined start has to be respected. Lately, the viral self-cleaving 2A peptides have gained a lot of popularity over the EMCV-IRES due to their small sizes and high expression rates of both proteins. A third IRES-like sequence, the “super IRES” (SIRES), was shown to be inferior. The SIRES sequence is a 10x repeat of 9nt identified in the 5′ leader of the Gtx homeodomain and binds ribosomes efficiently. Its repetitive nature might explain why it is not very suitable as bicistronic linker. Nevertheless, when cloned in front of an eGFP reporter gene, we found protein expression to be drastically increased compared to the control plasmid without SIRES. We hypothesize that the 5′CAP together with the ribosomal binding nature of the SIRES contributes to this higher expression. Next, we wondered if this approach could be suitable to boost expression from bicistronic expression cassettes. Indeed, expression levels of both proteins in 2A constructs were elevated. In all, we identified the SIRES-sequence as a mechanism to increase protein expression from both mono- and bicistronic lentiviral vectors. The resulting vectors generate higher protein levels and thus decrease the amount of vector to obtain the desired protein expression levels.
SIN lentiviral vector transduced hematopoietic cells establish polyclonal hematopoiesis in the C57Bl6 model
Session: Persisting transgenes (viral and non-viral)
The study of vector integration sites in hematopoietic gene therapy has largely focused on the emergence of clonal dominance and leukemia. New vectors and cell processing conditions may result in polyclonal rather than oligoclonal gene-modified hematopoiesis. However, with conventional LM-PCR methodology analysis of polyclonal samples is labour intensive and not always exhaustive. We therefore set up protocols and analysis tools to study polyclonal samples arising from C57BL6 mice (n = 56) transplanted with lentivirally transduced cells. We used LM-PCR and multiplexed 454 pyrosequencing of samples obtained at different time points (up to 9.5 months). In addition, we implemented an algorithm to determine which integration form statically significant common insertion sites (CIS). The clonal repertoire obtained for a given experimental condition ranged from 177 to 1162 integrations. Analysis of the retrieved lentiviral integrations showed that 15% occurred at least at 2 different time points, and 2.26% were within + /− 150 kb of oncogenes, which is comparable with gamma-retrovirally transduced cells studied in previous experiments (2.3%). Proto-oncogene integrations did however not result in outgrowth of dominant clones, since no increase in the number of reads of these clones was observed over time and no enrichment in either proto-oncogenes or CIS was seen. These experiments underline that polyclonal gene-modified hematopoiesis can be established in the C57BL6 model, and that LM-PCR in combination with pyrosequencing is a powerful tool to investigate hematopoietic reconstitution.
Clonal dominance with lentiviral-SIN-vectors containing an internal SFFV-promotor
Session: Persisting transgenes (viral and non-viral)
For γ-retroviral vectors insertional mutagenesis was shown to induce clonal dominance as well as malignant transformation of hematopoietic stem/progenitor cells in cell culture assays, animal models and gene therapy patients. Since lentiviral vectors were shown to have a different insertion pattern (no preference for transcriptional start sites of genes), they have been considered less genotoxic. We used our established serial bone marrow transplantation model to investigate whether a lentiviral vector with internal SFFV-promotor-enhancer sequences allows stable transgene expression (eGFP) in reconstituted mice without the side effects described for γ-retroviral vectors. To monitor (clonal) reconstitution, intermediate blood samples were taken every 4 weeks. All transplanted mice showed normal hematopoiesis at final analysis after serial BMT and approx. 1 year observation. LM-PCR was used to investigate vector integration patterns in all transplanted mice and revealed an oligoclonal pattern in both 1st and 2nd cohorts with indications for strong dominance of some clones in the second one. Two out of 9 integrations had occurred in growth-promoting genes (proto-oncogenes Mcts1, Trim32), which potentially contributed to their clonal dominance in the 2nd cohort. However, only for Trim32 transcriptional upregulation was found. In conclusion our data indicates that lentiviral vector with strong viral promotors have the potential to induce clonal dominance in a sensitive in vivo assay.
LBPA and CliniGene, Ecole Normale Superieure de Cachan , France
Session: Persisting transgenes (viral and non-viral)
In otherwise successful gene therapy trials insertional mutagenesis has resulted in leukemia. The identification of new short synthetic genetic insulator elements (GIE) which would both prevent such activation effects and shield the transgene from silencing, is a main challenge. Previous attempts with e.g. β-globin HS4, have met with poor efficacy and genetic instability. We have investigated potential improvement with two new candidate synthetic GIEs in SIN-gamma and lentiviral vectors. With each constructs two internal promoters have been tested: either the strong Fr-MuLV-U3 or the housekeeping hPGK.We could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro and lentivectors. In target cells a dramatic shift of expression is observed with an homogenous profile the level of which strictly depends on the promoter strength. These data remain stable in both HeLa cells over three months and cord blood HSCs for two months, irrespective of the multiplicity of infection (MOI). In comparison, control native and SIN vectors expression levels show heterogeneous, depend on the MOI and prove unstable. We have undertaken genotoxicity assessment in comparing integration patterns ingenuity in human target cells sampled over three months using high-throughput pyro-sequencing. Data will be presented. Further genotoxicity assessment will include in vivo studies. We have established insulated vectors which harbour both boundary and enhancer-blocking effect and show stable in prolonged in vitro culture conditions. Work performed with support of EC-DG research FP6-NoE, CLINIGENE: LSHB-CT-2006-018933
Institute of Virology, Technische Universität München , Munich, Germany
Session: Persisting transgenes (viral and non-viral)
In previous studies we have shown that the human hepatitis B virus (HBV) can be converted into a vector that allows hepatocyte-specific gene transfer. HBV vectors are produced by transfection of a helper-plasmid providing all viral proteins in trans and a transfer-plasmid encoding a pregenomic RNA, in which the genomic region of the small surface protein (S) has been replaced by a transgene. HBV gene expression was eliminated by knocking-out all HBV open reading frames. By deleting the 5′-core region downstream of the HBV encapsidation signal, we were able to enlarge transgene size up to 1250 bp. Additional 3′-core deletions allowed transferring a 1670 bp transgene, but resulted in reduced transduction capacity. Constructs comprising HBV core deletions synthesized mainly linear HBV genomes during reverse transcription. Linear HBV vector genomes, however, were still able to transduce primary human hepatocytes and expressed luciferase and GFP. Because they lack the naive HBV polyA-site, they either had to use an alternative polyA-site upstream of the original one or to re-circularize by recombination to allow RNA transcription. We identified two additional regions within the HBV genome conserved within the different HBV genotypes, which could serve as non-canonical polyA-sites upstream of naive polyA-site and upstream of motifs that could serve as cleavage factor binding sites. By systematic deletions of these regions in genotype D HBV, we were able to prove that both alternative polyA-sites were capable of initiating RNA termination and polyadenylation. Thus, gene expression from linearized HBV genomes may occur using alternative polyA-sites upstream of the naive one. Whether this plays a role in natural HBV infection remains to be determined.
Institute of Biotechnology, University of Lausanne , Switzerland
Session: Persisting transgenes (viral and non-viral)
Gene transfer that relies on integrating vectors often suffers from epigenetic or regulatory effects that influence the expression of the therapeutic gene and/or of cellular genes located near the vector integration site in the chromosome. Insulator elements act to block gene activation by enhancers, while chromatin domain boundary or barrier sequences prevent gene-silencing effects. At present, the modes of action of insulator and barriers are poorly understood, and their use in the context of gene therapies remains to be documented. Using combinations of reporter genes coding for indicator fluorescent proteins, we constructed assay systems that allow the quantification of the insulator or of the barrier activities of genetic elements in individual cells. This presentation will illustrate how these assay systems were used to identify short DNA elements that can insulate nearby genes from activation by viral vector enhancer elements, and/or that can block the propagation of a silent chromatin structure that leads to gene silencing. We will show that small elements of the order of 100–400 nucleotides can be designed to achieve both insulator and boundary function, as needed for safer integrating viral vectors.
G360, German Cancer Research Center , Germany
Session: Persisting transgenes (viral and non-viral)
A feasible option to overcome hematotoxicity is retroviral resistance gene transfer to hematopoietic CD34+ progenitor cells. Since γ-retroviral vector integration was found associated with adverse events, novel lentiviral self-inactivating (SIN) vectors represent a promising alternative. Using LM-PCR and 454 pyrosequencing we assessed the influence of BCNU-chemoselection on integration patterns in human CD34+ cells transduced with a lentiviral SIN vector overexpressing MGMTP140K. Integration site patterns of the BCNU-treated and an untreated group were analyzed and compared to an in silico set with 106 random integrations. We found different integration preferences of the lentiviral vector between the treated or the untreated group and the in silico set: preferential integration into genes and cancer genes were found in both groups. Integration patterns in BCNU-treated cells showed no significant differences to those found in untreated CD34+ cells.Additionally, the local influence of the provirus on the expression of 34 hit and flanking genes was analyzed on a clonal level of CD34+ colonies by quantitative real-time PCR. The expression profiling showed that integration of the provirus did not result in a significant deregulation of any of the integration-flanking genes. In conclusion, we could show that the general distribution of lentiviral integrations in BCNU-treated and untreated CD34+ cells displayed no profound differences. Furthermore, the used lentiviral SIN vector had no significant genotoxic potential. These findings demonstrate that SIN vector-mediated gene transfer displays a feasible and possibly safe option for MGMTP140 K-mediated chemoprotection of CD34+ cells.
Applied Virology and Gene Therapy, Georg-Speyer-Haus , Germany
Session: Persisting transgenes (viral and non-viral)
Mutations in the gp91phox gene, which codes for the large membrane-bound component of the superoxide producing NADPH oxidase lead to a defective respiratory burst in phagocytes and cause X-CGD in humans. Since insertional oncogenesis was observed in our current clinical trials, we developed a self-inactivating (SIN) vector (SERSfesgp91phox) with improved safety and efficacy profiles for the gene therapy of CGD. The genotoxicity profile of the SIN vector was tested using the in vitro immortalization (IVIM) assay of Modlich et al, which is based on the in vitro replating capacity of transformed primary mHSC after gamma retroviral transduction. In contrast to a SFFV-derived vector, the SIN SERSfes vector did not generate immortalized clones after transduction and plating of X-CGD Lin- cells. We also tested the SIN vector in a congenic mouse model and observed the animals for more than 30 weeks. As a control a SFFV driven vector expressing eGFP was used. The animals were monitored regularly for the development of leukemia and clonality by LM-PCR. Independently of the vector used, no leukemia was seen in the transplanted animals. However, animals transplanted with SFFV-eGFP transduced cells showed a clear tendency to oligo/monoclonality, while gene marked hematopoiesis in animals transplanted with the SIN SERSfesgp91phox vector were more heterogenous with multiple clones. In summary, both in vitro and in vivo genotoxicity assays revealed a favorable safety profile for our SIN gamma retroviral vector.
Human Gene Therapy Unit, Akdeniz University Faculty of Medicine , Turkey
Session: Persisting transgenes (viral and non-viral)
Kahraman S Molecular Imaging and Biology in press
Doerschug K The Journal of Immunology
Dajani R Am J Physiol Gastrointest Liver Physiol
Session: Persisting transgenes (viral and non-viral)
In gene therapy targeting hematopoietic disorders, insertional mutagenesis may provoke clonal dominance with leukemic progression. To identify the origin of insertional mutants, we transduced sorted hematopoietic subpopulations with γ-retroviral or lentiviral vectors and studied their expansion in C57BL/6J mice. Progressive clonal dominance only developed in the progeny of hematopoietic stem cells (HSCs), where expanding clones with insertional proto-oncogene activation (e.g., Evi1) were detected in ≈ 0.01% of treated cells. HSCs transduced with lentiviral vectors developed clonal dominance in the absence of proto-oncogene hits (Kustikova et al., Mol Ther 2009). We continued to study the “integrome” kinetics using 3 approaches: ligation-mediated PCR (LMPCR) with direct sequencing of dominant amplicons, LMPCR followed by 454 high-throughput sequencing, and locus-specific quantitative PCR (LsqPCR). 454 analysis mapped ≈ 4x more insertions than “manual” sequencing, and the 454 integrome was significantly less associated with proto-oncogenes. Dominant amplicons from “manual” analysis were often correlated with a higher number of 454 reads. LsqPCR showed up to 55-fold expansion and up to 20-fold reduction of individual clones (n = 27) over time. As the extent of clonal dynamics was not always accurately reflected by both dominant amplicon analysis and 454 sequence quantification, both should be complemented with LsqPCR to precisely quantify the clonal dynamics of insertional mutants.
Targeted gene delivery by AAV
Session: Persisting transgenes (viral and non-viral)
The potential utility for targeted gene addition has been recognized and a number of different approaches are currently under evaluation. A variety of viruses naturally establish latency by integrating their genome into the host genome. The non-pathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene (within the AAVS1 locus). To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved in spite of the resulting rearrangements. A latently infected, Mbs85-targeted mouse embryonic stem (ES) cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multi-lineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a novel viral strategy for targeted genome addition with the apparent absence of functional consequences.
Dep. of Experimental Hematology, Hannover Medical School , Germany
Session: Persisting transgenes (viral and non-viral)
Insertional activtion of proto-oncogenes by integrating gene transfer vector may cause clonal imbalance and even malignant cell transformation. We developed the in vitro immortalization (IVIM) assay that is based on in vitro replating of primary murine hematopoietic cells, to assess the genotoxic potential of integrating vectors. Up to today we performed approx. 200 assays. We found that self-inactivating (SIN) vectors have a lower transformation rate than gammaretroviral (GV) LTR vectors when using the same internal promoter. Lentiviral (LV) vectors with the internal SFFV enhancer/promoter transformed with 3 fold reduced incidence (number of positive assays within assays performed) compared to GV SIN vectors and also induced comparatively lower replating frequencies (an indicator of clonal fitness). The first intron of the Evi1 proto-oncogene was a common insertion site for both LV and GV vectors, and induction of Evi1 expression in mass cultures could be used to predict replating mutants. The use of internal cellular promoters instead of viral-derived promoters had a greater impact on safety than differences in the retroviral insertion pattern. Studies with different viral enhancer mutants support the conclusion that enhancer-mediated gene activation is the major cause for insertional transformation of hematopoietic cells. The use of vectors expressing therapeutic genes, in part analyzed in knockout cells, gave insights into potential transgene toxicities that influenced transformation events. In conclusion our assay is a powerful tool to assess vector genotoxicity and detects principal mechanisms underlying insertional mutagenesis observed in vivo in mice and in clinical trials.
Department of Human Genetics, University of Aarhus , Denmark
Session: Persisting transgenes (viral and non-viral)
Biological properties of the integration machinery of lentiviral vectors facilitate preferred insertion into actively transcribed genomic regions. We have previously shown that DNA circles generated during lentiviral transduction can serve as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) DNA transposase. Notably, insertion by this engineered pathway of integration was random and not biased toward genes. Here, we describe the enhanced gene transfer properties of a new combined HIV-1/SB vector system that allows efficient DNA transposition from non-integrating lentiviral vectors in a variety of cell types. Transposition directed by the hyperactive SB100X transposase is further enhanced by including improved SB T2 inverted repeats in the vector. With this vector system, we achieve a transduction rate that is increased up to 80-fold relative to background in human embryonic kidney cells (HEK293) and more than 30-fold in myelogenous leukemia cells (K562). Moreover, highly efficient gene insertion in both primary human fibroblasts and primary human keratinocytes, up to 160-fold above the background level, is reported. In summary, the combined HIV-1/SB vector system integrates genetic cargo with high efficacy, in some cases with almost the same efficiency as conventional lentiviral vectors. Our data suggest that the biological constraints on conventional lentiviral DNA insertion are resolved by the SB100X transposase, resulting in a random integration profile. We believe that such vectors may become valuable tools for genetic engineering and therapeutic gene transfer.
Hematopoiesis and Gene Therapy Division , Ciemat-Ciberer, Spain
Session: Persisting transgenes (viral and non-viral)
Gene therapy is a proficient and powerful tool to treat haematological inherited diseases. However, some leukemic events have been reported associated to this therapy. This major drawback discovery calls for studies regarding the implication of vector design in the gene therapy-linked risk allowing to highlight and prevent these hazards. Aiming to develop a sensitive assay to investigate the transactivating potential of gene therapy vectors, we set up an in vitro protocol based on high-MOI transduction of HeLa cells followed by integration and micro-array gene expression characterization. Different gamma-retroviral vectors and lentiviral vectors carrying different promoters with a wide range of strength were compared. In a first step, over one hundred clones were characterized by cytometry analysis and quantitative-PCR. Our data reinforce differences in expression due to the promoters. In most cases we failed to correlate expression levels of the transgene with the number of vector copies detected within the genome. Further studies, including Affymetrix arrays and LM-PCR have been initiated in selected clones. Transcriptional assays revealed differences in the expression pattern between vectors delivering different promoters while integration sites are currently under analysis. This assay will help to define vector design and to identify the transactivation potential of vector integration.
Institute for Biomedical Research, Georg-Speyer-Haus , Germany
Session: Persisting transgenes (viral and non-viral)
During the follow-up of our gene therapy trial aimed at the correction of X-CGD we observed clonal expansion and downregulation of transgene expression due to CpG methylation at the promoter region within the viral LTR. To counteract epigenetic effects on transgene expression we used ubiquitous chromatin opening elements (UCOEs), which are methylation-free CpG islands spanning divergently transcribed promotors of housekeeping genes with chromatin opening capability. For our studies we used a 1.5 kb DNA fragment derived from the core region of the UCOE from the human HNRPA2B1-CBX3 gene locus. This element includes the promoter region and the first exon plus part of the first intron of the HNRPA2B1 and CBX3 genes. Introduction of this element into a lentiviral vector resulted in high, persistent and copy number dependent expression of gp91phox. However, initial safety assays in growth factor dependent cell lines have revealed a potential for insertional mutagenesis caused by aberrant splicing of transcripts initiated from HNRPA2B1 promoter into the growth factor receptor gene generating fused HNRPA2B1 transcripts. To reduce the probability of insertional mutagenesis, we mutated the HNRPA2B1 splice donor site and added the polyadenylation signal of bovine growth hormone downstream of the HNRPA2B1 promoter. In a third construct, we included the target sequence of miRNA 142 within the HNRPA2B1 transcript. The effect of these modifications on virus titer and the genotoxicity potential of these constructs is currently being tested by RT-PCR and in cell culture assays.
Department of Translational Oncology, National Center for Tumor Diseases Heidelberg , Germany
Session: Persisting transgenes (viral and non-viral)
Gene therapy with oncoretroviral vectors caused insertional activation of protooncogenes and uncontrolled clonal proliferation in several clinical trials. Accordingly, large scale analysis of the clonal inventory of transduced samples is of decisive importance for the generation of safer vectors for future gene therapy trials. A genomic accessibility model established in our lab for the restrictive linear amplification-mediated PCR (LAM-PCR) showed that the performance of a comprehensive genome-wide integration site (IS) analysis is in the need of an actively designed validated strategy of restriction motif combinations. Thus, we established a novel non-restrictive (nr) IS analysis and sequencing assay enabling the complete identification of a transduced samples IS pool in one approach. To validate applicability in primary human cells, we performed nrLAM-PCR repeatedly on five primary human T cell clones, harbouring a total of 40 IS. We further analyzed different polyclonal preclinical and clinical samples. nrLAM-PCR analysis of a clinical patient sample, preclinical human HeLa cells and mouse hematopoietic lin- cell samples transduced with different gammaretroviral and lentiviral vectors resulted in the detection of 112 to 582 IS per single nrLAM-PCR set-up. Thus, nrLAM-PCR in combination with optimized high-throughput pyrosequencing quality preparation and software analysis allows to perform comprehensive genome-wide IS analysis.
Applied Biology, Institute for Biomedical Research -GSH Frankfurt , Germany
Session: Persisting transgenes (viral and non-viral)
The analysis of retroviral integration (RI) sites in our X-CGD gene therapy trial revealed an unprecedented clustering of integrations in or around the MDS/EVI1 and PRDM16 genes. Notably the same DNA regions have been found to be common targets of retroviral integration in murine cells. To define the molecular basis of RI clusters, we analyzed the chromatin status around the integration clusters. First, we did an in silico analysis of potential scaffold/matrix attachment regions (S/MARs) at the PRDM16 cluster and found a complete overlap between a predicted S/MAR and the integration cluster within intron 1 of the PRDM16 gene. Secondly, this region is contained within a DNA stretch with high DNA duplex destabilization potential, suggesting that the DNA topology around the PRDM16 integration cluster favors retroviral integration within this region. Furthermore preliminary data from chromatin immunoprecipitation (ChIP) experiments showed that the acetylation level of histone H3K9 is increased and the overall histone H3 load is decreased in this region. Therefore the primary determinant affecting retrovirus integration seems to be the topology of the chromatin at the integration site. According to this, retrovirus integration may be dictated by tethering of the virus preintegration complex to accessible chromatin regions like S/MAR sites, DNaseI hypersensitive sites, and/or to chromatin regions with a high degree of DNA bendability as they are typical for S/MARs and their associated chromatin structure.
Department of Pharmacy, Free University of Berlin , Germany
Session: Persisting transgenes (viral and non-viral)
Sleeping Beauty (SB) transposase and phiC31 integrase have emerged as potent tools for achieving long-term gene expression in various tissues. In previous studies, using either of the systems, long-term expression has been achieved in murine lungs. Activity of Tol2 transposase in the lung cells remains unexplored. The present study aimed at directly comparing these three different integration systems using an identical expression cassette. For each integration system, donor plasmids were constructed containing Ubiquitin B promoter and luciferase as a marker gene. Transfections were performed in murine (MLE12) and human (A549) alveolar epithelial cells and luciferase measurements were performed at different time points. No significant effect of any amount of Tol2 transposase could be observed on the resulting long-term expression. Among the other two systems, in MLE12 cells, there was no difference between the SB and phiC31 integrase mediated long-term expression. However, in A549, codelivery of SB expression plasmid resulted in higher and stable long-term expression values compared to those achieved with equal amount phiC31 integrase plasmid. Long-term expression studies with 5-Aza-2′deoxycytidine and Trichostatin A (inhibitors of DNA methylases and HDAC, respectively) provided evidence for lack of post-integrative gene silencing. These results, by directly comparing the three systems, present SB as the best candidate system for achieving stable long-term expression in the lungs.
Gastroenterology Hepatology Endocrinology, Hannover Medical School , Germany
Session: Persisting transgenes (viral and non-viral)
For severe monogenetic disorders of the liver, whole organ transplantation is often the only treatment option. Viral gene therapy vector systems, especially lentiviral vectors, could offer new therapeutic options. The patient's own hepatocytes could be genetically corrected ex vivo and transplanted back in to the liver. Alternatively, direct application of therapeutic vectors could serve as an in vivo gene therapy protocol. In the hematopoietic system however, integration of lentiviral vectors into the host genome has led to the development of premalignant and malignant clones. A systematic risk assessment of insertional mutagenesis in adult liver tissue has not been investigated. First, we established efficient transduction and transplantation protocols for primary hepatocytes based on optimised purification, enrichment and medium conditions. Lentiviral transduction efficiency was reproducibly increased to 35% using only 10 functional particles per cell and over 85% with a MOI of 100. LM-PCR and high throughput sequencing identified 2397 lentiviral insertion sites in in-vitro transduced primary hepatocytes. Correlation of integrome patterns with whole genome expression data of hepatocytes revealed preferential integration in areas of highly expressed genes. We currently perform serial transplantations of ex vivo and in vivo gene corrected hepatocytes into FAH(-/-) mice. Lentiviral insertion sites will be analyzed for putative clonal selections of transduced hepatocytes.
ASLV-derived vectors undergo extensive DNMT-dependent silencing in human cells, but can be reactivated by HDAC and DNMT inhibitors
Session: Persisting transgenes (viral and non-viral)
Vectors derived from avian sarcoma and leucosis viruses (ASLV) represent a potent gene delivery tool. These vectors are relatively safe among retroviral vectors due to their integration pattern, since they do not favour genes and promoter sequences and therefore exhibit low genotoxicity. We prepared ASLV-derived vectors pseudotyped with the VSV-G glycoprotein to enable them to infect various cell types of different origin. We used these vectors for infection of human cell lines to analyse the efficiency of infection and frequency of silencing. The majority of proviruses were silenced immediately after the integration into the host genome, but these silenced proviruses were efficiently reactivated with various histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors. During cultivation, the expressed proviruses underwent gradual silencing and epigenetic repression, which was highly dependent on the activity of DNMTs. Different DNMT knock-outs exhibited diverse silencing rates. The cells lacking DNMT1 and DNMT3B silenced the vectors only very inefficiently and even after long-term cultivation the majority of silenced proviruses retained the capacity to be reactivated. We analysed the capacity of previously described CpG island element (Senigl et al., 2008), which stabilizes expression during long-term cultivation, to protect the vector from early silencing. This element showed not to be very efficient in the early silencing protection, but stabilized the vector expression and retained the early-silenced vectors in a more easily reactivable state. The ASLV-derived vectors represent a relatively safe and efficient gene delivery tool, but several modifications protecting them from silencing in human cells are still needed.
Senigl J. Virol.
Session: Persisting transgenes (viral and non-viral)
Plasmid pEPI-eGFP is a prototype episomal vector, based on the transcription cassette CMV-eGFP-S/MAR and we have previously shown that it mediates efficient transfection of human CD34 + cells, albeit with very low long term eGFP expression and plasmid retention (1). To enhance its potential we substituted CMV by the promoter of SFFV virus (pEPI-SFFV) and the hybrid promoter EF1/HTLV (pEPI-EF1/HTLV), that, reportedly, function more efficiently in CD34 + cells. Additionally, the replication initiation region (IR) from the β-globin gene locus was added in one plasmid (pEPI-SFFV-IR), in optimal site determined by SIDD analysis (Stress Induced Duplex Destabilization), as we have recently shown that this bona fide mammalian replication initiator enhances plasmid's replication too(2). Plasmids were transferred into K562 cells and transfection rates were: for plasmids pEPI-eGFP as control, pEPI-SFFV and pEPI-EF1/HTLV, 25,6%, 25,6% και 26,4% respectively and for plasmid pEPI-SFFV-IR was 41,2%. All cell lines retained the episome for three months, with and without selection. Based on these data, we transfected CD34 + cells from adult peripheral blood with plasmids pEPI-SFFV, pEPI-SFFV-IR and control pEPI-eGFP. The transfection rate for EPI-SFFV-IR was slightly increased (30%) compared to pEPI-eGFP (26%) and pEPI-SFFV (24%). Importantly, intense fluorescence is detected in finally differentiated cells, BFU-E and CFU-E, in fluorescence microscopy, revealing strong expression of eGFP and significant increase in the level of retention of plasmid pEPI-SFFV (60% of the colonies) and pEPI-SFFV-IR (99% of the colonies) in these cells. This certifies the high potential of these two episomal vectors for gene transfer into hematopoietic progenitor cells.
Papapetrou E., et al., Gene Therapy,
Giannakopoulos A. et al., Journal of Molecular Biology,
Session: Persisting transgenes (viral and non-viral)
Integrase deficient lentiviral vectors (IDLV) demonstrated highly applicability for transient and stable gene delivery in proliferating and non-dividing cells, rendering them interesting for therapeutic applications. However, little is known about the existing residual integration events of IDLV, and comprehensive characterization of IDLV integration profiles in mammalian genomes is still missing. In this study, we used non-restrictive and standard LAM-PCR strategies to analyze IDLV integration in cultured cells. Our data revealed the lacking of representative 5-bp direct repeat of host DNA flanking the provirus, deletion of host DNA and LTR sequences, abnormal 3′-processing and concatemer integration. We retrieved 609 integrants from IDLV transduced SC-1 cells and 295 integrants from C1498 cells. Among them, 353 (SC-1) and 160 (C1498) integrants could be mapped to a definite position in the mouse genome. IDLV Integration pattern demonstrated random distribution of chromosomes, non-clustered around TSS sites, and less preference integration into Refseq genes compared to LV integration pattern (41.36% for SC-1, 46.27% for C1498, 70.55% for SC-1 transduced with LV, p < 0.001). No proto- and oncogenes as integration sites have been found in IDLV transduced cells. Taken together, the residual integration of IDLVs might not be mediated by an integrase-driven mechanism, the risk of insertional mutagenesis from IDLV is highly minimized and it provides one of the potentially safest tools for efficient gene delivery for clinical applications.
Mapping of transposon integration sites in murine and canine liver after transposition from a high-capacity adenoviral vector
Session: Persisting transgenes
Sleeping Beauty (SB) transposase represents a powerful tool for somatic integration with important implementations for experimental and therapeutic gene transfer. Herein, we analyzed transposon integration in murine and for the first time in canine liver after adenoviral delivery of a hyperactive SB (HSB) system. Mice and hemophilia B dogs were co-injected at optimized dosages with a canine coagulation factor IX (cFIX) encoding transposon donor and a second vector providing HSB in trans. After we observed stabilized cFIX expression levels in HSB treated animals, we analyzed fate of the transgene in murine and canine liver. To determine sites of insertion we performed PCR-based high-throughput sequencing. Of the 73 transposition events detected in liver of HSB treated mice, 74% (54/73) were found in non-gene areas and 26% (19/73) in genes indicating a random integration pattern. Of the transposition events detected in genes, 17 insertion sites were in introns and two in exons. In canine liver, we analyzed transposition events three years post-injection. Although we did not select for integration as we did in mice, we identified 4 unique transposition events of which 2 were intergenic, 1 was within a gene and one integration event hit a repetitive region. Moreover, we investigated which vector forms were responsible for persistent transgene expression. After performing in-depth PCR analysis, we found that episomal vector genomes including the HSB encoding vector were strongly reduced over time. In contrast, 0.01 copies/cell of the cFIX transposon were maintained in canine liver suggesting that cFIX expression was derived from integrated vector copies. Similar tendencies for the vector status were obtained in mice.
Transcriptional targeting of retroviral-mediated gene therapy by the synovial sarcoma, X breakpoint 4 promoter
Session: Persisting transgenes
Previously, we reported the eradication of mouse glioma by retroviral-mediated gene therapy using modified packaging cell line producing high-titer retrovirus. In this study, a tumor-specific targeting system was studied to develop the effective and safe gene therapy. We searched for genes expressing at high frequency in brain tumors but not in normal human astrocyte among cancer testis antigen genes. MAGEA3 and synovial sarcoma, X breakpoint 4 (SSX4) were identified as tumor-specific genes. The SSX4 promoter showed the tumor-specific activity. In order to define a useful tumor-specific promoter for targeting in context of retroviral-mediated gene therapy, the SSX4 promoter were used to restrict the expression of suicide gene HSVtk in retroviral vector. The glioma cell lines transduced with the retroviral vector were killed efficiently by addition of ganciclovir (GCV), but telomerase-immortalized fibroblast BJ-5ta cells were not. Mouse glioma RSV-M cells transduced with the retroviral vector were transplanted into S.C. of syngeneic mice. The administration of GCV into the mice suppressed the tumor growth completely. These results suggest that the promoter of SSX4 is useful for tumor-specific gene therapy, and promising the safe and effective retroviral-mediated gene therapy.
Gold nanoparticle bioconjugates by laser ablation - a novel method aiming at pure non-viral vectors for regenerative biology
Session: Physicochemical approaches
Bioconjugated gold nanoparticles are of increasing interest for biomedical applications such as gene- and drug delivery, therapy and diagnostics (1). But each biological or medical application demands a tailored design of the nanoparticulate vector. Since the relationship between design and function is often not predictable, a screening method providing various bioconjugates is required. Conventionally, the generation of nanoparticles is well established by chemical synthesis methods. The molecular design of the nanoparticulate carrier with required functions for targeting, enhanced cellular uptake etc. is enabled in a successive surface functionalization (after the nanoparticle generation). However, a screening of bioconjugates is often not enabled due to time constraints and limited adaptability of the multistep method to the conjugation of various biomolecules. In this context, we have established an in-situ bioconjugation during ultrashort pulsed laser ablation in liquids allowing both, the generation of pure stable nanoparticles and their simultaneous bioconjugation (2). This approach is demonstrated at the example of gold nanoparticle generation and conjugation with DNA oligonucleotides, DNA and RNA aptamers and a cell penetrating peptide. The process is pure, fast and allows for tunability in surface coverage of biomolecules on nanoparticles. First biomedical applications addressing sensing, imaging and cellular transfection are discussed as well. Since the mechanism of the in-situ bioconjugation during laser ablation is based on fundamental electron donor (biomolecule) - electron acceptor (nanoparticle) attraction, a huge variety of materials bearing these basic properties should be conjugable in future.
Han Nanobiotechnology
Petersen Advanced Functional Materials
Session: Physicochemical approaches
Plasmid AMEP is a medicinal gene product encoding AMEP (Antiangiogenic Metargidin Peptide), the disintegrin domain of human metargidin, which inhibits melanoma tumour growth and angiogenesis in melanoma mice models. In order to evaluate its safety, preclinical regulatory safety studies have been conducted on Swiss CD-1 immunocompetent mice after single or repeated (four at 7-day interval) muscle administration(s) of plasmid AMEP followed by electrotransfer. First, the single high-dose (400μg) toxicity study showed that plasmid AMEP was safe and well tolerated as no significant modification was observed on haematology, blood biochemistry, tissue pathology compared to the control group. Microscopic examination of injected muscles showed moderate signs of inflammation and muscle fibers regeneration. Four Repeated administrations at 7-day interval of increasing doses of plasmid AMEP (25, 100 or 400 μg) showed a dose-related decrease in white blood cells count at 100 and 400μg at day 29, which was not observed at the end of the recovery period (day 50), suggesting reversibility. No Plasmid AMEP related organ pathology was noted, excepted for the injected muscles, which demonstrated an increase in inflammation and regeneration at the highest dose-level. No other relevant findings were observed in ophthalmology (fundus) and in blood vessel density of organs after CD-31 immunostaining. The No Observable Adverse Effect Level was established at 25μg/injection administered in mice once a week for 4 weeks. In conclusion, Plasmid AMEP did not showed major adverse effects after four muscle administrations and did not seem to alter normal physiologic angiogenesis.
Gene Medicine Group NDCLS, University of Oxford , UnitedKingdom
Session: Physicochemical approaches
Q-PCR is a powerful tool for measuring gene transfer in cells and tissues, which we have used to measure gene delivery (DNA) and expression (mRNA) from plasmid vectors in lung tissues. Here, we report a biochemical anomaly that can lead to the detection of false positives after non-viral vector mediated delivery of plasmid DNA in animal models. A quantitative RT-PCR TaqMan assay (pCI-A) was designed across an intron boundary in a standard plasmid that had been aerosolized to sheep lungs and results expressed as % VE (% Vector/Endogenous CFTR mRNA). Initial results were encouraging, with vector-specific expression levels of 1510 % VE at day 1. Omission of reverse transcriptase (RT) from the assay resulted in negligible signal, thus the pCI-A assay was judged insensitive to DNA contamination. Unexpectedly, we then observed that RNase treatment of the total RNA samples did not result in a significant reduction in vector mRNA levels, whereas DNase treatment reduced RT-dependent TaqMan signal to < 1.65 % VE (Mann Whitney: p < 0.0001). This suggestedthat the pCI-A assay was in fact highly sensitive to DNA contamination, but in an RT-dependent manner. New primer sets were designed, which showed that the DNA- and RT-dependent signal was observed only when a specific reverse gene primer was used. In conclusion, our experience suggests that caution should be exercised when developing new TaqMan assays, particularly where DNA may contaminate samples expressing low levels of RNA.
Exploiting the virulence factor LLO for enhanced non-viral DNA delivery through forced lysosomal escape
Session: Physicochemical approaches
The introduction of therapeutic genes into cells by non-viral gene delivery systems is still limited by low transfection efficacy. The degradation of the introduced DNA inside the lysosome after uptake is one of the main limiting factors of non-viral gene delivery.The aim of this study was to exploit natural endosomal escape mechanisms to overcome this transfection barrier. Listeriolysin O (LLO), a pH dependent hemolytic and endosomolytic protein derived from Listeria monocytogenes was expressed in E. coli and purified by Ni-NTA chromatography. The pH restricted hemolytic activity of the purified protein in acidic (lysosomal) pH conditions (active only at pH < 6) was confirmed by lysis of red blood cells. In following in vitro experiments, C2C12 cells were transfected with fluorophore labeled DNA with or without LLO via poly-L-lysine. Using live confocal laser scanning microscopy, a substantial decrease of lysosomal trafficking of labeled DNA was observed compared to controls without LLO within 20min after transfection. The described results demonstrate the possibility of partially adapting the entry mechanism of L. monocytogenes to increase DNA release to the cytosol by disrupting the acidifying late endosomes of the target cells and hence potentially increase the efficiency of gene delivery. Additions of electrostatic or sequence-specific DNA binding motifs to LLO resulting in a more efficient co-delivery are currently under investigation. This project was partially funded by EXPERTISSUES.
Department of Biochemistry, Chungnam National University , Korea South
Session: Physicochemical approaches
In this report, we tried to synthesize new cationic lipid derivative of dexamethasone and to prepare liposome with DOPE for application to gene transfection in vitro. Polyamidoamine dendrimer (generation 0) was selected for the cationic head group because it displays lower cytotoxicity than other higher generations of dendrimers, and it contains primary and tertiary amine groups. Dexamethasone is the potent ligand of the glucocorticoid receptor which facilitates the transfer into nucleus, and it is known to enlarge the nuclear pore complexes. It is expected that single dexamethasone-conjugated PAMAM dendrimer could bind with pDNA because it has three primary amine groups on its hydrophilic head groups. The purified product was confirmed with MALDI-TOF mass spectrometry and 1H NMR. To make a liposomal structure, DOPE was used as a helper lipid. It was found that PAMAM-dexamethasone/DOPE liposome/pDNA complex was completely retarded at 8:1 N/P(nitrogen/phosphate) ratio. The mean diameter of lipoplex was 236.5 ± 5.82 nm and the zeta potential value was 36.52 ± 1.40 mV. From the results of the transfection study, delivery efficiency of PAMAM-dexamethasone/DOPE liposome was improved as N/P ratio increased. As expected, PAMAM-dexamethasone/DOPE liposome showed much enhanced transfection efficiency compared to native PAMAM. PEI25KD and Lipofectamine 2000 were used as control vectors.
Rebuffat, A Nat Biotechnol
Gruneich, J.A. Gene Ther
Bae, Y.M. Bioconjug Chem
Session: Physicochemical approaches
We have recently demonstrated induction of tumor-selective iodide uptake and therapeutic efficacy of radioiodine in neuroblastoma tumors after systemic sodium iodide symporter (NIS) gene transfer using non-viral gene delivery vectors. The aim of the current study was to evaluate the efficacy of pseudodendritic oligoamines (G2-HD-OEI) and polyamidoamine dendrimers (PAMAM) for systemic NIS gene transfer in a hepatoma mouse model by analysis of radioiodine biodistribution using 123I gamma camera imaging. For this purpose we used G2-HD-OEI and PAMAM vectors, which exhibit high intrinsic tumor affinity, to target a NIS-expressing plasmid to hepatoma (Huh7) cells in vivo. While systemic NIS gene delivery using PAMAM vectors did not result in specific iodide accumulation in Huh7 xenograft tumors, Huh7 tumors accumulated 6–11% ID/g 123I with a biological T1/2 of 11 h resulting in a tumor absorbed dose of 281 mGy/MBq 131I after G2-HD-OEI mediated NIS gene transfer. In contrast, tumors transduced with the control vector and non-target organs like liver, lung, and kidneys showed no significant iodide uptake. In conclusion, transduction efficacy and tumor selectivity of different dendrimeric polymers can vary in different tumor models and therefore have to be evaluated individually. After individual optimization, our results clearly demonstrate the feasibility of systemic NIS gene transfer using non-viral dendritic nanoparticle vectors in HCC followed by targeted radionuclide therapy.
Biomedical Optics Department, Laser Zentrum Hannover e.V. , Germany
Session: Physicochemical approaches
Cloning of several mammalian species has been achieved by somatic cell nuclear transfer (SCNT) in recent years. However, this method still results in very low efficiencies around 1% which originate from suboptimal culture conditions and highly invasive techniques for oocyte enucleation and injection of the donor cell. To overcome some of these problems, we present a new minimally invasive method for oocyte enucleation based on the application of femtosecond (fs) laser pulses. This method relies on focusing the laser beam onto the fluorescently labeled metaphase plate by a high-numerical-aperture objective. Three-dimensional scanning was achieved by lateral deflection of the beam using scan-mirrors and axial positioning of the objective. After having generated a three-dimensional stack of multiphoton images depicting the metaphase plate, our self-developed software automatically detected the bright fluorescent area of the metaphase plate in each plane. Afterwards, the fs laser beam was solely scanned over these marked areas with higher pulse energy and slower scan speed to create nanometer-sized cuts by ablation of DNA molecules. We show that fs laser based enucleation followed by artificial activation of porcine oocytes inhibited pronucleus formation and the first mitotic cleavage while maintaining intact morphology. In contrast, control groups without previous irradiation of the metaphase plate were able to develop to the blastocyst stage. As a next step, experiments clarifying the suitability of fs laser based enucleated oocytes for SCNT were performed. Furthermore, potential negative influence of photochemical side effects due to laser irradiation was investigated.
Dexamethasone loaded R3V6 peptide micelles as a gene carrier
Session: Physicochemical approaches
R3V6 peptides formed self-assembled micelles in aqueous solution with a hydrophobic core of valines (Vs) and hydrophilic corona of arginines (Rs). R3V6 peptides could bind to DNA by electrostatic interactions and deliver DNA into cells. However, compared with poly-L-lysine, the DNA delivery efficiency of the R3V6 peptide micelles was not high, due to the instability of the peptide micelles. To overcome poor DNA delivery efficiency of the R3V6 peptide and add anti-inflammatory effect, dexamethasone was loaded as a model hydrophobic drug into the core of the peptide micelles. First, it was confirmed that hydrophobic fluorescent dye 5-dodecanoylaminofluorescein (DAF) was loaded into the R3V6 micelles, but hydrophilic dye Oregon Green was not. The dexamethasone loaded R3V6 peptide micelles had higher DNA delivery efficiency than the R3V6 peptide micelle control. This enhanced efficiency may be due to higher stability of the drug loaded micelles in aqueous solution. Considering that dexamethasone is an effective anti-inflammatory drug, the dexamethasone loaded R3V6 peptide micelles are effective co-delivery carriers of drug and DNA and will be useful for inflammatory disease gene therapy.
Experimental Oncology, Klinikum rechts der Isar TUM , Germany
Session: Physicochemical approaches
Most efficient core-shell magnetic nanoparticles (MNPs) were selected and comprehensively characterized with respect to their core and coating composition using X-ray diffraction, magnetization measuruments and XPS data. Magnetic nucleic acid delivery vectors were optimized for DNAs, siRNAs, adeno- and lentiviral delivery. A simple method for evaluation of the magnetophoretic mobility and, hence, of the magnetic moment was used to evaluate the number of nanoparticles associated with each complex. Together with TEM and AFM data this approach delivers information on the morphology, composition and magnetic properties of the complexes. We suggest a “rule” to formulate virus magnetic complexes based on our fine-tuning of the nanoparticles-to-virus particles ratio in the range of 2.5–20 fg iron per physical virus particle depending on the nanomaterial used applicable to both adenoviral and lentiviral vectors. We show also that excess of magnetic nanoparticles, when preparing the complex, can inhibit infection efficiency. The developed magnetofection protocols with novel magnetic vectors are efficient to transfect difficult-to-transfect primary cells and improve significantly viral nucleic acid delivery with adeno- and lentivirus vectors. We have shown that the association of adenovirus with MNPs does not inhibit the inherent potential of the virus but that it rather enhance virus uptake into the cell.
Research and Development, Sonidel Ltd , Ireland
Session: Physicochemical approaches
Sonoporation-based gene transfection is emerging as an attractive means of facilitating site-specific gene transfer. This modality is particularly suited for use with naked DNA and uses approved ultrasound contrast agents (microbubbles) to enhance gene transfection. In employing naked DNA for the purposes of gene therapy it is advantageous to employ gene vehicles that are devoid of bacterial DNA and the use of minicircle DNA is one means of facilitating this. Here we describe the use of ultrasound to facilitate gene transfer and expression of a minicircle-encoded exogenous gene in murine tissues. In these studies C3H/HeN mice were employed as targets, as were RIF-1 tumours which are syngenic with this mouse strain. Microbubble preparations employed included MB101 and MB102 (Sonidel Ltd.) with neutral and cationic surfaces, respectively. The plasmid pCMV-luc and the minicircle MC07. CMV-luc were obtained from PlasmidFactory GmbH & Co. Ultrasound-mediated transfection (sonoporation) was performed using a Sonidel SP100 sonoporator, emitting ultrasound at a frequency of 1 MHz. Expression of luciferase was determined by in vivo bioluminescent imaging using a Xenogen Ivis Lumina system. In muscle, it was found that the level of gene expression achieved with the minicircle DNA was equivalent to that achieved using the plasmid pCMV-luc. The therapeutic potential of such an approach incorporating the use of minicircle DNA together with sonoporation will be discussed.
Arginine based peptide micelle for siRNA delivery
Session: Physicochemical approaches
In this research, RV peptides with 1 - 4 arginines and 6 valines were synthesized and evaluated as carriers for intracellular delivery of plasmid or siRNA. RV peptides formed micelles in aqueous solution with cationic arginines on the surface and hydrophobic valines in the core. Gel retardation assays showed that plasmid or siRNA was completely retarded at a 5/1 weight ratio (peptide/plasmid or siRNA). To evaluate the stability of RV peptide/siRNA complex, heparin competition assays were performed. The results showed that heparin replaced siRNA in polyethylenimine (PEI)/siRNA complex at a 4/1 weight ratio (heparin/siRNA). However, RV peptide/siRNA complex was stable up to an 8/1 weight ratio (heparin/siRNA), suggesting that RV peptide/siRNA complex is more stable than PEI/siRNA complex. In transfection assays with plasmid, pCMV-Luc, R3V6 peptide had higher plasmid delivery efficiency than any other RV peptides. R3V6 peptide was more efficient than PLL. However, PEI had much higher plasmid delivery efficiency than R3V6 peptide suggesting that RV peptides are not suitable for plasmid delivery. For siRNA delivery, anti-luciferase siRNA was complexed with RV peptides and delivered into luciferase-expressing 293 cells. R3V6 peptide was more efficient than PEI and PLL in silencing luciferase gene. In addition, MTT assay showed that all RV peptides are less toxic than PEI or PLL. The results suggest that R3V6 peptide will be useful for delivery of siRNA.
Biomedical Optics Department, Laser Zentrum Hannover , Germany
Session: Physicochemical approaches
Cell modulation by introduction of foreign molecules plays an important role in biomedical science. The transfer of membrane impermeable molecules such as DNA or RNA into cells is a key element to reprogram cells, knock-out and or overexpress proteins. For this reason new, alternative methods are required to penetrate the membrane avoiding negative side effects as seen in currently used methods e.g. electroporation and viral vectors. Herein we present the perforation of the cell membrane by two alternative approaches, inducing plasmonic effects in nanoparticles or at nanostructures. Therefore a weakly focused laser beam is used to treat nanoparticle labeled cells or cells growing on a laser nanostructured surface. The laser induced effects can permeabilize the cell membrane causing an exchange of intra and extra cellular molecules and thus lead to a diffusion of genetic material (e.g. GFP plasmids) or membrane impermeable dyes into the cells. Besides the characterization of the plasmonic effects and the cell-material interaction we present a high throughput method for perforation of the membrane of different cell lines, including primary and stem cells, based on the interaction of the nanoparticles or nanostructures with the laser radiation. We found high cell viabilities and efficiencies in various cell lines, using an optimized set of laser parameters.
Dep. of Biotechnology, Norwegian University of Science and Technology , Norway
Session: Physicochemical approaches
Chitosans are promising gene delivery vehicles with an attractive safety profile, but the understanding of structure-activity relationship and the criteria for the design of efficient chitosan gene delivery systems remain unclear. Here, we will show that the efficacy of gene delivery using chitosans is governed by the binding strength between the chitosan carrier and its DNA cargo. Several tailoring strategies were utilized to control the interaction between DNA and chitosan, including depolymerization to specific degrees of polymerization (DPn), glycosylation, self-branching and pegylation. The DPn of chitosan seems to be one of the most important parameters affecting the properties of the nanoparticles and the gene transfer efficacy. We were able to identify a narrow interval of chain lengths (DPn 30–40) that mediate maximum level of transgene expression in vitro. To improve the functional properties of the chitosan and to facilitate the unpacking of the polyplexes, chitosans were substituted with oligosaccharides containing N-acetylglucosamine. This substitution weakened the DNA binding strength through steric hindrance and facilitated the unpacking of the polyplexes. Moreover, self-branching of the chitosan backbone further improved the functional properties and these chitosans showed good serum compatibility, low cellular toxicity and high transfection efficacy in several cell lines, surpassing several commercial transfection agents. Tailoring of the chain length, substitution and self-branching of chitosan were shown to be feasible tools to modulate the electrostatic interactions between the chitosan and DNA in order to design chitosans with an optimized balance between polyplex stability and polyplex unpacking.
Dep. of Biotechnology, Norwegian University of Science and Technology , Norway
Session: Physicochemical approaches
Chitosans are attractive carriers for small interfering RNA (siRNA) but the critical requirements for efficient siRNA delivery with chitosans are poorly understood. In order to establish these and to identify the optimal molecular parameters of the chitosan carriers, we studied the effects of chain length, molecular architecture and charge density on the efficacy of siRNA delivery. Using a model series of well defined chitosan oligomers with constant charge density and degrees of polymerization (DPn) in the range of 18–85 monomer units, we first investigated what is the minimum chain length required to form physically stable nanoparticles that inhibit gene expression in vitro. We were able to show that the oligomers complex siRNA into physically stable nanoparticles (35–90 nm) that provided protection against RNase degradation. However, the gene knockdown depended strongly on the DPn of the carrier and the charge ratio of the formulation. In contrast to DNA delivery, longer chains and/or higher charge ratios were shown to be essential for successful siRNA delivery, indicating the necessity of strong electrostatic interactions. The chitosan with DPn85 clearly surpassed the shorter chitosans, showing 95% gene knockdown at a siRNA concentration as low as 45 nM that was sustained for 5 days in 293-Luc cells. Based on the structure-activity relationships obtained with the oligomers, we directed our further studies towards the determination of the chain length and charge density that mediate the maximum gene knockdown. We conclude that the superior chitosans for siRNA delivery in vitro are completely de-N-acetylated and have chain lengths in the range of DPn 250–300.
Non-viral carriers for optimal mRNA delivery in Hela cells
Session: Physicochemical approaches
Efficient packaging of nucleic acids is a necessity to accomplish their therapeutic effect. Viral carriers have already shown their capacity to transfect cells very efficiently but their toxicity is a serious concern. Alternatively, the ability of the less toxic cationic lipids and polymers to form complexes with nucleic acids and to deliver them into cells, is currently widely investigated. The use of mRNA has several advantages compared to pDNA; there are no immunogenic CpG motifs, there is no risk for insertional mutagenesis and mRNA does not need to cross the nuclear barrier to bring forth protein expression. mRNA encoding luciferase was complexed to the cationic lipids Lipofectamine or DOTAP/DOPE, and to the cationic polymers linear poly(ethyleneimine) (linPEI) or poly-beta-aminoester (PbAE). The complexes were incubated with HeLa cells and luciferase expression was assessed by a bioluminescence assay. Although all four cationic carriers resulted in significant luciferase expression, Lipofectamine was by far the most efficient. Transfection efficiency of the polymers was hardly sensitive to the presence of serum during their preparation, in contrast to the cationic lipids. We also assessed the proportion of cells that become transfected by means of transfection with mRNA encoding GFP and compared it with the pDNA analogue. For both cationic lipids as well as for the polymer PbAE, transfection with mRNA yielded a substantially larger fraction of transfected cells than transfection with pDNA. This and other advantages of the use of mRNA over that of pDNA may under certain conditions outweigh the disadvantage of the somewhat shorter expression period, in particular in cases where transient protein expression is required.
Laboratory Medicine, Karolinska Institutet , Sweden
Session: Physicochemical approaches
The usefulness of non-viral delivery vectors in the clinic is mainly hindered by their inefficient in vivo gene expression. Herein, we present a novel plasmid-based non-viral carrier that has shown to be promising for in vivo gene delivery. This new transport device is based on the assembly of DNA plasmids with an amphiphilic synthetic derivative of spermine molecules. A series of in vitro studies has enabled us to characterize several lipid-spermine conjugates and select one for in vivo administration - butanoyl-spermine. The novelty of this lipid-spermine conjugate lies in its short lipophilic tail, which confers self-assembling properties to the molecules and, yet, they retain their solubility capacity. The most promising pDNA particles resulted from the condensation with butanoyl-spermine at (+/−) ratios of 0.6:1 and 1:1. Such plasmid DNA nanoparticles presented the following properties: provide DNA protection against DNase I degradation, no cytotoxicity, a mild interaction with pDNA and an average diameter of 200 nm. pDNA/butanoyl-spermine complexes were administered to the M. tibialis anterior and the dermis of mice and gene expression was followed over 22 days by measuring the luciferase activity by bioluminescense. From day one, formulated pDNA resulted in a 5- and 10-fold increase in gene expression, as compared to naked DNA, in the muscle and skin, respectively. Moreover, this difference was maintained throughout the assessment period.
Microwave synthesis of calcium phosphate nanowhiskers as nonviral, inorganic gene delivery vectors
Session: Physicochemical approaches
Calcium phosphate nanowhiskers of hydroxyapatite (HA), tricalcium phosphate (TCP) and Biphasic (BCP) nanowhiskers were synthesized via a novel microwave assisted combustion synthesis as potential implant materials and inorganic, non-viral gene delivery vectors. Calcium phosphates, in particular hydroxyapatite, are known to be major constituents of bone and are bioactive both in vitro and in vivo. In addition, TCP is known to be biodegradable, while BCP has optimum biodegradability. NaNO3 , Ca(NO3 )2 4H2 O, KH2 PO4 , HNO3 , and urea were the reagents used in the synthesis. The different compositions vary from one another in their ratio of calcium to phosphate (Ca/P). The Ca/P of HA and TCP, are 1.67 and 1.5, respectively, while BCP nanowhiskers contain both HA and TCP. The materials were characterized using XRD, SEM, and TEM to confirm their compositions and morphology. SEM and TEM micrographs of the nanowhiskers show the crystallographic structure of the whiskers and suggest that the whiskers may be hollow tubes which would be ideal for an inorganic gene delivery system. FTIR analysis has been used to demonstrate the successful attachment of pDNA particles to the nanowhiskers. Mouse osteoblast cells were incubated with pDNA-nanowhiskers encoding for green fluorescent protein (GFP). After 7 days, GFP was visualized using fluorescence microscopy, proving the efficacy of these nanowhiskers as gene delivery vectors.
Hydrophobic modification of PAMAM dendrimer prior to amino acid conjugation to prepare novel gene carrier system
Session: Physicochemical approaches
In this study, we prepared poly (amidoamine) (PAMAM) dendrimer conjugate with 6-aminohexanoic acid and L-arginine using HOBt/HBTU coupling reagents. The 4th generation of PAMAM dendrimer was used because it displays relatively good transfection efficiency and low cytotoxicity compared to other generations. In our previous study, it was confirmed that L-arginine grafted PAMAM dendrimer displayed higher transfection efficiency. 6-aminohexanoic acid was selected as a spacer molecule to increase hydrophobicity and to introduce the flexibility of the conjugated amino acids. In addition, it is expected that this PAMAM dendrimer conjugates could form complexes with plasmid DNA because they have primary amine groups on their peripheral ends. The synthesized PAMAM conjugate was analyzed by 1H-NMR. It was confirmed that 98% of 6-aminohexanoic acid and 97% of L-arginine was conjugated to the surface of PAMAM G4, respectively. In agarose gel retardation assay, PAMAM-Ahx-Arg formed polyplexes at weight ratio of 8:1 (polymer : pDNA). In dynamic light scattering study, the mean diameter of polyplex was 177.5 ± 23.3nm. From the gene transfection experiments, PAMAM-Ahx-Arg showed much higher transfection ability compared to native PAMAM. Also, it was found that PAMAM-Ahx-Arg showed very low cytotoxicity compared with PEI25KD in WST-1 assay results. From these results, it is thought that the combination of 6-aminohexanoic acid and L-arginine with PAMAM dendrimer is beneficial to provide new PAMAM conjugate as an effective gene carrier system.
Choi, J.S. J Control Rel
Kim, J.B. J Control Rel
Kim, T.I. Bioconjug Chem
Session: Physicochemical approaches
Division of Physiology and Climatology, Indian Veterinary Research Institute , India
Session: Pluripotent stem cells
Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta superfamily, which plays a critical role in lineage determination and embryo patterning. Derivation of pure population of BMP2 + mesodermal cell lineage is very important in studying the mesodermal patterning and elucidating the phenotypic characteristics of these specific population of cells present in the early stage of embryonic development. In the present study we derived a transgenic BMP2 + cell lineages, which was microinjected into blastocyst. The microinjected blastocysts were transferred into surrogate mother. The chimeric mouse produced after microinjection was tested for evaluating presence BMP2 + cells or its derivatives in different organs by IVIS imaging of whole organ as well as by fluorescence observation in cryosectioned tissue sample. It was observed that mesodermal cell lineages penetrated heart, lung, liver, spleen, brain, kidney. We also microinjected differentiated cardiomyocytes into blastocyst and they were either cultured invitro or transferred into recipient. It was observed that within four days of in vitro culture, the cardiomyocytes disappear from blastocyst. They did not produce chimeric mouse either when pregnant mouse was allowed to give birth pups.
Involvement of glycosylation in notch signaling
Session: Pluripotent stem cells
Notch receptor activation by their ligands influences a wide range of developmental processes in metazoa like neuronal development, maintaining stemness, and T-cell lineage commitment. The extracellular domain of Notch consists of 36 EGF-like repeats, containing specific consensus sequences which are substrates for unusual post translational modifications in form of O-linked glucose and fucose. Recent studies revealed that these modifications are essential for Notch signalling and that differences in the extension of the glycan chains are a mechanism to modulate this pathway. It has been well established that O-fucosylation is necessary for the correct folding and expression of the receptor. The extension of O-fucose with N-acetylglucosamine by Fringe results in altered binding of the Notch ligands Jagged and Delta in mammals, which leads to different signal intensities. In contrast, only little is known about how O-linked glucose and its further elongation with two xylose residues influence Notch signalling. Acar et al. (2008) have recently identified the O-glucosyltransferase Rumi from D. melanogaster. Rumi deficient flies show a temperature dependent Notch phenotype. This led to the presumption that further modification of O-glucose with xylose may also result in a signal modulation comparable to the extension of O-fucose by Fringe. So far the genes encoding the xylosyltransferases are not known, hampering studies on the biological relevance of the Xyl-Xyl-Glc epitope on Notch.We now have identified two human genes (XylT1 and XylT2), encoding enzymes that transfer UDP-xylose in an α-1,3 linkage on an artificial glucose terminated acceptor, making them likely candidates for the unknown xylosyltransferase transferring the first xylose residue.
Stem Cell Biology Group, German Primate Center , Germany
Session: Pluripotent stem cells
Germ cell tumors of the testis are the most frequent tumors in men between 20 and 40 years. Their most common subtype is the seminoma, which arises from fetal germ cells that escaped from the control of the developing testicular stem cell niche, eventually leading to a fully developed seminoma. The molecular causes for the development of this type of germ cell tumor are still unknown. However, some types of testicular germ cell tumors share the expression of several factors with primordial germ cells and gonocytes and, interestingly, also with pluripotent embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. One factor playing important roles in both, iPS and ES cells is the transcription factor Krüppel-like factor 4 (KLF4). KLF4 expression data from totalling 179 human testicular samples including normal controls and seminoma, deposited in Gene Expression Omnibus repository for microarray data at the National Centre for Biotechnology Information, were analysed. Immunohistochemistry was used to detect KLF4 protein expression in carcinoma in situ (n = 6), seminoma (n = 14) and fetal human testes (n = 14). Normal spermatogonia, which are the stem cells of regular spermatogenesis, controlled by their stem cell niche, do not express KLF4. Remarkably and in striking contrast, carcinoma in situ and seminoma cells strongly express KLF4. This finding suggests that KLF4 may be an important factor for the maintenance of the developmental and the tumorigenic potential of testcular germ cell tumors as well as for the malignancy of seminoma.
Dedifferentiation of hMSCs by overexpression of Sox2
Session: Pluripotent stem cells
Characterization of pluripotent cells in macaque testes
Session: Pluripotent stem cells
Unipotent spermatogonia are derived from potentially pluripotent primordial germ cells. Primate testes contain two types of spermatogonia: a) reserve stem cells and b) self renewing progenitors. While rodents generate about 4000 sperm per initial stem cell division, in man an initial progenitor division creates only 16 sperm, leading to very different kinetics in spermatogenesis in rodents and primates (1; 2). So far, no systematic studies have explored the ontogenetic differentiation of male germ line stem cells in primates. In the present study we address whether pluripotent spermatogonial precursors in the Cynomolgus Monkey (Macaca fascicularis) are gradually eliminated from the developing testis by (potentially reversible) differentiation into unipotent spermatogonia. To this purpose, we use a panel of markers which allows us to histologically detect spermatogonia at various stages of differentiation in the adult monkey's testis. Using the expression of Oct3/4, Ap-2γ, VASA, GFRα1, Nanog, PGP 9.5, Tra − 1–81, and Tra-1–60 we identify sub-populations of spermatogonia, aiming to determine the total number of pluripotent cells in a non-human primate's testis. This project will provide novel insight into the mechanisms behind the differentiation of pluripotent germ line cells into unipotent stem cells in primates. Our data may enable us to select a target population in fetal, neonatal and adult non-human primate testes for the isolation and derivation of pluripotent stem cells.
Ehmcke J, Wistuba J, Schlatt S Hum Rep Update
Ehmcke S, Schlatt S Reproduction
Session: Pluripotent stem cells
ESC are a useful tool to investigate aspects of early embryogenesis and differentiation. Here, we employed a non-human primate (common marmoset, Callithrix jacchus) ESC line (Müller et al., Hum Reprod, 2009 24:1359) to study germ line stem cell marker expression likely representing the earliest stages of germ cell development occurring in culture. The first evidence of differentiation of the colonies was a pit-like structure in the center. The pits' bottoms consisted in the central part of flat, enlarged cells. This part was surrounded by smaller cells forming a multi-layered wall. The cells in the multi-layered wall maintained expression of pluripotency markers, e.g. OCT4, NANOG, and SOX2, while the central single-layered cells lost these markers. Individual central cells started expressing markers characteristic for testicular stem cells. We first observed coexpression of the germ cell marker VASA with the testicular stem cell markers CD9 and CD49f, respectively. Furthermore, we colocalized VASA and SSEA-4 in few individual differentiating cells. Interestingly, we have recently shown that adult spermatogonia also express SSEA-4. Later on, VASA/SSEA-4-positive cells detach from the pit bottom as small roundish cells maintaining this coexpression. These detaching cells also coexpress VASA and PGP 9.5. PGP9.5 is also expressed in spermatogonia. These immunofluorescence data were corroborated by RT-PCR. In summary, we provide evidence for spontaneous early germ cell development in this ESC line. These findings underline the close relation between ESC and testicular stem cells and may, at least in part, explain the ability of spermatogonia to spontaneously dedifferentiate into pluripotent cells in vitro.
Institute for Transfusion Medicine, University Hospital Essen , Germany
Session: Pluripotent stem cells
Embryonic stem cells (ESC) hold tremendous potential for therapeutic applications, including regenerative medicine, as well as for understanding basic mechanisms in stem cell biology. Many experiments cannot be conducted in human ESC because of ethical or practical limitations and thus nonhuman primate ESC serve as invaluable clinically relevant models. In regenerative medicine expression of the major histocompatibility complex (MHC) is a major problem for transplantation. To avoid rejection of transplant and overcome the immune barrier, recipients are typically treated with immunosuppressants. Disadvantages for recipients are different adverse effects including tumor formation. ESC display a relatively low expression of MHC class I and lack expression of MHC class II. During differentiation of ESC to other cell types MHC expression increases remarkably. An approach to avoid rejection of graft is silencing MHC expression in ESC with shRNA by RNA interference with lentiviral vector constructs. We expressed shRNA sequences targeting different regions of the marmoset β2-microglobulin (β2 m) in marmoset fibroblasts, B lymphoblastoid cell lines (B-LCLs) as well as in marmoset ESC. The down regulation of the MHC class I expression was followed on mRNA level by real time RT-PCR and on protein level by flow cytometry. The transduction of RNAi cassettes containing the sequences for shRNAs targeting β2 m suppressed MHC class I protein expression by up to 85%. These data demonstrate the feasibility of controlling MHC expression by genetically modifying non-human primate cells, setting the stage for a clinically relevant evaluation of this innovative approach in a large-animal model.
Feedback regulation in the “pluripotency network” of embryonic stem cells - insights from a mathematical model analysis
Session: Pluripotent stem cells
The expression of the transcription factors Oct4, Sox2, and Nanog is commonly associated with pluripotency of murine embryonic stem (ES) cells. So far the interaction between these factors has been characterized as self-enhancing by positive feedback regulations. However, new observations suggest that stem cell populations are heterogeneous with respect to the expression of Nanog. Moreover, Nanog-low cells are more likely to undergo differentiation, suggesting a functional role of this heterogeneity. Applying a mathematical transcription factor network model we explore mechanisms and feedback regulations to describe the observed variation of the Nanog-levels. In particular we show that these variations can occur under different assumptions yielding similar experimental characteristics. Based on model predictions we propose experimental strategies to distinguish between these explanations. Concluding from our results we argue that the heterogeneity with respect to the Nanog concentrations is most likely a functional element to regulate the propensity of a cell population for exiting from pluripotency and entering further differentiation.
Department of Cardiac-, Thoracic-, Transplantation and Vascular Surgery, LEBAO, Hannover Medical School , Germany
Session: Pluripotent stem cells
Induced pluripotent stem (iPS) cells may represent a favoured cell source for future regenerative therapies. One critical issue concerning the clinical use of autologous iPS cells is the accumulation of mutations in somatic (stem) cells over an organism's lifetime. Acquired somatic mutations are passed to iPS cells during reprogramming and may be associated with loss of cellular functions and cancer formation. Here we report the generation of human iPS cells from different cord blood-derived endothelial cell (CBEC) isolations. The reprogramming efficiency of different CBEC isolations correlates with their proliferation activity and their high expression levels of endogenous MYC. CBiPS cells show typical characteristics of embryonic stem cells and can be differentiated into derivatives of all three germ layers both in vitro and in vivo. For future therapeutic production of patient-specific and allogeneic iPS cell derivatives, CB may provide a juvenescent cell source with biological superiority, which is harvested routinely and without donor risk for public and commercial CB banks. CB could instantly become available for thousands of pediatric patients and, in particular, for newborns with genetic diseases or congenital malformations.
Department of Reprogramming, Hannover Medical School , Germany
Session: Pluripotent stem cells
Reprogramming of somatic cells to iPS cells holds great promises for stem cell research as well as regenerative medicine. The technology also allows to generate pluripotent cells lines from large animal species, for which ES cell lines have not been established. Here, porcine iPS cells are of particular interest as pigs represent suitable model systems for many aspects of human anatomy, physiology,or pathology. To generate porcine iPS, we have transduced fetal fibroblasts isolated from transgenic pig carrying the GFP reporter gene under the control of the murine Oct4 promoter with lentiviral vectors expressing human OCT4, SOX2, KLF4 and c-Myc from spleen focus forming virus (SFFV)-derived promoter/enhancer sequences. GFP expression indicating successful reprogramming at a frequency of approx. 0.05% was first observed around 15 days after transduction. GFP expressing cells were maintained in medium supplemented with FBS, Knock-out serum replacement, as well as MEK-ERK and GSK3-beta inhibitors. All GFP-positive colonies stained positive for alkaline phosphatase activity. So far cell lines could be passaged up to 6 times, whereas on further passaging cells started to differentiate. While more extensive characterisation of our GFP expressing cell lines is currently performed, these data clearly indicate the potential to generate porcine iPS by lentiviral transduction.
Department of Cardiac-, Thoracic-, Transplantation and Vascular Surgery, LEBAO, Hannover Medical School , Germany
Session: Pluripotent stem cells
Pluripotency of embryonic stem cells (ESC) is associated with the activity of a network of transcription factors including Sox2, Nanog and Oct4. To study the impact of Oct4 expression levels in induced pluripotent stem (iPS) cells regarding their (cardiogenic-) differentiation potential we have analysed iPS cells derived from OG2 mouse fibroblasts by retroviral overexpression of Oct4, Sox2, Nanog and Klf4. In the OG2 mouse model GFP expression is controlled by the Oct4 promoter. Heterogeneous GFP/Oct4 expression was observed in a batch culture of OG2-iPS cells. Via FACS 16 single cell-derived iPS cell clones were established, 9 displayed a high (H) and 7 a relative low (L) level of GFP expression; expression levels were stable for at least 20 + passages in individual clones. Using an embryoid body-based differentiation protocol, independent experiments revealed that the established L- and H-clones showed remarkable differences in their cardiac differentiation efficiency. Compared to L-clones, H-clones showed a delay in the onset of beating and formed less contracting areas, a marker of cardiomyocyte formation. To understand the molecular events underlying this phenotype we are analysing the integration sites and copy numbers of the four transcription factors used for reprogramming, the epigenetic modulation of gene expression, and other parameters. In parallel, OG2-derived ESC clones are analysed by the same approach. This will lead to a better understanding of iPS- vs. ESC and mechanisms involved in lineage differentiation.
Experimental pharmacology, Max-Delbrück-Center for Molecular Medicine , Germany
Session: Pluripotent stem cells
The study compares spontaneous and directed endodermal differentiation potential of human embryonic SA002 stem cells. Spontaneous differentiation of SA002 cells was induced by cell culturing on matrigel coated dishes without feeder cells in absence of bFGF. Co-culture systems with murine AML12 hepatocytes and their conditioned media were used for endodermal stem cell differentiation. Differentiation potential was evaluated by real-time PCR, immunohistochemistry and electron microscopy. Undifferentiated SA002 cells express typical pluripotent stem cell markers and possess a unique Cx43 expression, whereas Cx32 showed a sporadical expression. The cells were further characterized by low SOX17, CXCR4, AFP, HNF3b, C/EBPa expression, ALB was not detectable. Under in vitro conditions, spontaneous differentitation was distinguished by an increased expression of ALB, AFP, CK19 and C/EBPa. Cultivation of SA002 cell with conditioned media induced distinct changes in cellular and ultrastructure morphology towards an endodermal and hepatic lineage. On gene level only changes in ALB expression were noticed. Direct co-culture of SA002 cells with AML12 cells exerted several hepatic differentiation features of embryonic stem cells and showing morphological similarities to hepatic progenitor cells. High gene expression levels of early endodermal in the SA002 cells were detected. In vivo differentiation of SA002 cells, induced by sub cutaneous transplantation into immunodeficient NOD/SCID mice, revealed predominantly differentiation into cells of mesoderm and endoderm. After s.c.application no cell dissimination into other mouse organs was found by human PCR. The results suggest, that SA002 cells showed in vitro and in vivo an endodermal differentiation potential.
Centre of Reproductive Medicine and Andrology, Universitätsklinikum Münster , Germany
Session: Pluripotent stem cells
Developmental toxicity profiling of thalidomide upon randomly differentiated hESCs embryoid body
Session: Pluripotent stem cells
Human embryonic stem cells (hESC) are pluripotent cells, able to differentiate into 3 primary lineages like cardiac, hepatic, and neural cells. These differentiation studies were a potential tool to study the toxicity of drugs on those respective lineages. In the present study hESC randomly differentiated to study the developmental toxicity of Thalidomide. Various mouse and in vivo studies have showed the mechanism of teratogenicty for thalidomide. In our study we have done global transcriptional profiling of the effect of thalidomide during the embryoid body development at sub lethal concentration. Gene ontology analysis reveals thalidomide down regulated organ development, anatomical structure development, multicellular organismal development and circulatory system development in biological process and for molecular function we found thalidomide targets lipid and lipo-protein binding. Protein protein interaction showed different interacting genes among our down regulated genes. Pathway analysis demonstrates more number of genes from ECM receptor signaling. Collectively, thalidomide down regulated the endodermal and mesodermal markers during development and genes involved in angiogenesis and limb development. In summary this study shows developmental toxicity markers for thalidomide and provides the new tool for finding the developmental toxicity of any new clinical entity.
Department of Cardiac-, Thoracic-, Transplantation and Vascular Surgery, LEBAO, Hannover Medical School , Germany
Session: Pluripotent stem cells
Gene targeting by homologous recombination via customized zinc-finger nucleases (ZFN) is a powerful method to manipulate the genome and correct genetic defects. Although ZFN based homologous recombination has been shown to be significantly more efficient than conventional gene targeting, the selection of suitable clones still requires cells that proliferate in culture. Clinically applicable ZFN-based gene correction in patient-specific cells was so far not possible due to the inability to sufficiently expand most adult (stem and progenitor) cells in vitro. However, with the availability of human induced pluripotent stem (hiPS) cells with their almost unlimited potential for proliferation and differentiation, this method offers novel opportunities for the development of patient-specific regenerative therapies. As a first step towards ZFN-based gene targeting, we established non-viral gene-transfer in hiPS cells with transfection rates of up to 30% and high cell vitality. Aiming at the development of a general ZFN-based recombination approach in hiPS cells, we investigated the functionality of an eGFP specific ZFN in an eGFP transgenic hiPS cell clone. Finally, the development of a general applicable protocol for ZFN based site-specific recombination and gene correction in patient-specific hiPS cells may enable the development of cellular therapies for genetic diseases such as cystic fibrosis or hereditary cardiomyopathies.
Influence of microRNA-1 on cardiac differentiation of rhesus monkey embryonic stem cells
Session: Pluripotent stem cells
Contribution of microRNAs (miRs) to cardiac development and disease has been demonstrated. But only recent work reported that miR-1 directs murine and human embryonic stem cells (ESC) towards the mesendodermal lineage while inhibiting neuroectodermal differentiation at a progenitor cell stage. As an in vitro model for cardiomyogenesis we are using the rhesus monkey (Macaca mulatta) embryonic stem cell (RESC) line R366.4 which strongly favours neuronal differentiation but poor mesoderm and subsequent cardiomyocyte formation. Aim of this study is to investigate whether exogenous expression of miR-1 can directly enhance generation of mesodermal lineages, specifically cardiomyocytes in R366.4 cells. After lentiviral transduction of miR-1 in combination with the reporter gene GFP, expression of miR-1 in RESCs was demonstrated by qRT-PCR and cell differentiation was induced via embryoid body-formation; the expression of mesendodermal and early cardiac as well as neural markers was investigated. Our preliminary results do not indicate an efficient promotion of cardiomyocyte formation or the reversal of neuroectodermal lineage commitment in RESCs by exogenous miR-expression. However, the expression level of miR-1 is a critical parameter in our system, which is under ongoing investigation and optimization.
Generation of oct4 promoter EGFP transgenic pigs by somatic nuclear transfer
Session: Pluripotent stem cells
Porcine pluripotent cells are promising candidates for preclinical testing of regenerative cell therapies. However, little is known about pluripotent cell populations in the pig, and to date true porcine embryonic stem cells or induced pluripotent cells are not available. Here, we report the production and stem-cell-specific expression pattern of transgenic pigs carrying a mouse Oct4 promoter - enhanced green fluorescent protein (Oct4-EGFP) reporter construct. We used fetal porcine fibroblasts transfected with the mouse Oct4-EGFP construct for somatic cell nuclear transfer to produce 11 cloned piglets. The Oct4-EGFP construct was found to be expressed in cloned pig blastocysts and in primordial germ cells. We could also detect low expression of the Oct4-EGFP transgene in postnatal testis isolated from newborn transgenic piglets, suggesting that the Oct4-EGFP construct is expressed in the precursor cells of spermatogonia. Hybrid cells resulting from fusion of transgenic porcine fibroblasts with murine embryonic stem cells showed reactivation of the Oct4-EGFP gene. The fused hybrids displayed stem cell morphology, a high proliferation rate and formed teratomas in immuno-deficient mice. This transgenic pig line should simplify the derivation and maintenance of porcine embryonic stem cells and will be of utmost interest for reprogramming studies based on porcine cells.
RG Reprogramming, Hannover Medical School , Germany
Session: Pluripotent stem cells
Given their high abundance and easy accessibility, hematopoietic cells appear as a promising source for the generation of iPS cells. Therefore, we have investigated reprogramming of different populations of murine bone marrow cells utilizing lentiviral vector constructs optimized for transgene expression in hematopoietic cells. Bone marrow cells from OG2 mice (Oct4 promoter-driven GFP expression) were sorted into standard Lin- fractions (LSK, LK, LS) and Lin + cells. Cells were transduced with lentiviral vectors expressing human Oct4, Sox2, Klf-4 and c-Myc from the SFFV-derived promoter. Two days prior to and 7 days after transduction, cells were cultured with appropriate hematopoietic cytokines, thereafter cells were transferred to ES cell culture conditions. Seven days p.t., single GFP-positive cells were detected, which during the next 2 weeks gave rise to iPS cell colonies, characterized by GFP-expression, ES-cell morphology and alkaline phosphatase activity. Expression of pluripotency markers, such as Oct4, Nanog and SSEA-1, was demonstrated by qRT-PCR, Western blot analyses and flow cytometry. As shown by in-vitro differentiation and teratoma formation assays, BM-derived iPS lines were able to differentiate into all three germ layers, thus demonstrating their pluripotency. Our data indicate that both immature as well as more differentiated murine bone marrow cells can be reprogrammed, suggesting hematopoietic cells as a promising source for the generation of iPS cells.
Department of Genetics, Tarbiat Modares University , Iran
Session: Pluripotent stem cells
It has been established that the expression of four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) are enough to reprogram adult mouse and human cells to the embryonic state, generating induced pluripotent stem (iPS) cells. However, iPS technology not only as a cellular replacement therapeutic strategy but also as a key for understanding cellular developmental potentials has critical impediments. First among many important issues, the low efficiency of reprogramming is a major barrier to the generation and identification of iPS cell lines. Secondly, generation of iPS cells requires costly growth factors such as leukaemia inhibitory factor (LIF) or basic Fibroblast Growth Factor (bFGF) for mouse and human cells, respectively. The cytokine LIF integrates signals into mouse embryonic stem (ES) cells to maintain pluripotency. Here, we constructed an LIF-IRES-EGFP retroviral vector and utilized it in addition to the 4 conventional factors to support the induction of pluripotency in the donor mouse cells with in-situ LIF production. Additionally, the expression of EGFP in the target cells could be used as estimation for the efficiency of transduction. Moreover, retroviral vectors would be silenced in fully reprogrammed cells, and EGFP suppression, therefore, could be used to mark pluripotent stem cells. In conclusion, using LIF-IRES-EGFP retroviral vector, iPS cells can be derived from genetically unmodified donor cells discerningly and cost-effectively.
Efficient non-viral, multi-genetic modification of ESC and iPS cells for the enrichment and tracking of cardiomyocytes
Session: Pluripotent stem cells
Pluripotent stem cells with their ability to differentiate into cardiomyocytes (CMs) hold a great potential for replacement therapies and provide an in vitro model of human heart development. To date, the generation of stable transgenic hESC lines remains consuming and inefficient and hiPS lines with a transgene for the enrichment of specific progenies have not been reported. Herein, we describe a new one-step strategy for the efficient introduction of multiple plasmid-based constructs into hESC/hiPS. Stable clones carrying a combination of transgenes located on independent plasmids allow, for example, the antibiotic-based purification of stem cell-derived CM that additionally express reporter genes, enabling detection of transplanted cells in situ. Using our new approach enabling electroporation and subsequent clone establishment in a simplified and highly efficient manner, we succeeded in generating numerous stable transgenic hESC and hiPS clones within 12 days, with about 25% carrying both, a CM-specific selection construct and a vector-mediated fluorescence-label. Expanded clones were analysed for the expression of pluripotency markers, as well as the capacity to differentiate into derivatives of the three germ-layers. Stem cell-derived CM enrichment was initiated after beating clusters were identified in the differentiated cultures. These cells are tested for heart repair, tissue engineering, and electrophysiological assessment of drug toxicity in vitro.
Viral vectors facility, CNIC , Spain
Session: Pluripotent stem cells
Since the discovery of human pluripotent stem cells (hPSC), different methods to target specific genes involved in their biology have been developed. However, those methods are characterized by a low effectiveness rate. Even with the recently improved engineering tools such as the zinc-finger nuclease (ZFN) specific recombination and the possibility of transgenes generation in the Rosa26 locus, some problems are still found. Some of the obstacles are a poor recombination rate, toxicity of the reporter gene and target gene promoter silencing. Trying to solve these issues we are developing a Rosa26 locus genetically modified hPSC line, by means of integrative defective lentiviruses, with a double marker system and a Cre mediated recombination. This will allow an efficient site directed integration in human embrionic stem cells (hES cells). As a proof of principle, we will present data showing improved success rates for controlling several target genes. These genetically modified hPSC lines will allow the possibility to controlled overexppression and knockdown of genes during differentiation events.
Effect of different culture condition on development of stem cell like cells from early developing parthenogenetic embryos in buffalo
Session: Pluripotent stem cells
The present experiment was carried out to study the role of different culture conditions on multiplication of early stage parthenogenetic embryonic cells of buffalo. The parthenogenetic embryos of 32-cell stage were made zona free. The clumped blastomere were cultured on inactivated buffalo embryonic fibroblast (BEF) or murine embryonic fibroblast (MEF) with culture condition viz. (C-I) DMEM + ITS + FBS + BEF, (C-II) DMEM + ITS + BS + LIF + BEF, (C-III) DMEM + ITS + FBS + LIF + SCF + BEF, (C-IV) DMEM + ITS + FBS + LIF + SCF + IGF1 + BEF, (C-V) DMEM + ITS + FBS + LIF + SCF + IGF1 + BFGF4 + BEF and (C-VI) DMEM + ITS + FBS + LIF + SCF + IGF1 + MEF. The blastomere were cultured at 38oC, 5% CO2 and 90% relative humidity in CO2 incubator. Once the cells made stem cell clones, they were passaged. The cells clones formed were characterized by immunocytochemistry and real time PCR. A total of 5,3,5,4 and 21number of zona free morulae were cultured in culture condition C-I, C-II, C-III, C-IV and C-V, respectively. In all these culture condition blastomere could not be propagated not more than three passage. In C-VI, a total of 22 zona free morulae were cultured. In this culture condition the embryonic stem cell clone could be propagated up to 7th passage. In the present study parthenogenetic embryonic stem cells expressed pluripotent markers SSEA-1, 4, TRA 160, TRA181, Oct4, Nanog and alkaline phosphatase. The expression of pluripotent genes Oct4 and Nanog were also confirmed by real time PCR. The results indicated that multiplication of buffalo parthenogenetic embryonic cells from early stage embryos were better when they were cultured on MEF (C-VI) than on buffalo embryonic fibroblast cells.
Experimental Hematology, Hannover Medical School , Germany
Session: Pluripotent stem cells
Recently, Yamanaka and colleagues showed that the genetic modification of somatic cells with 4 reprogramming factors (RF) (Oct4, Sox2, Klf4, c-Myc) resulted in reprogramming into induced pluripotent stem (iPS) cells, which are very similar to ES cells. This finding offers new perspectives for the generation of patient- or disease-specific iPS cells as well as new avenues for disease models, drug discovery and regenerative medicine. Here we constructed a modular versatile lentiviral vector system for mono- and polycistronic expression of these 4 RF (of human or murine origin, optionally codon-optimized). To drive the RF, we used the strong retroviral promoter SFFV, which enables high transgene expression and is frequently silenced in ES cells. Using this system and taking advantage of an Oct4-GFP reporter model, we could generate murine iPS cells as early as 6 days post transduction and with high efficiency. To learn more about the kinetics of reprogramming, we linked each RF to a different fluorescent marker to be able to follow their gene marking and expression kinetics in relation to our Oct4-GFP reporter. Especially when using a 4-in-1 vector (harboring all 4 RF on one construct linked by 2A sites) we observed a clear gradual downregulation/silencing of RF expression after induction of reprogramming as indicated by Oct4-GFP. To avoid the risks of residual permanent RF expression and insertional mutagenesis, we further improved our system by the inclusion of FRT or loxP sites to be able to remove the SFFV-RF cassette through transient recombinase expression. In summary, the described vector system may ease the generation of clean murine and human iPS-like cells and also contribute to the identification of the underlying mechanisms.
Enhanced reprogramming of human somatic cells using small molecules and miRNAs
Session: Pluripotent stem cells
In 2006 it has been shown that the ectopic overexpression of defined transcription factors that are associated with embryonic development and/or the preservation of pluripotency in embryonic stem cells, including the transcription factors Oct4 and Sox2 in combination with either Klf4 and cMyc or Nanog and Lin28, induces somatic cells from mouse and human to become pluripotent stem cells named induced pluripotent stem (iPS) cells. We have recently shown that human cord blood endothelial cells can be efficiently reprogrammed into iPS cells. Similar to most other published protocols, these cells have been generated by means of retroviral vector techniques associated with potential risks including insertional mutagenesis and maintenance of transgene expression. Since 2006 progress has been made and different studies have now shown that application of proteins, small molecules and miRNAs can enhance the reprogramming efficiency and can partly substitute viral overexpression of the transcription factors. Based on these findings the aim of this ongoing study is to establish an optimized reprogramming protocol for different human somatic cell types to efficiently induce iPS cells without the need for viral overexpression. Therefore, the effect of small molecules like 5′azacytidine, trichostatin-A, valproic acid and BIX-01224, various miRNAs and antagomirs in different combinations are tested.
SEVI facilitates an efficient lentiviral transduction of murine and human ESCs
Session: Pluripotent stem cells
Lentiviral gene transfer systems are widely used for genetic modification of Embryonic Stem Cells (ESC). Recently, enhanced retroviral infection has been demonstrated using a naturally occurring fragment of the abundant semen marker prostatic acidic phosphatase (PAP). This peptide forms amyloid fibrils and is able to capture HIV virions and to promote their attachment to the target cells. The fibrils were termed Semen-derived Enhancer of Virus Infection (SEVI) and it was proposed that their effect also applies to further retroviruses. Our results show that lentiviral vectors pseudotyped with VSV-G, a foamyviral envelope, GALV or RD114 are able to bind to SEVI. The SEVI protein itself has no toxic effect on murine and human ESC and the cells do not lose their pluripotent state. Using mouse ESC a 1.7fold increase of the gene transfer rate was detected in SEVI treated cells for the VSV-G virus (MOI: 2). On human ESC 45,000 transduction units (TU) were used to transduce appr. 120,000 cells. For the VSV-G construct, the SEVI treatment only results in a 2fold increase and for the foamyviral envelope no enhancement could be detected. However, for the GALV envelope a 5fold enhancement and for the RD114 envelope even a 24fold enhanced transduction rate was achieved. The advantage of these two envelopes is that in contrast to VSV-G pseudotyped viruses they are not toxic. Furthermore, these envelopes are not able to bind to mouse cells and, thus, do not transduce mouse embryonic fibroblasts (MEF) often used as feeder cells. In conclusion, the addition of SEVI protein during transduction GALV and RD114 envelopes is a reliable and feasible tool to increase the gene transfer rate of human ESCs.
Institute of Virology, Technische Universität München, Munich , Germany
Session: RNA-based approaches
The pattern recognition receptor RIG-I, a cytosolic helicase, recognizes 5′-triphosphated RNAs (3p-RNA). This defined molecular structure of RNA is shared by all in vitro transcribed RNAs, is present in some viruses and induces an interferon (IFN) response. We used 3p-dsRNA recognized by RIG-I, which induced an antiviral IFN type I response and controlled HBV replication in vitro in stable HBV replicating cell lines as well as in HBV infected primary human hepatocytes (PHH). When complexed and i.v. injected, it transiently controlled HBV replication in HBV transgenic mice. To prolong and sustain this effect, we designed siRNAs targeting overlapping ORFs of HBV genome at the 3′-end of pregenomic HBV-RNA and demonstrated that they can be in vitro transcribed to become RIG-I ligands. 3p-siRNA induced INF type I and thereby 2′-5′-OAS in HBV replicating cells. Intracellular HBV pregenomic RNA, encapsidated DNA and cccDNA were significantly reduced and secretion of progeny HBV and viral antigens were diminished about 90%. Thereby, the antiviral effect of 3p-siRNA was superior to siRNA or 3p-RNA alone from day 8 onwards. All effects of 3p-siRNAs on HBV replication were confirmed in HBV infected PHHs. To proof the effect in vivo, we injected i.v. 3p-RNA, 3p-siRNA and non-phosphorylated siRNA, respectively, into HBV 1.3 tg BL-6 mice. In all cases, HBV viremia and antigen levels were reduced as well as pregenomic RNA. Hereby, the combinatory antiviral effect of 3p-siRNA was again superior to siRNA or 3p-RNA alone at day 9. Taken together, we designed HBV-specific 3p-siRNAs, which efficiently suppress HBV replication via a RIG-I dependent immune response combined with HBV gene silencing through RNA interference in vitro and in vivo.
Gene expression inhibition, induced by short interfering RNAs (siRNA) targeted to promoter
Session: RNA-based approaches
Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in many organisms by a process known as RNA interference (RNAi). The mediators of gene inactivation in the process are 21 base pair small interfering RNAs (siRNAs) generated from longer dsRNA by the RNase III-like enzyme, Dicer. RNAi can induce gene silencing via two pathways: post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). PTGS involves siRNA-mediated targeting and degradation of mRNA. However, siRNAs were demonstrated to induce TGS via DNA methylation at the targeted promoter. siRNA-directed TGS was first described in plants and yeasts and more recently in mammalian cells. Synthetic siRNAs can induce loss of gene activity comparable to long dsRNA. The limitation of this method is that the transfected synthetic siRNA works for only a few days. In this study, we used undifferentiated EC cell line, P19 to investigate the RNAi response to siRNA by using a transgene containing an enhanced green fuorescent protein (eGFP) gene as a target. To overcome the limitation of using siRNA, the cells tranfected with a vector, based on pSUPER system that directs the synthesis of siRNA-like transcripts. To investigate TGS pathway via RNAi, we also used a siRNA transcript against pgk-1 promoter. The data presented here showed reduction in eGFP gene expression as shown by fluorescence microscopy, flow cytometry and western blot. Furthermore, results of colony counting, after puromycin (puro) selection, indicated that puro gene expression was also reduced. However, southern blot analysis showed no methylation in pgk-1 promoter.
siRNA mediated knockdown of plasminogen activator inhibitor 1 in experimental sepsis
Session: RNA-based approaches
Plasminogen activator inhibitor 1 (PAI-1) is the primary inhibitor of plasminogen activators and is a key regulator of the fibrinolysis/coagulation cascade. Upregulation of PAI-1 in critical illnesses such as sepsis leads to coagulopathy, subsequent organ dysfunction and frequent death. We aimed to establish an effective siRNA treatment to reduce circulating PAI-1 in murine abdominal polymicrobial sepsis. First, knockdown efficacy of 3 different anti-PAI-1 siRNAs was assessed in vitro using lipofection and 50 nM siRNAs and the siRNA717 was selected for in vivo studies. Next, outbred female CD-1 mice were tail-vein injected with 10μg (0,62 mg/kg) of 717 or scrambled siRNA complexed with polyethyleneimine (PEI), N/P-ratio = 10. PEI alone and complexed with DNA oligos served as vehicle control. 72, 48 and 24 h later mice were subjected to caecal ligation puncture. Circulating PAI-1 was measured by ELISA 6 h, 24 h and 48 h and PAI-1 gene expression was monitored by RT-PCR in liver, kidney, lung and heart at 48 h post-CLP. CLP causes robust release of circulating PAI-1 (150x) at the protein level. 24 h pre-treatment with anti-PAI siRNA reduced circulating PAI-1 by 50% (compared to scrambled siRNA). This reduction was seen only at the 48 h post-CLP induction. Interestingly, the 72 and 48 h siRNA pre-treatment was ineffective in reducing circulating PAI-1. The reduction of circulating PAI-1 was concurrent with siRNA-mediated PAI-1 gene knockdown in liver and heart but not in lungs and kidney. siRNA-mediated PAI-1 gene knockdown translated into an effective reduction of sepsis-induced PAI-1 protein. The subsequent survival study will reveal therapeutic potential of siRNA mediated PAI-1 knockdown. Partially funded by IRG grant No. 203685.
Inhibition of HBV and HCV using long-hairpin RNAs
Session: RNA-based approaches
RNAi is a sequence specific mechanism, used to inhibit gene expression in mammalian cells. It has been described that viral replication can be inhibited by synthetic short interfering RNAs (siRNAs), gene constructs encoding short hairpin RNAs (shRNAs) or long hairpin RNAs (LhRNAs). The use of siRNAs or shRNAs as antiviral therapy is limited due to viral escape. As LhRNAs are processing in many siRNAs targeting different viral sequences this should avoid viral escape and provide a potent inhibition of viral replication. Previous studies have demonstrated that cotransfection of HIV-1 molecular clone pLAI together with a plasmid encoding HIV-specific LhRNAs, induce a potent and specific inhibition of HIV by LhRNAs. This prompted us to analyze HBV and HCV expression in cells expressing lhRNAs targeting specific viral sequences. Cotransfection of plasmids that express the HBV genome and specific LhRNA against HBV resulted in a drastic decrease of HBV SAg. Interestingly, inhibition was similar with lhRNAs or siRNAs targeting HBV. A decrease in SAg expression was observed in HBV constitutively expressing cells transfected with plasmids expressing lhRNAs. Preliminary results have also shown inhibition of HCV production in HCV-infected Huh7 cells transfected with plasmids expressing viral specific LhRNAs. Altogether, these results suggest that LhRNAs could be potent tools to inhibit viral production and therefore, base future therapies.
Pediatric HematolOncology, Heinrich Heine University Duesseldorf , Germany
Session: RNA-based approaches
Small nuclear U1 RNAs (snRNA) as components of the spliceosome specifically recognize 5′ splice sites (5′ss). Using lentiviral vectors as established gene delivery system to stably introduce expression cassettes for mutation-adapted engineered U1 snRNAs into primary human FANCC mutant cells, we demonstrate here the potential of this novel approach as curative therapeutic strategy for FA. A biallelic 5′ss mutation + 1G > T was identified in four Fanconi anemia (FA) patients from two pedigrees with germ-line mutations in FANCC. Sequencing of the RT-PCR-amplified endogenous FANCC exon 2 splicing products demonstrated that the mutant TT 5′ss led to exon skipping and to the activation of cryptic splice sites in exon 2 and intron 2, however at low efficiency also facilitated the use of the TT dinucleotide as splice donor, generating low levels of correctly spliced transcripts. Functional analysis of a heterologous three-exon, two-intron splicing reporter construct with FANCC exon 2 as the middle exon revealed that splicing at the mutant TT 5′ss is TT dependent, suggesting that the mutant 5′ss is recognized by a yet unidentified spliceosomal U1 snRNA. Using lentiviral vectors for stable expression of two TT-adapted U1 snRNAs, we finally showed that the DNA damage-induced G2 cell cycle arrest of primary patient fibroblasts can be corrected by TT-adapted U1 snRNAs. These results demonstrate that lentiviral delivery of engineered U1snRNAs is capable of restoring a functional FA pathway in primary cells of FA patients. This novel form of genetic therapy ensures that the corrected protein is still subject to the physiological gene expression control mechanisms.
Laboratoire dystrophies des ceintures , Genethon, France
Session: RNA-based approaches
Titin is a giant protein expressed in both skeletal muscles and heart. Several pathogenic mutations were identified in its last coding exon. The most frequent mutation commonly referred to as FINmaj, results in the replacement of 4 amino acids and affects a subset of patients in Finland. The mutation causes a Tibial Muscular Dystrophy (TMD) when present on one allele and a Limb Girdle Muscular Dystrophy phenotype 2J (LGMD2J) when present on both alleles. To obtain a model for studying the physiopathology of these diseases and evaluating therapeutic strategies, we introduced the FINmaj mutation in the murine genome by a knock-in strategy. This model reproduces symptoms of both the human TMD and LGMD2J. Considering the large size of the titin cDNA (about 100 kb), classical gene transfer strategies are not feasible. To bypass this hurdle, we are testing spliceosome-mediated RNA trans-splicing (SMaRT) to replace mRNA titin last exon. HER911 cells expressing a titin minigene with the FINmaj mutation in the last exon were transfected with a plasmid coding for a 3′pre-trans-splicing molecule encoding wild-type last exon. RT-PCR analyses showed that specific replacement of the mutated 3′portion of the minigene TTN transcript has occurred through trans-splicing. We could see the reduction of the level of mutant mRNA and the apparition of the wild-type corrected gene. Experiments to delivered trans-splicing RNAs in the FINmaj mouse model using AAV vectors are currently investigated.
Department of Dermatology, University Hospital Salzburg , Austria
Session: RNA-based approaches
RNA trans-splicing is a gene-therapeutical approach for the correction of large genes. The endogenous splicing machinery is utilized to recombine a target cellular pre-mRNA and a RNA trans-splicing repair molecule (RTM) by trans-splicing, replacing the disease causing parts of a gene by their wildtype copy. In a previous reported in vitro model we used SMaRT to replace upstream (5′) exons 2 to 9 of the plectin gene (PLEC1) on pre-mRNA level to correct a leucin insertion in exon 9 of PLEC1. This mutation underlies the rare variant of the blistering skin disease epidermolysis bullosa simplex with late onset muscular dystrophy (EBS-MD). To improve the pre-mRNA based RNA trans-splicing technology for the correction of the PLEC1 gene we have developed a high throughput screen to identify the best RTMs for improving gene correction. By using a fluorescent reporter system we are able to rapidly evaluate the effect of various RTM binding domains on trans-splicing functionality. After double transfection of single RTMs of a RTM library and a target molecule into HEK293 cells the best RTMs can be identified by “Fluorescence Activated Cell Sorting” (FACS) and semiquantitative real time PCR. The identification of the most efficient RTM will increase the endogenous correction of the plectin gene at mRNA level and should enable us to establish a gene therapy approach for patients suffering from EBS-MD. Supported by debra-austria.
Department of Dermatology, University Hospital Erlangen , Germany
Session: RNA-based approaches
A promising approach in the treatment of malignant diseases is the adoptive transfer of T cells engineered to express chimeric immunoreceptors which consist of a scFv directed against a cancer cell surface antigen and of the CD3-zeta and CD28 signaling domains. To avoid persistent auto-aggression, a reported life threatening risk of engineered T cells with constitutive immunoreceptor expression, we explored RNA electroporation for transient immunoreceptor expression in human T cells. CD4 + and CD8 + T cells were efficiently transfected by electroporation with RNA coding for immunoreceptors specific for carcinoembryonic antigen (CEA), expressed on colon carcinoma, for ErbB2/Her2neu, expressed on breast cancer, and for melanoma chondroitin sulfate proteoglycan (MCSP), expressed on melanoma cells, respectively. Half-maximal immunoreceptor expression was at day 2 and no expression detectable at day 9 after transfection. Upon specific stimulation with the respective target cells, transfected CD4 + and CD8 + T cells secreted the cytokines IL-2, TNF-alpha, and IFN-gamma. Moreover, RNA reprogrammed CD8 + T cells killed antigen-expressing target cells in vitro. Taken together, RNA electroporation of T cells provides a versatile tool for transient immunoreceptor expression with the substantial advantage of transient expression and thereby limited risk of unintended auto-aggression and, secondly, not being classified as gene therapy, thereby best suited to complement viral immunotherapy of cancer.
Tigar “loss of function” a therapeutical approach for glioma cells
Session: RNA-based approaches
TIGAR (TP53 induced glycolysis and apoptosis regulator). shows similarity to the bisphosphatase domain of the bifunctional PFK-2/FBPase-2 enzyme. TIGAR functions to lower fructose-2,6-bisphosphate levels in cells, resulting in an inhibition of glycolysis, and an overall decrease in intracellular reactive oxygen species (ROS) levels, correlating with a decreased sensitivity to apoptosis. Knock down of endogenous TIGAR expression sensitized cells to p53-induced apoptosis. Expression of TIGAR may therefore modulate the apoptotic response to p53 inducing signals, allowing survival in the face of stress signals that might be reversed or repaired. The decrease of the intracellular ROS levels in response to TIGAR may also play a role in the ability of p53 to protect from the accumulation of genomic lesions. This new gene TIGAR offers good prospects for metabolic regulation since p53 can modulate cell metabolism through its induction. A the moment neither this interaction nor the relationship between Tigar and radiosensitivity has been studied in glioblastoma. To study this interaction we tested the efficacy of Tigar siRNA in inhibiting Tigar expression and its effect on radiosensitivity in U87-MG cells wich possess wt p53. Tigar siRNA decreased Tigar protein to almost undetectable levels by 72-h post-transfection. Furthermore, Tigar siRNA transfection caused a significantly larger decrease in viability following irradiation, low the colony formation after the treatment and incressed significantely apoptosis. Anti-tigar strategies may, therefore, prove useful in improving the response of glioblastoma to radiotherapy, thus potentially improving patient survival.
Specific small interfering RNAs(siRNAs) targeted to CXCR4 and CCR5 confer HIV-1 resistance
Session: RNA-based approaches
Infection of a susceptible cell by HIV-1 required viral envelope interactions with the primary cell surface receptor, CD4, and a coreceptor, either CXCR4 or CCR5. Therefore, a potentially promising strategy is to exploit siRNAs to prevent viral entry at the cell surface by down-regulating essential cell surface HIV-1 coreceptors. In our studies, we designed specific siRNA oligonucleotide and establishment of short hairpin RNA expressed plasmid constructs against HIV-1 cell surface coreceptors, CXCR4 and CCR5, to inhibit viral entry. These siRNA oligonucleotides prepared using synthetic RNA duplexes consisting of two unmodified 21mer oligonucleotides annealed together to from siRNAs for CXCR4 or CCR5. These plasmid constructs were designed to high-level express shRNA under the control of U6 promoter that employs RNA polymeraseIII in mammalian cells. HeLa-TZM cells transfected with siRNA oligonuceotide or shRNA expressed plasmid constructs showed significant down-regulation of their respective coreceptors. Also, we established the optimal condition of transfection for each siRNA & shRNA oligonucleotide expression plasmid construct and confirmed the effective reduction of CXCR4, CCR5 expression levels by each coreceptor specific siRNA. Specially, effective reduced levels of CXCR4 and CCR5 expression showed at 5nM siRNA concentration and 48 hours after transfection. These results showed that the possibilities of the promising siRNA for in vivo gene therapeutic applications.
Department of Laboratory Medicine, Karolinska Institute , Sweden
Session: RNA-based approaches
Splice-correcting phosphorotioate RNA anti-sense oligonucleotides (ASO) with 2′-O-methyl modifications are promising therapeutic agents for several disorders caused by aberrant splicing. However, their usefulness is hindered by the lack of efficient delivery. Unmodified 25 K polyethylenimine (PEI) has shown potential for plasmid delivery but seems to be less efficient for short nucleic acid sequences. Herein, we have evaluated several amino acid-modified PEI molecules as carriers for ASO. By characterization of their properties, such as size, stability and transfection into mammalian cells we have identified tyrosine-modified PEI (PEIY) as an efficient ASO delivery system. Hela705 cells containing an aberrant luciferase gene, interrupted by a mutated β-globin intron, were used to assess the splice correction mediated by the various modified PEI/ASO polylexes. PEIY has a self-assembly nature, as opposed to the highly cationic parent polymer, which is relevant for the stability of the ASO/PEIY complexes. As a result, at an optimal ratio of 20:1 (+/-), PEIY/oligo particles were able to significantly correct the splicing in Hela705 cells both on the mRNA and protein level. ASO formulated by PEIY could enhance luciferase activity up to 450-fold. This increase was three times higher than that produced by the commercially available transfection agent Lipofectamine. PEIY/ASO polyplexes resulted in at least 80% correct splicing of the transcript. Moreover, extremely low doses of ASO (0.025 μM) could still show significant splice correction represented by 150-fold increase of luciferase activity and 47% mRNA correction. Our findings reveal a new platform that can be further developed for ASO in vivo targeting.
Berlin-Brandenburg Center for Regenerative Medicine, Charite University Hospital Berlin , Germany
Session: Safety, regulation, clinical translation
Legislation in individual member states of the European Union on human embryonic stem cell (hESC) research is as divergent as the different cultural, ethical, and religious views on the issue. Differing views in the individual EU member states are described in a multitude of national legislations on stem cell research and are reflected by the European human embryonic stem cell registry (hESCreg). Despite the divergent hESC legislation in Europe, the ethical concern in Europe is unifying. European countries in which hESC research is allowed carry out experiments in accordance with the fundamental ethical principles and human rights laws of the European Union. Here we describe the actual legal situation of stem cell research in the EU and Europe-associated countries as well as in the United States and in Asian-Pacific countries.
Elstner A. Stem Cell Research
Session: Safety, regulation, clinical translation
Critical limb ischemia (CLI) is the severe endpoint of peripheral artery occlusive disease (PAD). Based on a preclinical proof of concept of administration of allogeneic placental-derived mesenchymal-like stromal cells (Pluristem Therapeutics Inc.) in a standard limb ischemia model in mice a regulatory strategy was developed to get the regulatory approval to start a clinical phase I trial in patients with critical limb ischemia. Using the scientific advice procedure of the Paul Ehrlich Institute (PEI) at an early stage of development the preclinical program was defined to demonstrate safety and efficacy and to support the clinical study design. Pharmacodynamic, pharmacokinetic and toxicology as well as tumorigenicity studies according to the EMEA guideline on human cell based medicinal products have been performed and a consistent manufacturing procedure had to been proven. An adaptive phase I/II clinical trial design was developed according to EMEA and ICH guidelines and finally regulatory approval was given by the PEI and the FDA
Stimulating translational adult stem cell therapy in the netherlands
Session: Safety, regulation, clinical translation
In 2007 the Dutch administration decided to substantially stimulate research with adult stem cells. In response to that the ministry of Health asked ZonMw to conduct an exploratory study in the field of adult stem cell research in the Netherlands. The study, presented to the State Secretary in April 2008, revealed that translational adult stem cell research in the Netherlands is very promising. The transition from preclinical to clinical research in the field of adult stem cell therapy is however above all hampered by a lack of funding. ZonMw therefore recommended the ministry to set up a grant programme with the goal to specifically stimulate the transition from preclinical to clinical research in the field of adult stem cell therapy. In September 2008, in response to the proposal for a research programme in ZonMw's report on the exploratory study, the Ministry of Health undertook to provide the necessary funding for the implementation of the ‘Translational Adult Stem Cell Research’ programme for the period 2008–2022. The 22.4 million euro programme is subsidized by the Ministry of Health and matched with 1 million euro by the Netherlands Organisation for Scientific Research. The main criterion for funding in this programme is that projects aim at performing a clinical phase I/II trial within six years. Research projects on preclinical research questions only are therefore excluded. The first grants will be allocated October 2009 and presented at the ESGCT meeting. These grants will substantially stimulate Translational Adult Stem Cell Research.
Stimulating translational gene therapy in the netherlands
Session: Safety, regulation, clinical translation
The Dutch funding organisation ZonMw developed a funding programme to improve the transition from preclinical to clinical research in the field of gene therapy. Funded projects may consist of a preclinical phase, a pharmaceutical and a (obligatory) clinical phase. The maximum time frame of the projects is 6 years. The 15.6 M Euro programme is fully subsidized by the Dutch Ministry of Health and started in 2004. Table: Overview of the status of the fifteen translational projects in the ZonMw gene therapy programme; due to space restrictions, four projects are presented in the manner directly below. Principal Investigator Method Disease Cinical trial Prof.dr. J.J.P. Kastelein AAV LPL deficiency Completed Prof.dr. A. Hagenbeek Retrovirus Hematological malignancies Ongoing Dr. V.W. van Beusechem Adenovirus Glioblastoma multiforme Awaiting regulatory approval Dr. J. Haanen Naked DNA HPV positive penile cancer Expected 2010 The programme is currently evaluated and the results of this evaluation will be presented to the Ministry later this year. During the first five years of the programme, two clinical trials have successfully been executed and two more are ongoing. So far the results of completed clinical trials have been encouraging. The other funded projects are in the preclinical or pharmaceutical phase. In general the preclinical phase and the pharmaceutical phase take longer than expected. From the programme ZonMw has learned that translational research is complex and difficult and needs long-term support.
Institute for Multiphase Processes, Leibniz Universitaet Hannover , Germany
Session: Tissue engineering
In tissue engineering the combination of appropriate cells on a suitable biomaterial as a carrying structure poses a particular challenge. In this regard mesenchymal stem cells (MSCs) have manifold application and a high therapeutic potential. It results from their ability to differentiate into multiple tissues when cultured under specific conditions. However, before clinical application of MSCs in cell replacement therapy can be achieved, the safety and feasibility must be preclinically tested in a preferably non-human primate animal model. The common marmoset monkey (Callithrix jacchus) is such a model readily used in biomedical research due to its genetic and physiological similarities to humans. Therefore, MSCs isolated from bone marrow of 14 animals were seeded on collagen scaffolds [1,2]. These scaffolds consisting of porcine collagen 1 (diameters: 15 mm, thickness: 3 mm) exhibit a homogeneous pore structure and a defined pore size of 65[mu]m. The porosity is 98%. On day 1 after seeding the culture medium was completed with dexamethasone, indomethacin, IBMX and insulin as adipogenic supplements. Usage of CalceinAM and ETh demonstrated the cell viability after differentiation. Detection of intracellular lipid vesicles of the cell seeded scaffolds during adipogenic development was done with Oilred dye. After a cultivation period of 4 weeks the cell distribution of viable cells in the scaffold was dense and equally, the amount of death cells was negligible low. The generation of fat vacuoles was extensive distinguishable. Intracellular lipid vesicles in the cells were clearly stained by Oilred and prove the differentiation capability of these MSCs in collagen scaffolds. The project was supported by the Cluster of Excellence “REBIRTH” (DFG).
Kuberka M. et al. Int J Artif Organs
Kuberka M. et al. Biomed Tech
Session: Tissue engineering
Angiogenic potential of conditionally proliferating human endothelial cells
Session: Tissue engineering
Endothelial cells play a wide variety of roles in control of vascular function. However primary ECs are inhomogeneous, the number and their ex vivo expansion are limited. Recently, we proposed reversible expression of proliferation inducing genes as a tool to expand cells in vitro. The strategy is based on a Tet-on autoregulatory expression unit driving expression of an expansion gene. Strictly Dox dependent human endothelial cell lines (CI-HECs) were established. In vitro the cells maintained relevant phenotypic properties, such as expression of CD31, CD34, up take of acLDL and tube-formation in matrigel assay. Several models were established to evaluate the angiogenic potential of cells. Upon s.c. injection of tagged CI-HECs into RAG2yC mice only a transient engraftment of 8 days could be observed. In contrast, upon co-injection with murine CT26HA tumor cells, CI-HECs efficiently grafted and proliferated in vivo as documented by an increasing bioluminescence signal. Histological analysis of tumors confirmed that the cells participate in formation of vessels. CI-HECs were engrafted as spheroids in matrigel plugs. A complex 3-dimensional network of human neovessels was formed in mice. The CI-HECs could be also re-isolated from Matrigel implants and used for serial transplantations. Importantly, upon reisolation, the cells maintained Dox-dependent growth and endothelial properties. The experiments show that the conditionally proliferating human endothelial cells efficiently engraft in mouse models and are capable to contribute to vessel formation in vivo. This indicates that the concept of conditional proliferation is compatible with a short term expansion in vitro under conditions that do not compromise the biological potential of the cells.
Engineering of three-dimensional bioartificial cardiac tissues using a defined hyaluronic acid-based matrix
Session: Tissue engineering
Chondrogenic differentiation of porcine adipose derived mesenchymal stem cells (MSCs) in fibrin glue
Session: Tissue engineering
The main foci of tracheal Tissue Engineering are the functional epithelium and the stabilizing cartilage clamps. Adipose-derived MSCs have the potential to differentiate into multiple mesenchymal lineages including chondrocytes. To this point, these cells can be used to reconstruct cartilage clamps of the trachea. To enhance the chondrogenic differentiation of MSCs, growth factors including TGF-β1 and bFGF, in combination with an appropriate matrix, for example fibrin glue, can be employed. Following isolation from porcine adipose tissue and subsequent cultivation of MSCs, the cells were mixed with fibrin glue. This mixture was cast into teflon/silicon molds in the shape of native trachea and thereafter cultivated in differentiation medium containing 10 ng/ml TGF-β1 and 10 ng/ml bFGF. The medium was supplemented with the proteinase inhibitor aprotinin to reduce fibrinolysis. After 4 weeks of differentiation, it was observed that aprotinin supplementation enhanced the stability of the fibrin and histological examinations revealed the MSC production of the extracellular matrix component, collagen II, confirmed by PCR. Haematoxylin/Eosin staining revealed that the MSCs produced cartilage-like lacunae within the fibrin glue. Live/Dead assay indicated that a large amount of cells survived in the matrix after 4 weeks of cultivation. In conclusion, these data suggest that fibrin glue can be a suitable matrix for the viability and chondrogenic differentiation of porcine MSCs.
Division of Experimental Orthopaedics, Orthopaedic University Hospital Heidelberg , Germany
Session: Tissue engineering
Little is known about the potential of growth factor-augmented biphasic implants composed of a gel and a solid scaffold to enhance chondrogenesis of mesenchymal stem cells (MSC). We analyzed whether a collagen-type I/III carrier and fibrin glue (FG) combined to a biphasic construct support in vitro chondrogenesis of MSC and allow for local release of bioactive TGF-β1. Furthermore, a possible advantage of partial-autologous fibrin glue (PAF) over commercial FG was assessed. Collagen carriers seeded with 5 × 105 human MSC with or without FG, PAF, or TGF-β1- upgraded FG, were cultured for 6 weeks in chondrogenic medium with or without TGF-β1. Pellets with or without FG/PAF served as controls. Fibrin glue and collagen-carriers allowed strong up-regulation of COL2A1, AGC and COL10A1 mRNA, deposition of collagen-type II, and mediated a significantly higher proteoglycan content compared to biomaterial-free pellets. Collagen-carrier groups contained significantly more proteoglycan than FG and PAF pellets, while biphasic PAF-carrier constructs were inferior to FG-carrier constructs. Upgrading of biphasic FG-carrier constructs with 50 ng TGF-β1/construct mediated chondrogenesis as successfully as supply of TGF-β1 via the medium. In conclusion the biphasic carrier constructs showed a high biofunctionality by continous form stability with improved chondrogenesis and long-term local supply of bioactive TGF-β1 which may be useful to enhance matrix-assisted repair strategies for damaged cartilage.
Tet-inducible co-expression plasmid for non-viral tissue regenerative gene therapy approaches
Session: Tissue engineering
Inducible expression systems for co-expression of 2 therapeutic genes could maximize safety and efficacy of gene therapy approaches for tissue regeneration. The aim of this study was to design and evaluate doxycycline inducible co-expression systems. A Tet-inducible co-expression system was modified to contain both the response and activator cassette. First, EYFP and dsRED were tested as reporter genes instead of therapeutic genes. Time and dose dependent expression kinetics were monitored in transfected C2C12 cells by fluorescence microscopy and RT-PCR. A system for angiogenesis and one for osteoinduction were designed. The angiogenic system contained VEGFA and PDGFB. The osteoinductive system employed BMP2 and BMP7. The angiogenic system was tested by ELISA for VEGFA and PDGFB expression. The osteoinductive system was tested by differentiation assays and a reporter gene assay both in 2D and 3D culture. Gene expression kinetics were dependent on the applied amount of inductor without basal expression. VEGFA and PDGFB expression levels correlated with the applied amount of inductor. The osteoinductive system generated a strong inductor dependent differentiation, both in 2D and 3D culture. The results prove the feasibility of Tet-inducible co-expression systems for the regulated delivery of up to 2 genes. Co-expression of BMP2 and 7 produced a superior induction of osteogenesis compared to single gene delivery. The lack of basal expression without inductor proves the unique safety features, enabling complete expression shutdown in the case of adverse effects of the therapy. This work was partially funded by EXPERTISSUES and ANGIOSCAFF.
Leibniz Research Laboratories for Biotechnology and Artificial Organs LEBAO, Hannover Medical School , Germany
Session: Tissue engineering
Klinik und Poliklinik für Herz- und Thoraxchirurgie, Universitätsklinikum Köln , Germany
Session: Tissue engineering
Effect of hypoxia on chondrogenic differentiation of human bone marrow MSCs in hydrogels
Session: Tissue engineering
Reconstitution of functionality in degenerated intervertebral discs may require the application of mesenchymal stromal precursor cells (MSCs). Micromass cultures are standard models for chondrogenic differentiation of MSCs. Because of their dense cell package they are not suitable for clinical application. Here we present evidence for successful induction of chondrogenesis in gelatin hydrogels at low cell density. In 0.5 ml hydrogels 0.4 million MSCs were embedded and chondrogenesis was induced by addition of 10 ng/ml transforming growth factor ß3. Standard micromasses served as positive controls. Furthermore, to mimic in vivo oxygen conditions, we lowered the oxygen concentration in our cell culture system to a final concentration of 4%. After two weeks, progress of differentiation was determined by analysing, via qRT-PCR, the mRNA expression of matrix molecules such as aggrecan and collagen type II. The influence of hypoxia on differentiation was determined by comparing hypoxic with normoxic cultured cells. Hydrogel embedded cells show an increase in chondrogenic marker expression after culturing under hypoxic conditions. The average expression level for those markers was slightly lower than in standard micromass cultures. Hypoxia does not influence the marker gene expression in micromasses. Remarkably, chondrogenesis took place under all conditions. So, hydrogels are attractive alternatives regarding micromasses for future applications in regenerative medicine.
Hematology Oncology and Stem Cell Transplantation, Hannover Medical School , Germany
Session: Tissue engineering
Previously shown, lentiviral vector (LV)-mediated programed mouse DCs (SMART-DCs) efficiently enhanced anti-tumor T cell responses in vivo. Here, we evaluated the engraftment and bio-distribution of human SMART-DCs and further assessed the potential to support engraftment and expansion of human T cells. CD14 + monocytes were transduced with a bicistronic LV expressing hGMCSF and hIL4 and co-transduced with LV expressing the firefly luciferase (fLuc). In vitro SMART DCs showed persistent viability (3 weeks) and stable DC immunophenotype (CD209, CD86, CD83, HLA-DR). SMART DCs were subcutaneously injected into NOD/Rag-/-/IL-2rg-/- or NOD/SCID/IL-2rg-/- mice. In vivo engraftment and bio-distribution were assessed by bioluminescence image analyses. SMART-DCs/fLUC engrafted in mice and showed high luminescence at day 7 after injection and persisted > 6 weeks. In order to evaluate the capacity of SMART-DCs to stimulate engraftment and expansion of T cells in vivo, pp65-expressing SMART-DCs were generated from PBMC obtained from a CMV-reactive donor and injected into NOD/Rag-/-/IL-2rg-/- mice at day − 7. Autologous T cells were transduced with fLuc-LV and injected at day 0 in the lateral tail vein of vaccinated mice. In vivo engraftment and expansion of T cells evaluated by bioluminescence image analyses demonstrated higher engraftment of fLuc-T cells in SMART-DC/pp65 vaccinated mice, compared with non-vaccinated controls, confirming the potency of SMART-DCs in humanized mouse models.
Institute for Biological Interfaces 1, Research Center Karlsruhe KIT , Germany
Session: Tissue engineering
Differentiation experiments using embryonic stem (ES) cells often not consider the 3D organization of in vivo differentiating cells and the influence of a cell specific extra-cellular matrix (ECM) composition. For engineering mature and long-term functional tissues in vitro, sophisticated cultivation systems are required that mimic the spatiotemporal regulation of tissues developing in vivo. It is well known that endothelial cells in the heart exhibit the property of close anatomical and functional interaction with cardiomyocytes. Therefore, our co-cultivation studies will focus on the influence of primary microvascular endothelial cells (MVECs) on the differentiation and maturation of ES cell-derived cardiomyocytes. For the generation of cardiomyocytes we used a transgenic ES cell line exhibiting puromycin resistance allowing progressive enrichment of beating clusters. MVECs were isolated from murine hearts using endothelial cell specific antibodies and immunomagnetic beads. To optimize the co-cultivation conditions different media compositions were tested. Therefore both cell populations were analysed by RT-PCR and immunocytochemistry at various time points.Further studies will focus on the influence of the ECM secreted by organ specific MVECs on the differentiation of ES cells and their maturation into functional cardiac phenotypes. By using this cultivation system fundamental aspects of cell-cell interactions during stem cell differentiation can be investigated.
Tissue engineering of human oral mucosa on different scaffolds for clinical applications
Session: Tissue engineering
In oral and maxillofacial surgery, there is a great demand for oral mucosa for intraoral grafting after tumor dissections or traumas. In this study, three different scaffolds were tested for suitability as an underlay for a three-dimensional reconstruction of human autologous oral mucosa for clinical applications.For this purpose, primary human oral fibroblasts and keratinocytes were cultured and increased in vitro. They were seeded on matrices: TissuFoil E (TFE) Dermal Regeneration Template Single Layer (DRT) and Vicyl to create a three-dimensional reconstruction of oral mucosa. First only fibroblasts were seeded on scaffolds to obtain a dermal equivalent after seven days. The seeding was analyzed and characterized using electron-microscopy and immunostaining. In the experiments that followed, keratinocytes were seeded on dermal equivalents to develop organotypic co-cultures. They were cultured for seven and fourteen days to get full-thickness oral mucosa substitutes. Frozen sections of the probes were stained against CD90 (fibroblasts) and cytokeratin 14 (keratinocytes) with fluorescent dyestuffs. The results showed that all of the matrices were biocompatible because cells were able to grow on them. In oral mucosa substitutes on Vicryl, no natural stratification of the cells could be shown, but there was good biostability. TFE provided the best cell growth with good stratification and natural arrangement of epithelial and dermal components, but bad biostability of the membrane. The combination of good biostability and good cell growth with natural stratification could be seen only on the DRT-construct. For this reason, we consider DRT to be the most suitable for its intended use.
University of Veterinary Medicine Hannover , Small Animal Clinic, Germany
Session: Tissue engineering
In tissue-engineering, 3D scaffolds are of particular interest for in vivo as well as in vitro tissue regeneration. Two-photon polymerization (2PP) is a unique technology for the fabrication of complex 3D scaffolds from photopolymerizable materials. In order to access general possibility of BMSC cell growth on produced scaffolds, long term proliferation on two different 2PP compatible materials is studied. Flat samples from an organic-inorganic Zr-based hybrid (2% of Milcher's ketone), and a poly(ethyene) glycol diacrylate (PEGda) material (2% Irgacure 2959), are prepared by photopolymerization with UV light. Canine bone marrow was obtained by aspiration from the posterior iliac crest. After isolation MNCs by Ficoll-Paque density gradient centrifugation, the MNCs were cultured in sterile flasks containing Chang medium and were incubated in 5% CO2 air at 37°C. Each sample was seeded with 1 × 106 cells in 2 ml growth medium. For three weeks cellular organization was analysed daily using light microscopy. Afterwards the samples were fixed and analysed by scanning electron microscopy. The growth on the material samples was compared to that on glass and plastic. The Zr-based hybrid allowed adherence and a high proliferation of the BMSCs. The BMSCs on the PEGda sample showed rapid adherence and grew initially well, too. Later spreading decreased and a loss of cells from the material was observed. Thus, the results of this initial study demonstrate that the Zr-based hybrid material can be considered as a compatible material, making it attractive for settlement with mesenchymal stem cells. In order to confirm this observation, further experiments i.e. for the genetic stability are necessary.
Surface and structure analysis of microencapsulated chitosan/carboxymethyl-b-cyclodextrin nanoparticles
Session: Vector targeting
We developed a gene delivery system consisting of chitosan/carboxymethyl-ß-cyclodextrin nanoparticles (CS/CD NPs) which elicited low toxicity and high gene expression. In this work, we propose microencapsulation of the CS/CD NPs in mannitol to facilitate their administration by lungs. It is known that particle surface chemistry interfaces with biological environments, and this governs its biological behavior. Therefore, we conducted surface and structure analysis of these microspheres using confocal laser scanning microscopy (CLSM) and the surface-sensitive analytic techniques; X-ray spectroscopy (XPS) and time of flight secondary ion mass spectrometry (TOF-SIMS). Microencapsulated NPs (M-NPs) were prepared through spray drying of mannitol labeled by Bodipy flourophore with NPs prepared by ionic gelation. Mannitol microspheres (M) and isolated NPs were used as controls. The CLSM micrographs obtained show that labeled mannitol forms a continuous matrix in which NPs are homogeneously distributed. According to XPS analysis, the ratio carbon/oxygen and the absence of phosphor and sodium signals in the case of M-NPs and M indicate that NPs are embedded inside the mannitol matrix. The mass spectra obtained by TOF-SIMS prove that the characteristic mass of mannitol m/z 183(molecular ion + H+) is present in the case of M-NPs and M but not in the case of isolated NPs, suggesting that NPs are totally covered with mannitol. CLSM, XPS and TOF-SIMS analysis prove that NPs are efficiently encapsulated and homogeneously distributed within M-NPs.
Gene Therapy Program, Dept. of Cellular Biology and Anatomy, LSU Health Sciences Center , Shreveport, LA, 71130, United States of America
Session: Vector targeting
Gene therapy relies on efficient and targeted viral delivery. Retargeting is particularly important based on limited success of adenoviruses (Ads) for metastatic cancer. A major hurdle for systemically administered Ads is promiscuous tropism leading to transduction of non-target cells, notably liver. This can be circumvented by modifying Ad native tropism and redirecting to different receptors expressed on target cells. Many tumors overexpress the chemokine receptor CXCR4, a G protein-coupled receptor that plays a major role in cancer metastasis. We explored retargeting Ads to CXCR4 overexpressed on breast cancer cells. We tested a recombinant bispecific adapter fusing the extracellular domain of the Ad receptor CAR to CXCL12, the chemokine ligand for CXCR4. This adapter dramatically enhanced Ad transduction of CXCR4-positive breast cancer cells. In contrast, the adapter protected normal cells and liver from Ad transduction. The adapter is a powerful agent to retarget Ads to metastases. However, while efficient for retargeting, the adapter approach is not feasible for replicating Ads. A separate adaptor is also technically cumbersome, requiring production of two components and separate Ad conjugation. This could be avoided by genetically modifying an Ad, resulting in a one-component agent. Based on the modular structure of the Ad fiber protein, we generated a ligand-presenting molecule. The native fiber gene was replaced with a recombinant gene consisting of the fiber tail domain, the shaft and trimerization domains from T4 bacteriophage fibritin, and the CXCL12 ligand. With nearly ubiquitous CXCR4 overexpression in breast tumors, we expect this genetic retargeting could be exploited in Ad transduction for virotherapy of metastatic disease.
Antibodies from immune phage display libraries for regenerative medicine applications and anti-metastasis therapy
Session: Vector targeting
We have generated a panel of high affinity scFv antibodies using chicken immune phage display technology that bind to the primary cilium of a quiescent population of lineage −ve CD45 + ve Mesenchymal Stem Cells (MSCs) that reside in human bone marrow. This MSC subset can be readily isolated using Miltenyi MACs systems, expanded rapidly in culture and can differentiate into bone, cartilage and fat tissue, and are therefore of particular interest in regenerative cell therapy. This panel of recombinant antibodies also bind to a small number of CD14 + ve MCSF + ve, RANK + ve CD51/61 + ve pre-osteoclasts circulating in peripheral blood. Both MSCs and Osteoclasts are cellular targets in anti-metastatic therapies, being implicated in cancer metastasis from its primary site (in the case of MSCs) and in colonisation and survival of metastatic cancer cells in the bone marrow (MSCs and Osteoclasts). We have engineering both humanised scFv and chimeric full length chicken/human IgG1 and are testing the stability and specificity of these antibody constructs. We currently aim to produce stable functional antibody for the isolation of MSCs for regenerative cell therapy, and to demonstrate the anti-metastatic potential of humanised antibodies in antibody-dependent cellular cytotoxicity (ADCC) assays.
Institute of Virology, University of Cologne , Germany
Session: Vector targeting
Conditionally replicating adenoviruses represent a promising tool to target cells cancer killing. They exert their antitumor activity through selective cancer cell replication and lysis. In this context, the our study uses a strategy involving triple level targeting of cancer cells by enhancing Ad transductional efficiency and providing Ad E1A transcriptional and mRNA translational regulation of expression. We generated a modified CRAd (Ad5/3-CXCR4-UTR) incorporating a serotype 5/3 chimeric fiber gene to increase cancer cell transduction, and the CXCR4 promoter and the 5′-untranslated region (UTR) from the Fibroblast Growth Factor-2 mRNA to regulate E1A. We hypothesized that this novel vector would improve CRAd infectivity and replication specificity, thus enhancing therapeutic efficacy by selectively targeting of cancer cells. Oncolytic activity of Ad5/3-CXCR4-UTR was tested in a panel of breast cancer cell lines and primary cells. Early gene transcription and viral replication were assessed in cells by measuring E1A mRNA levels and E4 copy number. Expression of E1A protein was examined by immunoblot analysis In breast cancer cells, Ad5/3-CXCR4-UTR demonstrated enhanced oncolytic activity compared with WT and dual-level targeted CRAds, while in normal cells oncolytic activity was absent. In addition, there was a marked increase in the viral transcription and replication in breast cancer cells that was restricted in normal cells. In this study, we demonstrate targeted replication of Ad5/3-CXCR4-UTR in breast cancer cells. Thus, this novel construct is a potentially powerful cancer therapeutic agent. Our future goal is to evaluate the therapeutic efficacy of this CRAd using in vivo models of breast cancer.
Division of Gene Therapy, University of Ulm , Germany
Session: Vector targeting
Until today numerous targeting approaches have been developed to redirect adenovirus-based gene transfer vectors specifically to certain surface receptors on the target cells in order to increase gene transfer efficiency. However, an increasing number of recent publications has evidenced that the success of adenovirus targeting does not only depend on receptor binding and cellular uptake of the virions, but that capsid modifications (while permitting efficient receptor binding) may critically interfere with intracellular trafficking. Therefore, improved knowledge on the intracellular fate of targeted Ad vector particles is mandatory for a rational design of successfully targeted Ad vectors. However, fluorescent particle labeling as the method of choice to elucidate intracellular trafficking has significant limitations: (i) capsids can only be labeled all over the particle surface and this imposes the risk of interference with particle infectivity, (ii) capsomere-specific labeling has only been demonstrated for the capsomere IX and requires extensive genetic modifications, (iii) two-color labeling approaches are not available. Here we present a novel, robust and straight forward labeling procedure that overcomes these limitations. It allows for specific labeling of the capsomeres fiber, protein IX, or hexon and permits two-color labeling. Using this technology we analyzed two different bioresponsive bonds that can be used for the attachment of shielding or targeting moieties to the capsids: disulfide and hydrazone bonds. We demonstrate that in contrast to disulfide bonds, hydrazone bonds become quickly hydrolyzed after uptake of the virions and are thus favourable for the generation of bioresponsive vectors.
URIA-Centro de Patogenese Molecular , Faculdade de Farmacia de Lisboa, Portugal
Session: Vector targeting
A crucial factor for successful gene therapy is the efficacy of specific gene transfer, which is usually done by lentiviral vectors. Fusion and specificity of lentiviral vectors must be provided by envelope glycoprotein domains. Sindbis envelope can pseudotype lentiviral particles and display exogenous protein domains. Previous results demonstrated that Sindbis envelope can accommodate anti-receptor single-chain antibodies (scFv) and target cell-specific viral infection. Additionally, it was shown that protein A-chimeric Sindbis envelope can specifically target cells immunolabelled with anti-receptor IgG via Fc recognition. However, these strategies might present some problems for in vivo applications, since there may be non-specific reactions with plasma antibodies and the need for cloning a receptor specific antibody each time a new molecule is to be targeted. To overcome these problems we developed a new lentiviral vector capable of transducing several cell types in a specific manner without the above constraints, that consists of a chimeric scFv-Sindbis virus envelope that binds fluorescein (FITC) with high affinity and consequently recognize FITC-conjugated proteins. Using this targeting strategy we were able, in vitro, to target efficiently and specifically cells expressing a receptor labelled by a FITC-conjugated antibody. Moreover, we could specifically kill those transduced cells by Sindbis-mediated lentiviral delivery of a diphtheria toxin. To demonstrate in vivo the efficiency of this gene therapy proposal we will use NOD/SCID mice injected with human T-cell acute lymphoblastic leukaemia cells. The strategy herein proposed has the potential to be applied to a broad range of diseases.
Department of Pediatrics, Ludwig-Maximilians-, University Munich , Germany
Session: Vector targeting
Molecular conjugates comprising targeting ligands hold great promise for site-specific gene delivery to distant tumors and individual organs including the lung. Here we show that a specific agonist of a certain G protein-coupled receptor can be used to improve gene transfer efficiency of polyethylenimine (PEI) gene vectors on bronchial and alveolar epithelial cells in vitro and lungs of mice in vivo. Receptor expression was confirmed in pulmonary epithelial cell lines by western blot. Two different agonists (LIG1 and LIG2) were conjugated to fluorescein-labeled BSA (FLUO-BSA) and compared for receptor binding/uptake in different lung cell lines. Binding of FLUO-BSA-LIG1 was 2–4-fold higher than for FLUO-BSA-LIG2 and could be specifically inhibited by free LIG1 and specific antagonist. Internalization of FLUO-BSA-LIG1 was confirmed by confocal microscopy. Molecular conjugates of PEI and LIG1 (PEI-g-LIG1) were synthesized with increasing coupling degree (FLIG1 (LIG1:PEI) = 2, 5, 8, 16) and analyzed for DNA binding, particle formation and transfection efficiency. At optimized conditions (N/P 4, FLIG1 = 5), gene expression using PEI-g-LIG1 was significantly up to 46-fold higher than for PEI gene vectors and specifically inhibited by antagonist. Gene expression in the lungs of mice after aerosol delivery was 14-fold higher with PEI-g-LIG1 FLIG1 = 5 than for PEI. We suggest that targeting of this receptor using LIG1 represents a promising approach to improve pulmonary gene transfer.
Department of Pediatrics, Ludwig-Maximilians-University Munich , Germany
Session: Vector targeting
Insufficient specificity of gene therapeutic approaches may be the cause of limiting side effects. Either local delivery or cell specific gene expression has been suggested to overcome these problems. In the present work we show, that localized delivery to the lung by aerosol administration together with tumor specific transgene expression is therapeutically effective in a mouse model of non-small lung cancer (NSCLC). Tumor bearing mice were treated with BC-819, a plasmid, that is undergoing clinical trials for use in humans suffering from recurrent and BCG resistant bladder cancers. In BC-819 expression of Diphtheria Toxin A is driven by the H19-promotor, which is only active in embryonal and various tumor tissues, including lung tumors. Expression of Diphtheria Toxin leads to immediate cell death. In the present study lung tumors were generated in immunocompromised mice by orotracheal application of A549 cells stably transfected with the luciferase gene (A549-luc). Tumor take and growth was monitored via in vivo imaging. H19 expression and BC-819 activity was confirmed on A549-luc cells. Beginning from day 14 mice (n = 5) were treated weakly with 1 mg of BC-819 plasmid, which was complexed to branched PEI 25 kDa by aerosol application in a whole-body-exposition chamber. BC-819 treated mice showed decreased tumor growth and significantly improved survival compared to the untreated control group. We suggest that BC-819 holds promise for the treatment of lung cancer.
Lab. prenatal diagnostics of inherited diseases, Otts Institute of Obstetrics and Gynecology , Russia
Session: Vector targeting
Desirable feature of synthetic DNA carriers is their ability to provide cell and tissue DNA uptake specificity by means of receptor-mediated endocytosis. CXCR4 is a receptor of chemokine SDF-1 and is expressed on some types of stem and cancer cells. CXCR4 also interacts with vMIP-II (viral macrophage inflammatory protein II). The aim of this project is to design and to characterize the group of chemokine derived peptides (CDPs) for receptor-mediated gene delivery. Our present study focused on three peptides, two of them is derived from N-terminal sequences of the chemokine SDF-1 and one - from vMIP-II. All peptides are modified with DNA-binding sequence (K8). Control peptide consists of only K8 sequence. Gene delivery by CDP/DNA complexes is glycerol-dependent and the level of luciferase expression with CDPs was comparable with the efficacy of PEI in CXCR4 expressing cell lines (A172, HeLa) and was 10–50-fold higher compared to unmodified peptide. On the contrary CDP transfection efficacy on CXCR4-negative cells (CHO) was much lower than in PEI. Intracellular uptake analysis of biotin-labeled peptides indicated that CDPs entered cells more efficiently than oligolysine. Addition of free CDP to cells during the uptake analysis blocked peptides transport significantly. In summary, the results presented herein show that oligolysine polyplexes can be specifically targeted to cells expressing CXCR4 receptor, by using peptides derived from chemokine SDF-1 and viral protein vMIP-II.
Muscle-targeted gene transfer enables continuous secretion of GLP-1 and exendin-4 in mice
Session: Vector targeting
Glucagon-like peptide-1 (GLP-1) is a potent insulinotropic hormone with an important role in maintaining normal blood glucose but the short half-life of the active peptide, necessitates continuous infusion or regular self-injection limiting clinical use. Exendin-4 is a GLP-1 homologue resistant to DPP IV mediated degradation, increasing its half-life and making it an attractive alternative for Type 2 diabetes therapy. However, two injections are still required every day. The aims of the study were to generate plasmid constructs enabling constitutive secretion of GLP-1 / Exendin-4 and evaluate expression, biosynthesis and secretion in murine muscle in vitro and in vivo. GLP1(7–36); Exendin-4; and reporter eGFP cDNAs preceded by signal sequence were sub-cloned into pVR1012 plasmid. In vitro transfections were undertaken in the C2C12 muscle cell line. In vivo gene transfer was performed in male CD1 mice by direct plasmid injection into hind limb muscles. GLP-1 and Exendin-4 expression/biosynthesis in vitro was confirmed by RT-PCR/cytoplasmic immunofluorescence staining, and constitutive secretion of bioactive GLP1 (260 ± 20 pmol/l) and Exendin-4 (2433 ± 585 pmol/l) demonstrated by specific immunoassay. Biosynthesis following plasmid-mediated gene transfer in mice was confirmed by immufluorescence staining; with glucagon-like peptide secretion into systemic circulation at 7 days (GLP1: 56 ± 28 pmol/L vs eGFP: 13 ± pmol/L; p = 0.01), (Exendin-4: 847 ± 295 pmol/L vs eGFP: 6.6 ± 1pmol/; p = 0.0002). Glucose levels were unaffected but weight lower with Exendin-4 (40 ± 1 g vs eGFP: 44 ± 1 g; p = 0.001). Constitutive secretion of GLP-1 and Exendin-4 has been attained following plasmid-mediated gene transfer to muscle with bioactivity confirmed in vivo.
Pegylated cationic liposome mediated targeted gene delivery
Session: Vector targeting
Surgical Oncology, ECRC, Charite Berlin and Max-Delbrück-Center for Molecular Medicine , Germany
Session: Vector targeting
The bacterial Clostridium perfringens enterotoxin (CPE) is produced by the Clostridium strain type A. The transmembrane tight junction proteins claudin 3 and 4 are the specific receptors for CPE, which are overexpressed in human epithelial tumors such as colon, breast, pancreatic and ovarian cancer. CPE binding to the claudins forms membrane pore complexes resulting in rapid cell death. The tumor-specific claudin 3 and 4 overexpression can be used for targeted cell killing by CPE. This study aimed at tumor cell killing by CPE gene transfer. We generated expression vectors carrying the CPE cDNA, which were tested for in vitro gene therapy of claudin 3 and 4 overexpressing human cancer cell lines HCT116, MCF-7 and Panc-1. CPE expression analysis by real-time PCR at mRNA and by Western-blotting at protein level revealed efficient in vitro CPE expression leading to cytotoxic activity after transfection. The highest cytotoxic effect of up to 70% cell killing was observed 48 h after transfection and was dependent on the claudin 4 expression level. Therefore, MCF-7 cells with the highest claudin 4 expression showed the highest sensitivity to CPE.The antitumoral effect of CPE gene transfer in vivo was tested in MCF-7 tumor-bearing nude mice. The in vivo electroporation mediated gene transfer of the CPE-expressing vector led to reduced tumor growth in tumor-bearing mice compared to the untreated control group. These results provide first evidence, that CPE gene transfer can be used for a novel suicide gene therapy of claudin 4 and 3 overexpressing tumors, leading to a rapid and efficient cell killing in vitro and in vivo.
Ternary PEI/PDNA/insulin complexes increase nonviral gene delivery to pulmonary epithelial cells
Session: Vector targeting
Polyethylenimine (PEI) has been used for nonviral gene delivery in many preclinical studies. However, one major drawback is its unspecificity towards cell types. The aim of this study was to investigate the effect of human insulin as a targeting ligand for receptor-mediated gene transfer to human lung epithelial cells. A high number of insulin receptors could be detected on alveolar epithelial cells (A549) with FACS measurements and fluorescence microscopy. To avoid loss of function of the targeting ligand through biochemical reactions, insulin was introduced into polyplexes by electrostatic interactions, forming ternary PEI/pDNA/Ins particles. Surface adsorption was confirmed by particle size, surface charge and fluorescence resonance energy transfer (FRET) measurements. Transfection experiments showed an up to 16-fold increase in gene expression on A549 cells after coating with 5 μg insulin per μg of plasmid DNA but not on bronchial epithelial cells. Furthermore, cytotoxicity could be significantly reduced due to insulin addition. Our results demonstrate that insulin represents a potent targeting ligand for receptor-mediated gene delivery to alveolar epithelial cells.
Centre de Genetique Moleculaire et Cellulaire, UMR5534 CNRS Universite Claude Bernard Lyon1 , France
Session: Vector targeting
Our long-term purpose is to develop transcriptionally targeted oncolytic HSV-1 to fight against hepatocellular carcinomas (HCC). The rational of transcriptional targeting relies on the use of selective promoters, able to mimic the activity of viral promoters specifically in HCC, while being silent in non-HCC cells, when expressed from the virus genome. Towards this end, we have identified a number of human liver promoters displaying HCC specificity, and confirmed that they remain active in hepatoma-derived cell lines upon HSV-1 infection. The promoters of alpha-fetoprotein (AFP), angiopoietin-like 3 (ANGPTL3) and alcohol dehydrogenase 6 (ADH6) fulfilled these criteria and were thus cloned into the HSV-1 genome, where they drive luciferase expression. Viral multiplication and luciferase activity of recombinant HSV-1 vectors, expressing luciferase under the control of HCC-specific promoters, were assessed in human liver cancer-derived cell lines, in cell lines derived from other tumours, and in primary cells. Our results show that AFP and ANGPTL3 promoters drive luciferase expression specifically in HCC cells. Other promoters, such as 9xHRE and E2F1 promoters, are active in HCC but also in other cancer cell lines. Infection of human HCC xenografts subcutaneously implanted in nude mice confirmed that AFP and ANGPTL3 promoters drive HCC-selective expression also in vivo and are thus potential candidates to allow selective HSV-1 multiplication and spread in HCC.
WITHDRAWN
Session: Persisting transgenes
Extensive efficacy and safety data of self inactivating (SIN) lentiviral vectors (LV) for gene therapy applications was shown in in vitro and in vivo preclinical models using mouse cells. With human cells, safety studies are performed by comparing the profile of vector integration in cultured cells and at different time points after transplantation. Appearance of common integration sites (CIS) targeting specific genes or the emergence of dominant clones with integrations targeting cancer related genes may be triggered by vector genotoxicity. However, for reported LV-specific CIS it is unknown whether their enrichment is the result of a preferential integration bias or a subtle genetic selection or a combination of both. Here we devised an experimental strategy aimed to identify genes whose deregulation may lead to enhanced survival/proliferation of human hematopoietic cells in vivo. Human cord blood-derived CD34+ cells were transduced with a previously developed genotoxic LV or the standard SIN LV. The integration profile of these vectors was generated from in vitro cultured cells and after expansion and differentiation in vivo from transplanted Rag2-/-,gamma chain-/- mice (20 mice at 13 and 20 weeks). More than 200′000 Linear Amplification Mediated PCR products were sequenced by 454-pyrosequencing. Comparison of the different integration datasets will show if significant differences in CIS-gene candidates will emerge between these two vectors and/or during time and it will be instrumental to pinpoint their role in genotoxicity. The integration profiles of genotoxic and SIN LV in human cells could be used as reference against which to assess integration data obtained from the growing number of clinical trials using LV for gene therapy.
Treatment of metastatic renal cell cancer with autologous T cells genetically retargeted against carbonic anhydrase IX – first clinical experience
Session: Clinical trials
Adoptive transfer of autologous T lymphocytes that are gene-transduced to express antigen-specific receptors (‘genetic retargeting’) represents an experimental therapy to provide tumor-specific immunity to cancer patients. We studied safety and proof of this concept in patients with metastatic renal cell carcinoma (RCC).
A single-chain antibody-type (scFv) receptor based on the monoclonal antibody (mAb) G250 was constructed. This mAb recognizes an epitope on carboxy-anhydrase IX (CAIX), an antigen expressed on metastatic RCC. Three patients with CAIX+ metastatic RCC were treated with a maximum of 8 infusions of 2 × 107 to 2 × 109 gene modified T cells.
We successfully generated functional scFv(G250)+ T cells ex vivo. Infusions of the gene-modified T cells were initially well tolerated. However, following 4 to 5 infusions liver enzyme disturbances reaching toxicity grades 2–4 developed, necessitating cessation of treatment in 2 patients. Examination of liver tissue from 1 patient revealed T cell infiltration around the bile ducts and CAIX expression on bile duct epithelium. Circulating scFv(G250)+ T cells and scFv(G250) DNA copies were transiently detectable in all patients. In addition, peripheral blood lymphocytes collected from the patients following treatment were able to ‘kill’ and produce IFN-γ upon stimulation with CAIX+ target cells. All 3 patients developed anti-scFv(G250) antibodies and showed progressive disease between 36 and 106 days after start of treatment.
The scFv(G250)-retargeted T cells exerted receptor-specific function in vivo. However, reactivity of these cells most likely with CAIX expressed on bile duct epithelium hinders administration of numbers expected to yield anti-tumor activity. Strategies to attenuate activity of the retargeted T cells against normal tissues expressing the target antigen need to be developed to circumvent such observed adverse events, and will be discussed.
Gelatin Nanoparticles for Ocular Gene Delivery
Session: Eye and brain
The gene therapy is promising for the treatment of several eye diseases. The development of safe and effective delivery vehicles represents a major challenge for its clinical application. Cationized gelatin has been described as an interesting biomaterial in complexes formation for gene delivery that possesses interesting properties as biodegradability and biocompatibility. Moreover, the cationization process confers to this polymer more positive charges that are necessary to interact with negatively charged genetic material. The main goal of the present study was the development of cationized gelatins-based nanoparticles and the evaluation of their efficacy as pDNA delivery vehicles to corneal epithelium. Ionic gelation technique allows the production of nanoparticles, through an electrostatic interaction between cationized gelatin and TPP as crosslinker. After the physicochemical characterization (size and zeta potential), developed nanoparticles were able to efficiently associate the model pDNA (pEGFP-c1), as indicated by the gel electrophoresis technique, these results were further confirmed by the PicoGreen® test which revealed an association efficiency from 85 to 98%. The cytotoxicity and transfection ability of the developed nanoparticles was assayed in the HCE (Human Corneal Epithelial cell line). The cationic gelatin based nanoparticles successfully transfect the pEGFP to corneal cells in HCE cells, exhibiting a very low toxicity profile. Therefore, these results suggest that this new nanoparticles can be interesting nanocarriers for ocular gene delivery.
Natural-occurring polymers nanoparticles for gene delivery
Session: Physicochemical approaches
The present work describes a novel approach to develop new nanoparticulated systems based on natural components and their use as gene delivery systems. Using natural polymers of different sources, chondroitin sulfate, hyaluronic acid, dextran sulfate, carragenann and colominic acid it was investigated their ability in form nanoparticles to associate siRNA and pDNA. The nanoparticles were formed by a previously described modification of the ionic gelation technique that comprises the use of low molecular weight polyamines as reticulant. It was carried out the physico-chemical characterization of nanoparticles by the measurement of their size and zeta potential, the morphology by transmission electronic microscopy and the association efficiency was perfomed by the use of agarose gel electrophoresis. Its internalization profile was investigated by using fluoresceinamine-marked polymers in rat skin fibroblasts. All polymers originated spherical nanometric systems with negative zeta potential that can efficiently associate both siRNA and pDNA at a 2.5 and 5.0 % (w/w) respectively. The cell uptake studies showed that the systems can be efficiently internalized by fibroblasts. The results suggest the great potential of such systems as new non-viral transfection agents.
Baculoviruses as vectors for gene transfer into human prostate cancers
Session: Vector targeting
The FP6 BACULOGENES project aims to create improved human gene therapy (GT) vectors using the insect type baculovirus (BV) species, AcMNPV. BV has several advantages over existing vectors. To construct safer clinical vectors and to expand their capacity, the program seeks to produce a minimal genome virus, by eliminating unnecessary genes and identifying the BV encapsidation signal(s).
The susceptibility of normal, benign, prostate cancer cell lines and primary cultures to BV has been tested. BV retargeted to neurotensin receptor on the cell surface of prostate epithelium resulted in rapid and efficient attachment of retargeted BV to malignant cell lines relative to normal cells. Primary malignant prostate cell cultures were also infected and the highest transduction efficiencies were seen in undifferentiated epithelium and in primary prostate stromal cells. Using a BV expressing bacterial nitroreductase from a strong constitutive promoter, the transduction efficiency of the most metastatic prostate cell lines was very high (70-90%), compared to non-malignant prostate epithelial cells (2-20%). This rendered the cancer cells susceptible to CB1954 prodrug treatment. Similar results were obtained with primary epithelial cultures. Finally, to prevent complement mediated destruction of BV following intravenous administration we have exploited an ex vivo human blood loop system in which co-inoculation with complement inhibitors suppressed inflammatory cytokines and prolonged BV survival.
Thus vector re-targeting, transduction efficiency and efficacy of therapeutic gene delivery suggest BV can fulfil its promise as an alternative human GT vector.