Abstract
INSERM U935, University Paris 11, Villejuif, France .
Ectopic expression of defined sets of genetic factors can reprogram somatic cells to induced pluripotent stem cells (iPS) which are almost indistinguishable from embryonic stem (ES) and offer a novel therapeutic strategy to generate patient-specific stem cell lines. Human iPS were establish by reprogramming human ES (H9 and SA01)-derived mesenchymal cells with Oct4, Sox2, Nanog and Lin28. After 10–21 days in ES culture conditions, the ES-like colonies were plucked, amplified and characterized. We confirmed that human iPS clones were pluripotent and share identical pluripotent markers and gene expression profile with hES lines. IPS can be induced to differentiate in NOD/SCID mice to generate teratomas. We characterized the haematopoietic potential of these iPS in comparison to ES cell lines (H9 and SA01).
We generated hematopoietic progenitors by two methods, co-culture on the murine Op9 cell line and from embryoid bodies (EBs). We followed the appearance of CD34, CD43, and CD45 and progenitors by CFC assays. Typical hematopoietic colonies were identified including CFU-E, BFU-E and CFU-GM in methylcellulose. Interestingly, iPS generated a higher proportion of CD34+CD43+CD45+ cells and the iPS from H9 cells a higher number of CFU-C's as compared to parental ES cell line, indicating that epigenetic factors are involved in the lower hematopietic potential of H9. Transplantation of hematopoietic cells derived from EBs from human iPS and ES into irradiated NOD/SCID mice are currently in progress. Our data demonstrate that hematopoietic potential can be induced in reprogrammed terminally differentiated tissues with high efficiency, paving the way for future hematopoietic cell therapy applications.
INSERM U972; Université Paris 11; Hôpital Kremlin-Bicêtre , 80, rue du Général Leclerc 94276, Le Kremlin-Bicêtre Cedex , France .
Cellular therapy is now an alternative to the transplantation of the whole liver for treatment of liver metabolic diseases. However the lack of donors and the difficulty of manipulating isolated adult hepatocytes in vitro have encouraged the use of stem cells including human embryonic stem cells (hESCs). However, generating fully functional hepatocytes from hESCs and achieving this goal using clinically-compatible conditions remain major challenges. We have developed chemically defined culture conditions to achieve specification of hESCs into definitive endoderm and to differentiate them further into immature hepatic progenitor cells. These cells co-expressed AFP and CK19, or HNF4alpha. They also expressed apolipoprotein A-II (ApoAII), the epithelial cell surface markers EpCam, and E- cadherin.
We have also defined conditions to differentiate further the progenitors. The cell population generated expressed functions of more mature hepatocytes including albumin, tyrosine aminotransferase, tryptophane oxygenase, alpha-1-antitrypsine, and detoxification enzymes. Surface markers such as cMET, asialoglycoprotein receptor, Low Density Lipoprotein receptor were also expressed in 30 to 40 % of the cells as assessed by FACS analysis. The ES-derived hepatic cells had also the ability to internalize DiL and to store glycogen.
After hESC cell transduction by lentiviruses carrying the GFP gene under the control of the hepatic promoter of ApoA-II gene, differentiated cells expressed GFP. After transplantation into immunodeficient uPA transgenic mice, hESC-derived hepatocytes transduced with a GFP-expressing lentivector engrafted into the liver and expressed human albumin and alpha-1-anti-trypsin for at least 8 weeks without forming any tumour.
Conditions to purify and expand differentiated hepatocytes are underway.
This work was supported by ANR-RIB-05, Pôle de compétitivité “Ingecell”, Région Ile de France and EC FP7”LIV-ES”.
CNRS UMR 7211 &, Université Pierre et Marie Curie-Paris6 , 75013 Paris, France .
Adoptive transfer of natural regulatory T-cells (nTregs) may have a great therapeutic potential for the induction of tolerance in allo-transplanted patients. This concept was demonstrated in murine models of graft versus host disease (GVHD), but alloantigen-specific nTregs were always more efficient to control GVHD than polyclonal nTregs. Recently, we reported a procedure for expanding human polyclonal nTregs in RPMI medium containing interleukin (IL)-2, anti-CD3/anti-CD28 coated beads and rapamycin (namely TCM for Treg Culture Medium) during 3 weeks. Whether or not alloantigen-specific nTregs could be generated was investigated here. Using FACS-sorted nTregs (CD4+CD25high T-cells) stained with CFSE, we first studied cell divisions of nTregs stimulated by immature (i) or mature (m) allogeneic monocyte-derived dendritic cells (DCs), and cultured in TCM + IL-15. Since mDCs yielded higher nTreg divisions than iDCs, nTregs were stimulated twice (day-0 and day-10) by mDCs (mDC1). At day-21, mDC1-stimulated nTregs were tested for their ability to inhibit the proliferation of autologous effector T-cells (Teff) stimulated by either mDC1 or a third party of allogeneic mDCs (mDC2) used as control for specificity. Data showed that mDC1-stimulated nTregs more efficiently suppressed the proliferation of mDC1-stimulated Teff than mDC2-stimulated Teff. Whether or not alloantigen-specific nTregs rapidly divided upon stimulation was examined by sorting CFSE-stained nTregs into CFSElow and CFSEhigh fractions at day-6 of mDC-stimulation. Each fraction was assayed for suppressive activity at day-21 of culture. Results showed that CFSElow cells, by contrast to CFSEhigh cells, contained alloantigen-specific nTregs. Overall, our data provide evidence that alloantigen-specific nTregs can be generated.
LBPA/ CLINIGENE, Ecole Normale Superieure (ENSC) , 94235 CACHAN Cedex, France
In otherwise successful gene therapy trials for the treatment of SCID patients and others, insertional mutagenesis has resulted in leukemia development. Besides the integration of vectors that including strong enhancers, more recently, SIN-vectors have been shown to partially retain oncogenic potential. The identification of genetic elements which would both prevent such activation effects and shield the transgene from silencing, is a main challenge. Previous attempts met with difficulties in producing the vectors and poor efficacy of the insulators (GIE). The improvement of integrating vectors safety has been investigated using new candidate synthetic GIEs. The latter have been introduced in retroviral and lentiviral vectors. Native LTRs, SIN-LTRs, and SIN-insulated constructs have been designed and compared, using two sets of internal promoter, i.e. strong and housekeeping. We could establish that a specific insulator translates at best into functional activity and boundary effect in both vector types. We could also determine that other genetic elements are key determinants in order to achieve accurate expression and viral titre, from these insulated vectors. A dramatic shift in the expression profile is observed in target cells, with a homogenous pattern including data on both cell-lines and primary HSCs from cord blood. The assessment of potential genotoxicity will be presented, based on the comparison of the integration patterns ingenuity in human target cells sampled over a three months period with both reference LTRs and SIN versus test insulated vectors, using high-throughput pyro-sequencing.
Work performed within the EC-DG research FP6-Network of Excellence, CLINIGENE: LSHB-CT-2006-018933.
Inserm, U758, Human Virology Department, F-69007; Ecole Normale Supérieure de Lyon, F-69007; Université Lyon 1, F-69007, Lyon, France .
B-lymphocytes are attractive targets for gene therapy of diseases associated with B-cell dysfunction and also for immunotherapy by their potential to induce specific immune activation or tolerance. However, up to now lentiviral transduction of true quiescent B-cells has not yet been achieved.
Recently, we generated high-titer HIV-based lentiviral vectors (LVs) pseudotyped with measles virus glycoproteins (H/F-LVs) that allowed the transduction of completely quiescent human T-cells after a single exposure (1).
Here we demonstrate for the first time that H/F-LVs can efficiently transduce quiescent B-cells in the absence of any stimulus. We systematically achieved up to 50% of transduction of resting adult B-cells with H/F-LVs where VSVG-LVs remained completely refractory. Most importantly, the naïve phenotype of transduced B-cells was conserved and no upregulation of early or late activation markers was detected. No differential expression of the B7 molecule CD86 was shown, implying that B-T interactions should be maintained.
In addition, we demonstrate that H/F-LV-binding/entry to both SLAM and CD46 receptors on the quiescent target cell are essential for an efficient transduction where SLAM seems to be especially important in determining vector tropism. Finally, we co-transduced quiescent B-cells with H/F-LVs and VSVG-LVs observing that H/F-LV does not trigger or facilitate VSVG-LV entry, pointing out a differential entry mechanism between the two vectors. In conclusion, these novel H/F-LVs represent excellent tools to study B-cell gene functions for research purposes and gene therapy applications.
(1) Frecha C, Costa C, Nègre D, Gauthier E, Russell SJ, Cosset FL, Verhoeyen E. Stable transduction of quiescent T cells without induction of cycle progression by a novel lentiviral vector pseudotyped with measles virus glycoproteins. Blood. 2008 Dec 15;112(13):4843-4852.
Centre de Biophysique Moléculaire UPR 4301 CNRS, Université d'Orléans and INSERM , rue Charles Sadron 45071 Orléans Cedex 2-, France .
Tissue degeneration is the common clinical feature found in tendon disorders due to injuries or as a consequence of rare connective diseases. Tendon regeneration is always difficult and requires a very long period. Moreover, the return at the initial state of the biomechanical parameters is never reached. The mechanism of tendons remodelling is controlled by growth factors acting at precise phases of regeneration.
The aim of this study is to evaluate ultrasound-assisted microbubbles to mediate gene transfer in tendon. Our set-up was consisted of a 1 MHz piezoelectric ultrasound transducer connected to a power amplifier and an arbitrary waveform generator. Mice Achilles tendons were transfected with 15 μg of plasmid DNA encoding luciferase gene. Different ultrasound parameters were tested to determine optimal acoustic setting for tendon gene transfer. Our data clearly indicate that the transfection efficiency was dependent on acoustic pressure, time exposure, and microbubbles number. Ultrasound combined with microbubbles improved the plasmid DNA transfer in Achilles tendon. No increase of gene expression was obtained with ultrasound alone. The highest luciferase activity (100-fold) was achieved with the following conditions: 200 kPa, 40 % DC, 106 microbubbles (BR14) and 10 minutes of exposure time. The kinetic of gene expression evaluated by in vivo bioluminescence revealed that the luciferase gene expression was stable up to 80 days.
Overall, our data show that ultrasound and microbubbles is a promising method for efficient gene transfer in tendons. Its efficacy to transfer therapeutic genes is currently evaluated on injured tendons and on transgenic mice model.
Administration of miRNA142.3p-Regulated rAAV2/1 Vectors in Skeletal Muscle Reduces the Initial Anti-Transgene Immune Response
Recombinant AAV2/1 vector (rAAV2/1) has emerged as a strong candidate for gene transfer into skeletal muscle. However its efficacy is partially limited by the development of anti-transgene immune responses.
In a murine model of limb-girdle muscular dystrophy type 2D (Sgca-null), administration of rAAV2/1 expressing the alpha-sarcoglycan (sgca) transgene under control of the ubiquitous CMV promoter results in a transient expression of the protein in skeletal muscle whereas a more stable expression was obtained using a muscle-specific promoter. To better understand the impact of restraining transgene expression to non-hematopoietic cells, its expression was prevented in haematopoietic cells by RNA interference after introduction of miRNA142.3target (mir142.3pT) sequences into the CMV-sgca cassette of the rAAV2/1 vector. To study the immune responses induced by such vectors, we developed a reporter Sgca-HY transgene to follow specific anti-HY CD4+ and CD8+ T cell responses in mice.
We observed that a single i.m. injection of the rAAV-CMV-sgca-HY vector in C57BL/6 and Sgca-null mice induced strong cellular immune responses 14 days post-administration. Both CD4+ and CD8+ T cells were activated in lymphoid organs and an extensive mononuclear cell infiltrate was observed in the injected muscle associated with muscular destruction. The introduction of mir142.3pT decreased the initial anti-transgene CD4 and CD8 responses but only delayed the functional activation of CD8+ T cells, which correlated with evidence of muscle disruption.
These data suggest that miRNA142.3p-regulated expression systems are useful to reduce the initial immunogenicity of the transgene after rAAV-mediated gene delivery into skeletal muscle, but may not be sufficient to elicit long-term tolerance.
Cellular Therapy Using Regulatory T Cells in Uveitis
Treg cellular therapy is a promising approach in autoimmune and chronic inflammatory diseases. However, Tregs specific for the target tissue, but not polyclonal Tregs, have a strong therapeutic potential when they are administered systemically. This approach is not clinically applicable yet since it is not possible to obtain clinical-grade auto-antigen-specific Tregs. Only polyclonal Tregs can be used clinically after purification with magnetic beads coated with anti-CD25 antibodies. A therapeutic alternative to systemic injection of auto-antigen-specific Tregs would consist in injecting clinical grade pre-activated purified polycloncal Tregs directly into the site targeting by the autoimmune attack. The rationale for this approach is that in vitro pre-activation of polyclonal Tregs will trigger their suppressive function, which will take effect in the inflamed site after their injection. Indeed, since Tregs are capable of bystander suppression exerted on Teffs with different antigen specificities, Tregs could inhibit the activation of all T lymphocytes present in the inflamed tissue. This approach can be evaulated in uveitis since cells can be injected directly into the vitreous of the eye. Furthermore, considering the small size of this organ, a therapeutic effect could be obtained with small cell numbers that are achievable without expansion in vitro. We first tested this cellular therapy strategy in a murine model. The pre-clinical data fully support this approach and lead us to propose a clinical trial in patients with severe bilateral uveitis and poor visual acuity who are refractory to other treatments.
Towards HIV Gene Therapy in Humanized Mice
The search for new therapeutic strategies against HIV is prevented by the lack of a suitable animal model. We showed that immunodeficient NOD.SCID.γ c-/- mice (NSG) humanized with cord blood hematopoeitic progenitor cells (HPC) preferentially reconstituted CD4+ T cells. Our anti-HIV strategy is based on the transfer of therapeutic transgenes in HPC to confer a selective advantage to « HIV-resistant » T cells. To transfer the genes, we built lentiviral vectors modified to specifically express the transgenes in CD4-T cells. Two fusion inhibitors are used in combination: one is the membrane-bound form of an analog of gp41, derived from the T20 inhibitor and the other is a partial agonist of CCR5. We then evaluated the expression and the potential of the vectors to inhibit HIV replication in vitro. Then HPC from cord blood were transduced with the vectors before their injection in NSG mice. The impact of gene therapy was measured after infection with HIV Bal using CCR5 as a coreceptor for entry. We showed that humanized mice were indeed susceptible to HIV infection and that a high viral load correlated with CD4-T cell deletion. Some mice treated by gene therapy reconstituted their CD4 T cells after depletion without viral rebound whereas the virus came back in control animals. These preliminary results indicate that humanized mice are a good model to assess HIV gene therapy strategies. This project should lead to clinical trials using gene transfer vectors optimized in a new model of HIV infection of human cells in vivo.
INSERM U948, Biothérapies Hépatiques , EA4274, CHU Hotel-Dieu, Nantes.
INSERM , U563, Toulouse
Dystrophic Epidermolysis Bullosa (DEB) is caused by mutations in COL7A1 encoding type VII collagen, and is inherited in a dominant or recessive manner. DEB patients suffer since birth from severe blistering of the skin and mucosae after mild trauma. Type VII collagen assembles into anchoring fibrils, which are key dermo-epidermal adhesion structures. No specific treatment is available for DEB but the targeted removal of exons carrying recurrent mutations shows therapeutic potential. Among the 118 exons of COL7A1, 84 exons encoding the central collagenous domain are in frame. Of these, exons 73, 74 and 80 are of particular interest because they carry many recurrent recessive or dominant mutations. We have demonstrated the dispensability of these exons for type VII collagen function, and have shown their specific skipping (with a low efficacy) using antisense oligoribonucleotides (AONs) masking exonic splicing enhancer (ESE) sequences. To increase the efficacy, we have fused the antisense sequences to the snRNA-U7 in lentiviral vectors. Surprisingly, these vectors failed to induce exon skipping, despite expression of the snRNA-U7. Therefore, we targeted other key splicing sequences (splice donor/acceptor and ESEs) with weaker predicted secondary structures using AONs and obtained higher skipping efficiency (up to 45%). The next step involves the production of lentiviral vectors expressing these antisense sequences fused to the snRNA-U7 to achieve long-term expression of the antisense and demonstrate the feasibility of this approach on animal models. If successful, this approach offers potential for treating patients using local or systemic administration of AONs or vectors.
Antiangiogenic Gene Therapy: An Application of Plasmid Vector Encoding Soluble FLT-1 Receptor for Locally Advanced Vulvar Cancer
One of the cancer gene therapy strategy is the inhibition of tumor angiogenesis. sFLT-1 is an alternatively spliced soluble form of FLT-1 receptor that effectively inhibits vascular endothelial growth factor (VEGF) and therefore blocks the process of angiogenesis in vivo and tumor growth.
The antiangiogenic properties of the cloned plasmid expression vector—psFLT-1 - has been previously verified in vitro conditions as well on laboratory animals. In the treated animals the inhibition of skin angiogenesis and tumor growth were observed. This work documents the successful use of psFLT-1 plasmid in patients with primarily non-operable invasive carcinoma of the vulva. So far, 30 women were twice injected, directly to the tumor, with 800 μg of psFLT-1. The preliminary outcomes show that the applied gene preparation is save and well tolerated by patients. In majority of the patients the significant reduction of the tumor size was observed that allowed for surgical removing slightly shrank cancer. The studies are still in progress. It seems to be worth underlining that antiangiogenic gene therapy as neo-adjuvant treatment may be useful for cancer therapy in the future.
Targeted Genome Editing in Mammalian Cells Using Engineered Zinc Finger Nucleases
Rational genome engineering in mammalian cells is of enormous potential across basic research, drug-discovery as well as cell-based medicines. To this end, Sangamo Biosciences and Sigma-Aldrich have recently partnered to commercialize a novel technology that enables high-frequency genome editing via the application of designed zinc finger nucleases (ZFNs). Within these chimeric proteins the DNA binding specificity of the zinc finger protein determines the site of nuclease action. Such engineered ZFNs are able to recognize and bind to a specified locus and evoke a double-strand break (DSB) in the targeted DNA with high efficiency and base-pair precision. The cell then employs the natural DNA repair processes of either “homology-directed repair (HDR)” or “non-homologous end joining (NHEJ)” to heal the targeted break. These two pathways provide the investigator with the ability to provoke three unique outcomes in genome editing—gene correction, gene deletion and targeted gene addition. Furthermore, the speed and efficiency of this process enables us to knockout multiple genes in the same cell. Drawing from our work with transformed cell lines, primary human cells, and multi-potent stem cells, we will present several examples of single, double and triple gene knockout, as well as targeted gene insertion into native chromosomal loci.
Institut de Génétique et Microbiologie , UMR 8621, Batiment 400, 91405 Orsay Cedex et
Using gene therapy requires to identify the most useful and appropriate gene(s). Its identification may be straighforward (the wild-type version of the deficient gene) but some cases require more sophisticated or indirect approaches. For example, mitochondrial mutations which lead to severe neurodegenerative diseases cannot be corrected directly since the genetic manipulation of the mitochondrial genome is not possible yet, except in yeast.
We have used the yeast S. cerevisiae to mimic mitochondrial diseases due to mutations in mitochondrial tRNA genes and in a second step, the powerful genetic properties of this yeast to select suppressors (correcting genes).
Several mutations have been created in the tRNALeuUUR, the tRNAVal and the tRNAIle mitochondrial genes, which are quite conserved with respect to their human counterparts. The mutant phenotypes were variable, from completely defective (which for a yeast appears as unability to grow on respiratory substrates and loss of mitochondrial DNA) to moderate ( conditional or leaky), with an excellent relation to the respective severity of the human syndrome. Multicopy suppressors (increasing copy number of a wild type nuclear gene) have been searched and a general suppressor- the translation mitochondrial elongation factor Ef-Tu- was effective on all of them. The cognate tRNA synthetase was also efficient in several situations.
This fast procedure in yeast allowed selecting a nuclear gene which corrects all tRNA mutations identified up to now. This gene was in turn shown to be efficient on a patient cell line by Sasarman et al., 2008, confirming the success of the yeast tool.
Généthon, CNRS FRE3087, 1, rue de l'Internationale , 91000 Evry, France .
Dysferlinopathies are recessive muscular disorders caused by defects in dysferlin. Genetic mutations are responsible for two major phenotypes: Limb Girdle Muscular Dystrophy type 2B and Distal Miyoshi Myopathy. These skeletal muscle diseases are characterized by progressive loss of muscle integrity and strength. Recently, dysferlin was demonstrated to be involved in membrane repair process, providing a preliminary understanding of the pathophysiological mechanism in these diseases.
Currently, no treatment is available. Considering the recessive nature of dysferlinopathies, a possible therapeutic strategy is gene transfer. To date, the best vector for gene transfer in muscle is Adeno Associated Virus (AAV). However, the human dysferlin cDNA size approximates 7 kb, preventing its direct incorporation into a single AAV vector, since the encapsidation limit is around 4.7 kb. In order to bypass this limitation, we set up a strategy taking advantage of the concatemerization ability of AAV vectors. The dysferlin cDNA was separated into two AAV2/1 vectors, one carrying a muscle specific promoter followed by the 5′ half of dysferlin cDNA and a 5′ splicing signal and another one carrying a 3′ splicing signal, the remaining dysferlin sequence and a polyadenylation signal. We demonstrated the ability of these vectors to produce a human full-length dysferlin mRNA in a cellular model. To test this approach in vivo, intramuscular injection of both vectors was performed into dysferlin deficient mice. Dysferlin mRNA was correctly spliced and was expressed at least for one year. At the protein level, dysferlin was detected at 237 kDa, the size of the expected full-length protein. This expression was associated with an improvement of the histological aspect of the muscle, a reduction in the number of necrotic fibers, restoration of membrane repair capacity and a global improvement in locomotor activity. Altogether, these data support the use of such a strategy for the treatment of dysferlin deficiency.
Development of a Therapeutic RNAi Delivery System Using Nonpathogenic Bacteria Expressing inv and hly: Transkingdom RNA Interference (tkRNAi)
Footnotes
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Presentors are underlined for all abstracts.