Twelfth Annual Meeting of the French Society of Cell and Gene Therapy

The French Society of Cell and Gene Therapy (SFTCG) was very pleased to welcome scientists to the beautiful city of Marseilles in the south of France to attend the 12th annual meeting of the society, from March 9 to 11, 2016. Nearly 200 scientists from the United Kingdom, Italy, Spain, the United States, Germany, Japan, and France attended this meeting, held at the Palace of Arts. In particular, Spanish students and lecturers were welcomed as part of a joint exchange program between the Spanish and the French Societies. For this occasion, a free copy of the special issue “French Gene and Cell Therapy in 2016,” edited by Nathalie Cartier and Pierre Cordelier and published in collaboration with Human Gene Therapy, was distributed to all participants.

This meeting was preceded by the morning Public Session, allowing high school students and patients' associations to interact with researchers involved in gene and cell therapy programs. These “speed dating” sessions and roundtables were organized by Sophie Gomez and the French Society of Cell and Gene Therapy in partnership with INSERM and AVIESAN ITMO Cancer. Associations represented during this Public Session were SOS rétinite-Association Nationale de Lutte contre la Cécité (SOS-Retinitis National Association Against Blindness), Collectif Solidarité Charcot (Charcot initiative), AFM-Téléthon, AFAF (Association Française de l'Ataxie de Friedreich, French Association of Friedreich's Ataxia), and Vaincre La Mucoviscidose (Defeating Cystic Fibrosis Association). Researchers involved were Nathalie Cartier, Sophie Gomez, Vincent Mouly, Michel Pucéat, Patrick Midoux, and Els Verhoeyen. They answered questions on the pertinence of the DNA sequences used in gene therapy programs and why translation to the clinic is taking so long. This session started with the broadcast of the movie Dr. Virus and Mr. Hyde, directed by A. Saïb and J. Crépu, in collaboration with the France 5 television network. In the afternoon, Nicolas Boisgérault (University of Nantes, France) gave an overview during the Educational Session, “On Focus,” of recent successes in the field of cancer virotherapy, which have confirmed the clinical potential and safety of oncolytic herpes, vaccinia, and measles viruses. These viruses have the capacity to induce strong and specific antitumor immune responses by promoting both immunogenic death of tumor cells and efficient priming of immune cells. Clinical trials have now been approved for melanoma, hepatocellular carcinoma, and multiple myeloma. The hope is that these new therapeutics will open up a new era in the treatment of cancer.

During the Opening Session chaired by Nathalie Cartier, the first invited keynote speaker, Marc Kay (Stanford University, Stanford, CA), described how murine–human xenotransplant models for muscle and liver represent a robust animal model that may more closely predict clinical trial outcomes than previously used systems. Such new models in combination with multispecies DNase-shuffled AAV capsid libraries have permitted the selection of novel rAAV vectors, allowing enhanced transduction of human muscle and liver. However, the episomal nature of the vector genomes in transduced cells restricts classical rAAV-mediated gene transfer to quiescent tissues. High rates of hepatocellular carcinoma resulting from promoter activation of oncogenic loci in young mice, and loss of episomal AAV genomes with normal growth and development, have raised concerns about treating infants. To provide a viable approach to treating infants, Marc Kay's group has developed an AAV promoterless gene-targeting approach without the use of nucleases and used this tactic to successfully treat mice with hemophilia B. In conclusion, the discovery and characterization of novel AAV vectors and their use in both classical gene transfer and genome-editing approaches broaden their application in both biological discovery and therapeutic applications. This Opening Session also featured Nicolas Levy (Inserm UMRS910 AIX-Marseille University), who presented a new potential therapeutic target for Hutchinson–Gilford progeria syndrome. This genetic disease, responsible for premature aging, is caused by mis-splicing of lamin A/C mRNA responsible for the production of the toxic protein, progerin. Protein degradation pathways are affected in this disease. Nicolas Levy's group investigated the protein degradation pathway as a tool to act on the level of the toxic progerin. They found that a proteasome inhibitor decreases the level of progerin in cells, indicating a potential therapeutic intervention for progeria. A lively discussion ensued, which carried over to the following day, after the yet much appreciated gala and dancing dinner, which took place at the “Villa Gaby,” one of the most beautiful Italian-style buildings erected along the “Corniche” of Marseille at the end of the 19th century.

The next morning, Pietro Genovese (HSR TIGET, Milan, Italy) reported during the Gene Editing Session chaired by Els Verhoeyen that he has successfully achieved homology-directed DNA repair in the primitive hematopoietic stem and progenitor cell (HSPC) subset, and confirmed the therapeutic potential of this strategy by targeting a corrective cDNA into the IL2RG gene of a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited hematopoietic stem cells (HSCs) gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. The gene targeting protocols were refined and scaled up to develop a robust, scalable, and clinically ready process for ex vivo editing of HSPC. This process may possibly enable the first clinical testing of an HSPC gene correction approach for the treatment of SCID-X1. After this presentation, Jean Paul Concordet presented an overview of the tools engineered in the last few years to edit the genome, a topic that is currently highly debated between the SFTCG and the French Society of Human Genetics. Dr. Concordet more specifically focused his talk on CRISPR/Cas9 technology. He discussed the specificity of the editing process according to the RNA guides and the stability of guides chemically modified by phosphorothioate. He presented the two modes of DNA repair (homology-directed repair and nonhomologous end joining repair) after a CRISPR/Cas9 DNA break, and the dependence of these active processes on the stage of the cell cycle. Finally, he introduced likely the most useful web platform to design RNA guides to direct Crispr/Cas9 to the genomic region of interest. This platform is open to the scientific community.

The Cancer Therapy Session sponsored by Sanofi Oncology and chaired by Georges Vassaux and Pierre Cordelier featured Prof. Ugur Sahin from TRON (Mainz, Germany), who presented very interesting results on mRNA-based vaccines against cancer. The mRNA-encoding antigen appears to be a very promising tool to induce a specific immune response against cancer cells in preclinical and clinical experiments. The presentation covered the in vivo targeting of dendritic cells and the induced, specific immune response obtained according to the route of mRNA-encoded antigen. Data on mRNA uptake by dendritic cells showed that micropinocytosis is the preferred uptake pathway. The presentation highlighted the promising therapeutic benefits of mRNA and also the need to develop smart vectorization systems for systemic delivery to increase vaccine efficacy. The latest innovation in the field of chimeric antigen receptor therapy for cancer was then presented by Sandra Guedan (IDIBELL-ICO, Barcelona). Dr. Guedan reported dramatic tumor regressions in patients with B-cell neoplasms, such as chronic and acute lymphoblastic leukemia and non-Hodgkin lymphoma, using CARs targeting CD19. She also presented preliminary data on targeting solid tumors, such as pancreatic adenocarcinomas, using this approach. She discussed the design of CARs and the requirement for optimal costimulation, as well as the selection of the correct cell type for engineering.

During the Cell- and Gene-Mediated Immunology Session, sponsored by Genosafe and chaired by Federico Mingozzi and Els Verhoeyen, Naomi Taylor (IGMM, Montpellier, France) stressed that optimal antitumor immunity depends on T-cell metabolic fitness. As the energetic requirements of T-cells upon antigen stimulation are met by an increased rate of glycolysis, the group of Dr. Taylor assessed whether glucose transporter (Glut1) levels could be used to distinguish T lymphocytes with distinct features. Glut1-Hi lymphocytes exhibited increased effector phenotype acquisition, a skewing toward a cytotoxic fate, and augmented proliferation. Thus, separating T lymphocytes on the basis of Glut1 levels may allow the development of new T-cell immunotherapy strategies. Nutrients are also important for regulatory T-cell induction. Reduced glutamine levels are observed in the intratumoral environment and induce a marked augmentation of regulatory T-cells. Glutamine deprivation can be exploited to treat a model of colitis by increasing Tregs while maintaining a Th1 environment. Thus, while the cytokine environment modulates T-cell fate, nutrients seem to guide T-cell fate and this concept can be exploited for therapies. Later this day, a relaxed and friendly “Speaker's Dinner” was held in a private apartment, and was highly appreciated by all the participants.

Back to work, the Recent Advances in Vector Development Session, sponsored by InCellArt and chaired by Patrick Midoux, took place on the morning of Friday, March 11, 2016. During this session, Axel Schambach (Hannover Medical School, Germany) reported on a new vector system derived from alpharetroviruses, characterized by a more random and potentially safer integration pattern than gammaretroviral or lentiviral vectors. In addition, a stable producer cell line was recently generated in his laboratory, providing a basis for upscaling of vector production for clinical trials. In particularly, as they allow efficient gene transfer into human T-cells and HSCs, alpharetroviruses represent promising tools for clinical application in gene therapy of inherited diseases and cancer immunotherapy. In the same session, Daniel G. Anderson from MIT (Cambridge, MA) described nucleic acid delivery systems for RNA therapy and gene editing. Prof. Anderson has developed an original method for the synthesis of a great number of degradable polymers and lipid-like materials for nucleic acid delivery. He has used a combinatory approach allowing the synthesis of multiple vectors by using simple molecules of high reactivity. Some of these vectors demonstrated good in vivo efficacy to deliver siRNA or mRNA to treat viral infection and cancer. Lastly, Prof. Anderson presented promising results of gene editing using CRISPR/Cas9 mRNA nonviral delivery.

Later the same day, during the Innovative Therapies Session chaired by Anne Galy and Vincent Mouly, Elvire Gouze (C3M, Nice, France) proposed a new therapeutic strategy for a rare genetic disease, achondroplasia, which is characterized by abnormal bone growth and short stature. A single-point mutation in the fibroblast growth factor receptor 3 (mFGFR3) gene results in aberrant, excessive intracellular signaling. Dr. Gouze demonstrated that administration of soluble recombinant FGFR3 can act as a decoy and prevent FGF from binding to the mutant FGFR3, resulting in normal skeletal growth in a preclinical mouse model. These remarkable results may signify a big step forward toward a potential treatment for patients with achondroplasia.

The Manufacturing Session chaired by Eduard Ayuso was dedicated to process development and quality control of AAV and lentiviral vectors. Matthias Hebben (Généthon) presented data comparing the manufacturing process for AAV8 vectors using transient transfection of HEK293 and baculovirus infection of insect cells both cultivated in stir-tank bioreactors. The purification yields were higher for AAV8 vectors produced by transfection, but more importantly the infectivity of the vectors was also better using the transfection method. Indeed, AAV8 vectors produced in insect cells showed a reduction on the VP1 protein that explained the lower infectivity. Nonetheless, it was discussed that production costs would be a limiting factor for using transfection technology in applications for which large amounts of vectors are required, such as the treatment of muscular diseases. James Warren's (bluebirdbio) presentation was focused on the manufacturing of lentiviral vectors for ex vivo applications. Lentiviral vectors in this case are used as starting material to genetically modify HSCs for the treatment of B-thalassemia, adrenoleukodystrophy, or sickle cell disease. The manufacturing method for lentiviral vectors used in these studies consisted of a quadruple transfection of plasmids into HEK293T cells that were grown on adherent support (cell factories). This method produced enough vectors for ex vivo applications in rare diseases applications, but might be limited for treating a larger number of patients. In that case, scalable technologies such as wave bioreactors, stir-tank bioreactors, or fixed-bed bioreactors should be implemented. Particular attention was given to the quality control tests and validation of analytical methods when the investigational product is moving toward phase III and commercialization, such as LentiGlobin for the treatment of B-thalassemia. Eduard Ayuso (INSERM 1089, Nantes) discussed recent data on the characterization of DNA contaminants in AAV stocks using next-generation sequence (NGS) technologies. For the first time, NGS technology was used to identify and quantify DNA contaminants in AAV stocks produced in insect cells infected with baculovirus expression vectors. Illegitimate DNA originating from baculovirus and host cells was low, corresponding to ≤1.5% and ≤0.02% of the total reads, respectively. Also, the sequencing coverage showed that the proximity to the ITRs increases progressively the probability for baculoviral DNA to be encapsidated. A global conclusion of the session was the need for the development of accurate quality control methods to support the commercialization of gene therapy drugs based on viral vectors.

The scientific meeting ended with two remarkable lectures from pioneers and rising stars in the field of cell and gene therapy. Dr. M. Cavazzana (Necker Hospital, Paris) reported data from four thalassemia major patients treated with HSC gene therapy, with positive results. She presented very promising data on the first sickle cell disease patient treated with a lentiviral vector encoding an antisickling form of beta-globin (HbAT87Q). This vector was made by bluebirdbio. The patient received 5.6 million CD34+ cells per kilogram with approximately one vector copy per cell. Twelve months after gene therapy, the patient was producing 50% antisickling hemoglobin and no longer required transfusion, his reticulocyte count was 143 g/liter, and his lactate dehydrogenase (LDH, a biomarker of hemolysis) was low, at 274 U/ml. The oxygenation curve was normal. These results are very encouraging and open up new perspectives for gene therapy of hemoglobinopathies. Finally, Takanori Takebe, a young researcher from Yokohama University in Japan, gave an enthusiastic talk. He reported the generation of diverse organ buds from stem cells, showing how epithelial cells such as hepatic or renal progenitors differentiated from pluripotent stem cells organize themselves in organoids in the presence of mesenchymal and endothelial cells. The organoids become vascularized when grafted in mice. Prof. Takebe further presented a high-throughput platform to generate these organoids to an extent to be used in regenerative medicine. Finally, Pierre Cordelier closed the meeting with the Best Poster and Best Presentation awards. The Best Poster award went to Manuela Romano from Naomi Taylor's group in Montpellier, for her work on modulating cell fate decisions via manipulation of metabolic pathways for HSC therapy. Later, Sara Fañanas (CIEMAT/CIBERER; IIS-FJD, UAM, Madrid) received the Best Presentation award for her work on gene editing of the PKLR gene in human hematopoietic progenitors. During his closing comments, Pierre Cordelier stressed that this meeting was a tremendous opportunity to better identify new technological, methodological, and regulatory challenges. The program of the event, which included internationally renowned researchers and clinicians, was a unique opportunity for participants (students and young researchers) to deepen their knowledge in areas at the forefront of therapeutic innovation and to access the knowledge of recognized experts.