I-Stem grows along defined strategic plans
After an initial plan drafted in 2004 which laid the framework of a two-year phase (2005-2006) during which we conducted feasibility tests prior to the construction of the Institute, the second phase was prepared by a strategical plan elaborated in late 2006. It lasted three years (2007-2009) and was marked by growth of the structure and successful demonstration of the pilot programs. Phase III, which responded to a strategic plan elaborated in late 2009, run from 2010 to 2015. It aimed at fulfilling clinical expectations, enlargement of pathological indications and industrialization processes. The current strategic plan runs from 2016 to 2020. It is scheduled in order to bring the clinical proofs of concept of the two main therapeutic axes developed by I-Stem, namely, regenerative medicine and pharmacology based on pathological modelling.
I-Stem Phase I, 2005-2006: the introductory period, team training and open lab
The first team, consisting of twenty researchers, corresponded to the first phase, “launch”, which lasted two years. It was funded by four partners including 3 associated institutions (Inserm, UEVE, AFM) and a fourth (Génopole) that gave its support to the operation. For two years – in fact rather 18 months – we have compiled a “commando team” to address three essential questions and define the principles on which to base the Institute building. The three questions were respectively on our ability to access lines of stem cells (embryonic) necessary to our research, the applicability of automated technologies to these cells, and finally the acquisition and development of scientific and technical expertise. The main principles were to define the structure of the Institute, its financing and the terms of its public and private partnerships. The construction phase of I-Stem has resulted in a work performed by a sociology lecturer and researcher in Evry, Dr Philippe Brunet, published under the title: “The trial of technoscientific work in biotechnology: the case of the Stem Cell Institute. ”
This validation focused on the following aspects:
- build research teams of internationally recognized scientific level
- develop research programs respectful of French law,
- convince key financial partners of the interest and the relevance of the investment in the exploitation of the therapeutic potential of stem cells.
I-Stem-Phase II, 2007-2009: growth and success of pilot demonstration
During 2006, positive assessments of Inserm and AFM led to the creation of two administrative entities that are still running, Inserm Unit 861 (joint with the University of Evry, UEVE) and the Centre for the Study of Stem Cells (CECS). Both structures were positively reassessed at the end of Phase II, a timetable consistent with the development of the third strategic plan of the Institute.
Phase II was marked by strong growth in both biological resources in equipment, number of teams and programs.
- In addition to imported embryonic stem cell lines, a campaign was carried out in collaboration with the team of Professor Stéphane Viville in Strasbourg, which has enabled us to bank 19 lines from embryos discarded during a preimplantation diagnosis because of the presence of a genetic defect responsible for a severe disease.
- In parallel, since 2008, I-Stem teams have taken the opportunity of gene reprogramming techniques developed by Shinya Yamana (published November 2007) to create many iPS cell lines carrying genetic defects.
- Many colleagues have turned to us to have access to iPS cell lines exhibiting mutations they studied. In response, we have created the “iPS workshop “, which ensures at the same time the production of the cell lines and the formation of a member of the external team for a period of three months full-time at I-Stem. To date, more than twenty laboratories have benefited from this workshop.
- The start of Phase II coincided with the relocation of I-Stem in 1600 square meters, gradually extended to 2300, located within theGenopole campus I. Research space consists mainly pf confined Level II areas required for work on human cells and unconfined laboratories for biochemical experiments, molecular biology, histology and analyses.
- The platform for high-throughput screening of compounds was installed in 2007 and implemented from mid-2008. Screening campaigns have been increasing in number every year. Itshowed itsfull power at the end of the second phase of I-Stem through allowing screening – within an industrial collaboration with Hoffman LaRoche – of 220,000 compounds.
- The measurement tools for high content screening were installed, especially an automated imager and quantitative PCR on 384-well plates.
- An area dedicated to animal studies, preclinical research in rodents has been implemented.
While Phase I was conducted by a small “commando”-like team without formal internal structure, phase II, marked by a doubling of the number of I-Stem members in 2007 and another doubling in 2008, led to the organization of research teams, each responsible for developing a program and under the responsibility of a team leader.
- The majority of research teams are organized around a disease or group of diseases affecting a single organ or a single cell type. Description of the objectives and activities of the R & D is offered to the reader in a specific folder.
- Three technological research teams are organized around major methodologies and technologies Institute (production of cells in bulk, high and medium speed screening, functional genomics)
- In parallel, both management teams have been structured: a scientific office, responsible for the coordination and facilitation of scientific and technical activities, as well as partnerships and the administrative, financial and logistics office, dedicated to all support functions.
Team building, access to biological resources and implementation platforms have diversified approaches and programs.
- Phase II has seen, since 2007, the first patents and the first international publications on technological programs (cell production, identification of genomic abnormalities) and biological research (identification of several differentiation protocols)
- The arrival of several teams has diversified pathological indications in the context of two major research established at the outset, cell therapy and disease modeling paving the way for drug screening.
- Several research collaborations have been established with academic partners and industrial pharmas and biotechs.
I-Stem Phase III, 2010-2013: towards therapeutic application
From 2010, the development of I-Stem continued on the basis of a stabilized workforce (between 70 and 80 employees) reached in 2009. The priority objective highlighted in this strategic plan was the preparation of the clinical trial of pre-clinical results by I-Stem teams. It was precisely a phase of “translational research”.
- For cell therapy, we used three main types of protocols to efficiently produce epidermal cells, striatal neurons and retinal pigment epithelium. Translational research has sought to establish the conditions so that, without losing effectiveness, these cells can be implanted – respectively in patients with cutaneous ulceration related to sickle cell disease (PACE program), Huntington’s disease (HD-repair program ) and retinitis pigmentosa (STREAM program) – with the utmost respect for safety.
- Regarding drug discovery, several molecular mechanisms involved in various pathologies have been revealed by our cell studies derived from ES or iPS lineages. On these models we are looking for the normalization of these pathological mechanisms using chemical compounds using the drug screening techniques we have imported from the industrial world. The identification of effective compounds in vitro allows, after verification in relevant animal models, to consider the establishment of a clinical trial. This is what we were able to achieve successfully for Metformin, an anti-diabetic drug whose corrective effect had been observed on cells carrying a mutation responsible for type I dystrophic myotonia (experimental results published in 2015, results of MYOMET clinical trial published in 2018).
Over the 5-year period covered by this strategic plan, we have been able to develop some key principles on which to base our business in a stable way, which we detail below.
Human cell models
The discovery of protocols to direct the differentiation of human pluripotent stem cells to a specific cell fate has been a priority goal of the I-Stem teams during the first ten years of the Institute’s operation, because access to these cells clearly determined our ability to use them for our approaches to cell therapy, disease modeling, and pharmacology of monogenic diseases. This research has been successful with the development of specific protocols for many specific neuronal populations, astrocytes, retinal cells, vascular smooth muscle, mesenchymal stem cells, skeletal muscle cells, keratinocytes, and the melanocytes of the epidermis, the dermal fibroblasts. This aspect of our activity is gradually decreasing, however, as many protocols are now available either internally or by importing protocols developed by other teams.
We have been working since the beginning on embryonic stem cells (ES) and, since their appearance at the end of 2007, on pluripotency-induced cells (iPS). These two populations of human pluripotent stem cells have the cardinal characteristics that underpin the interest, the ability to proliferate identically (each mother cell giving birth to two identical daughter cells) without limit, without ever entering senescence as all other cells in the body do, and the ability, under other culture conditions, to differentiate to give rise to any of the cellular phenotypes of the body. ES and iPS, however, are not entirely similar and comparative studies show that iPS-GMO cells produced from adult cells by gene transfer coding for proteins acting directly at the DNA level have a number of characteristics. which makes them potentially less reliable for the studies we are conducting, an obstacle that we are circumventing by multiplying the controls, notably thanks to the use of genome editing techniques by CRISPR / Cas.
We need to obtain specific cell populations homogeneously, or at least very predominantly. The problem is not trivial, and most of the teams that work with pluripotent stem cells today do not pose it the same way as we do because they stay in the field of basic research. The homogeneity of the cell populations is an obligation for us because it is essential to carry out comparative studies of subtle molecular mechanisms, such as those that we explore in search of anomalies related to genetic diseases or potentially therapeutic effects of pharmacological agents that we test. It is also necessary in cell preparations intended for therapeutic applications, which must respond very precisely to regulatory quality control constraints.
Genetic Disease Models and Pharmacological Approaches
The generation of cellular models of monogenic diseases, which can be analyzed in vitro on demand, has been at the heart of I-Stem’s activity since the beginning. Initially only based on the rare ES cell lines derived from PGD embryos, this activity has vastly expanded based since 2008 on iPS lines that allow access to any of the pathologies. Pathologies explored in recent years by teams of I-Stem are very diverse, myotonic dystrophy type 1, Duchenne myopathy , various limb girdle muscle dystrophies, spinal muscular atrophy, Huntington’s disease, autistic Phelan McDermid syndrome, Lesch Nyhan syndrome, epidermolysis bullosa simplex, neurofibromatosis type 1, progeria, Wolfram syndrome, retinitis pigmentosa, adenomatosis polyposis coli…
The main foundation of our pathological modeling activity is the multi-parametric, morphological, genic, protein and functional comparison of differentiated cells in exactly the same way from healthy patient-derived lines and controls, or created by genome editing thanks to the CRISPR tools (so-called “isogenic” controls). These comparisons should allow us to identify anomalies potentially due to the presence of the mutation, which we verify through a battery of experiments involving in particular gene correction techniques and the use of other models of the same pathology.
This paradigm allowed the I-Stem teams to discover many pathological mechanisms. However, it has some limitations, biological and technical. As far as biological resources are concerned, it must first be pointed out that certain differentiation protocols have long escaped us, the most frustrating example being the differentiation into skeletal muscle fibers, which we only mastered at the end of the period covered by this 3rd strategic plan by adapting the protocols developed by the Australian company Genea Biocells. We have already pointed out above the relative reliability of iPS cell lines linked, notably, to the existence of epigenetic abnormalities associated with reprogramming, which sometimes randomly deform certain molecular mechanisms that we must not confuse with pathological damage. Another problem that may arise in certain pathologies is related to the theoretical “age” of the cells, which represents a very early developmental stage, which can be roughly characterized as fetal. Certain monogenic pathologies result from abnormalities that only appear later, or even in adulthood. This does not necessarily preclude looking for very early abnormalities, which the cells would manage for a while to control thanks to compensating mechanisms whose identification is interesting because it points to potential therapeutic paths. In some cases, however, we have failed to characterize early molecular mechanisms associated with the disease and this has led us to discontinue programs (e.g., Leigh syndrome or Friedreich ataxia).
The search for pharmacological compounds capable of positively modifying the activity of differentiated cells from pluripotent stem cells derived from samples taken from donors carrying monogenic diseases is at I-Stem the logical consequence of the exploration of the pathological mechanisms. This orientation has been very structuring for the Institute both scientifically and technically, since all the teams are pursuing at least one program of this type. Very heavy investments have been made to support them, giving access on the site to a very large set of tools, including protein analysis (Odyssey, MacQuant, Biotek Synergy, Clariostar, Ventana), molecular analysis (Ion Proton , Qiacube, Tape, QuantStudio), microscopy (MetaSystem, spinning disk, confocal, incucyte, etc.), cell imaging (Bravo, ImageXpress, Leap, Arrayscan, CX7, Hammamatsu), to the semi-industrial high-throughput screening (Biocel 1800, BenchCell) and bioproduction platforms (CompacT SelecT, Fill-It, Cryomed) that are emblematic of I-Stem’s combination of scientific innovation and technological innovation. One of the main originalities of I-Stem since its creation is the combination of advanced biological research on pluripotent stem cells, and research and technological development programs for the creation of the most powerful platforms developed, selected and implemented by technological research teams. The “platform engineers” of I-Stem are at the same time researchers, trainers and managers of the instruments for which they are responsible. They also perform a technological watch in each of their fields, in order to be able to propose to the Board of Directors of the Institute the adjustments and, sometimes, the necessary changes. Some equipment is fully managed by these specialized engineers. However, a constant effort has been made to ensure that the teams themselves have access to the instruments through special training that often requires careful supervision by the platform engineers.
The search for methods to replace cells lost due to a genetic pathology by healthy ones, produced entirely in the laboratory has been, since the birth of I-Stem, a very active line of work. Cell therapy requires the identification of culture protocols that result in the production of cell populations that are exactly similar to those that are to be replaced. The quality of differentiation is therefore an absolute prerequisite. This objective is all the more complicated to achieve because it must be combined with an imperative of homogeneity that is not, more often than not, addressed as such in the protocols developed by other teams. Added to this is the need for often massive cell production. Some differentiation techniques do not allow this “amplification” which is mandatory for cell therapy approaches, when it is intended to provide a treatment that can be applied to all patients who need it, even in the case of most rare diseases. Finally, and this is a particularly acute concern for cell therapy applications, production protocols must be transferable to pharmaceutical establishments that are governed by GMP (Good Manufacturing Practice) rules in clinical grade. This requires in all cases a systematic adaptation of the protocols defined in the conditions of the experimental research -which aims first to obtain effective products- so as to make them compatible with a use in humans -that is to say perfectly harmless- without loss of biological and clinical efficacy.
I-Stem teams have matured in two programs: epidermis for the treatment of sickle-cell-related skin ulcers (PACE program) and retinal pigment epithelium for treatmentof retinitis pigmentosa (STREAM program). A third product, the medium spiny striatal neurons for the treatment of Huntington’s disease, is less advanced in pre-clinical studies. Current programs are based on embryonic stem cell lines. This choice was imposed until recently because of the acknowledged reliability of these cell lines compared to iPS cells. There is, however, serious doubt about the potential of these cells after transplantation in the medium and long term, related to the absence of any consideration of a possible immune rejection of the transplant. If such rejection would jeopardize the survival of the grafted cells at a time when it would compromise their beneficial effect, then alternative strategies should be considered. We have been concerned during the period covered by this third strategic plan within the global alliance for iPS therapy (GAiT) network, which aims to create banks of iPS cell lines from donors that their genetic heritage makes particularly useful to bypass the immune response (so-called “triple homozygous” haplotypes that have only 3 antigenic cell markers instead of 6). I-Stem is associated with AnneLise Bennaceur-Griscelli’s E-STEAM team which is building such a “haplobank” in Genopole.
I-STEM phase IV (2016-2020): towards clinical proofs of concept validating our pharmacological and regenerative medicine approaches
The 4th strategic plan of I-Stem coincided with the migration of the Institute in brand new premises, offered to us by the Genopole within the framework of the CRCT (Clinical and Translational Research Center). We occupy 1600 square meters, half of which divided into 4 experimental zones of 200 square meters each, devoted respectively to the culture of human cells (L2 confined laboratories), to “dry” biology (biochemistry and molecular biology), robotic technologies of production and analysis, and support sectors (microscopy, cryopreservation, servers, etc …). The other half of the laboratory is occupied by offices (80 people), storage, and living and circulation areas. Access is, moreover, free to a large conference room of 200 square meters and several meeting rooms.
Development of cell therapy products
As regards regenerative medicine, the activities of I-Stem’s biological research teams can be grouped into three categories: completion of ongoing programs, innovation for improvement of ongoing programs, innovation for future programs.
First of all, of course, we are completing our current programs, that is to say in the period covered by this 4th strategic plan, until the complete technology transfer to the pharmaceutical establishment in charge of the production of ATMPs, and the implementation of clinical trials. In the absence of establishments with the requisite skills for the production of cells derived from pluripotent lines, we first went through an exploratory phase during which we tested several potential providers. Only Atlantic BioGMP (ABG), the contract manufacturing organisation of the EFS in Nantes, has fully met the specifications of STREAM and PACE. The close collaboration with EFS-ABG teams has already allowed us to submit a clinical trial application for the treatment of retinitis pigmentosa, which has been successful with an authorization delivered to us on January 23, 2019 by the regulatory authorities. The clinical trial itself, sponsored by CECS/I-Stem and supervised by the teams of the Clinical and Regulatory Management Department of the Institute of Biotherapy, will be carried out by our collaborators from the Institute of Vision and Health. Hospital XV-XX (Paris). The GMP production program is currently underway at EFS-ABG for the PACE (epidermis) program, for a clinical trial in collaboration with AP/HP teams in 2020.
The developed cell therapy products can also be modified to better meet the needs. We have launched this program of improvement on the epidermis. Because it is composed only of keratinocytes, the PACE leaflet does in fact reproduce this tissue imperfectly, let alone the entire skin coverage. Our work has led in recent years to the identification of differentiation protocols for complementary cell populations, melanocytes that pigment the skin and protect it against the genotoxic effect of UV, and fibroblasts that are the main element of the dermis and ensure, in particular, the interface with blood vessels. The “skin” cell therapy program at I-Stem will therefore be continued with a view to creating a more complete product, which will be pigmented and associated with a dermal sub-layer. This will enable us to initiate a “PACE 2” clinical program whose indications can be extended to a spectrum of pathologies beyond cutaneous sickle-cell ulcers.
We are looking to develop new cell products, this time derived not from ES cells but from iPS cells. For years I-Stem has been focusing on the complementary potential of cell therapy products derived from iPS cells, because the making of these lines can be done from selected donors. But cell therapy, like any biological transplant, faces the difficulty created by the immune system whose activity aims to reject any intrusion of biological material with an immune identity recognized as different from that of the recipient. A solution devised by British scientists fifteen years ago is to find particular donors in the general population who possess what is called a “triple-homozygous haplotype”, that is they only have one type of HLA-A antigen (the 2 A’s are identical), one type of HLA-B antigen and only one type of HLA-DR antigen. These donors should thus be compatible for heterozygotes with the same 3 markers and 3 others whose precise identity no longer has any consequence. We determined and published (in 2013) that the triple-homozygous donor alone with the most common haplotype in the Caucasian population (A1, B8, DR3: so it is A1A1, B8B8, DR3DR3) could serve up to 14.5 % of the population (heterozygotes A1Ax, B8By, DR3DRz) in France and in all countries of Caucasian population. We collaborate closely in the production of such iPS lines with AnneLise Bennaceur-Griscelli’s team in France, the Global Alliance for iPS Therapies (GAiT) worldwide.
There are several other ways of circumventing the immune system, which are grouped under the terms “ghost cells” (or “stealth cells”). The I-Stem teams are now exploring these avenues in close collaboration with Thierry Heidmann and his teams who have identified the molecular basis of such bypasses observed in physiological conditions (e.g. pregnancy) and have offered to put them to good use for regenerative medicine.
Pathology modeling and pharmacology programs
We are continuing programs undertaken during the third strategic plan, including Steinert’s myotonia and Duchenne muscular dystrophy (with a specific new program on the neurological impact of dystrophinopathies), spinal muscular atrophy, several limb girdle myopathies (program launched at the end of the 3rd plan), Wolfram syndrome, epidermolysis bullosa simplex, neurofibromatosis type 1 and Huntington’s disease. We refer readers who wish a detailed presentation of these research projects to the pages that specifically present them elsewhere on our site.
In parallel, through the MyoPharm program, we are now opening collaborations on several ultra-rare neuromuscular diseases (whose prevalence does not exceed a few hundred patients in France) around programs sponsored by other specialized institutes (GIPTIS, NeuroMyogene, Genethon, Institute of Myology, etc …). It seems to us technically possible today to systematically study the pathological mechanisms that accompany the mutations at the origin of any neuromuscular disease and to use these data to identify pharmacological compounds capable of oppose it. The scientific and technological underpinnings of such a program can be summarized, schematically, in a nutshell: 1. iPS cells can be derived from cells of any patient carrying a neuromuscular pathology. 2. Cell culture protocols make it possible to obtain motor neurons and myotubes, which are the most relevant in vitro models for studying neuromuscular diseases. 3. A technique (CRISPR/Cas) is used to modify the cell genome to create or correct a point mutation, making it possible to establish corrected control lines for the causal mutation of the pathology that is needed to identify by comparison the molecular and cellular abnormalities associated with the mutation. 4. High throughput sequencing (in its applications to gene expression analysis) allows the molecular functioning of mutated cells and controls to be compared and thus to identify molecular abnormalities. 5. High-content analytical platforms allow the systematic multi-parametric study of cellular and functional phenotypes arising from these molecular abnormalities. 6. High throughput screening tools are applicable to mutated cell populations, opening the way for the identification of repurposable drug candidates that can then be rapidly tracked -as a result of their repositioning- through the entire pathway to clinical trials.