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.

Regenerative medicine

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.