7 research outputs found
An Orthotopic Model of Glioblastoma Is Resistant to Radiodynamic Therapy with 5-AminoLevulinic Acid
Radiosensitization of glioblastoma is a major ambition to increase the survival of this incurable cancer. The 5-aminolevulinic acid (5-ALA) is metabolized by the heme biosynthesis pathway. 5-ALA overload leads to the accumulation of the intermediate fluorescent metabolite protoporphyrin IX (PpIX) with a radiosensitization potential, never tested in a relevant model of glioblastoma. We used a patient-derived tumor cell line grafted orthotopically to create a brain tumor model. We evaluated tumor growth and tumor burden after different regimens of encephalic multifractionated radiation therapy with or without 5-ALA. A fractionation scheme of 5 Ă 2 Gy three times a week resulted in intermediate survival [48-62 days] compared to 0 Gy (15-24 days), 3 Ă 2 Gy (41-47 days) and, 5 Ă 3 Gy (73-83 days). Survival was correlated to tumor growth. Tumor growth and survival were similar after 5 Ă 2 Gy irradiations, regardless of 5-ALA treatment (RT group (53-67 days), RT+5-ALA group (40-74 days), HR = 1.57, p = 0.24). Spheroid growth and survival were diminished by radiotherapy in vitro, unchanged by 5-ALA pre-treatment, confirming the in vivo results. The analysis of two additional stem-like patient-derived cell lines confirmed the absence of radiosensitization by 5-ALA. Our study shows for the first time that in a preclinical tumor model relevant to human glioblastoma, treated as in clinical routine, 5-ALA administration, although leading to important accumulation of PpIX, does not potentiate radiotherapy
Mutation-Specific Guide RNA for Compound Heterozygous Porphyria On-target Scarless Correction by CRISPR/Cas9 in Stem Cells
International audienceCRISPR/Cas9 is a promising technology for gene correction. However, the edition is often biallelic, and uncontrolled small insertions and deletions (indels) concomitant to precise correction are created. Mutation-specific guide RNAs were recently tested to correct dominant inherited diseases, sparing the wild-type allele. We tested an original approach to correct compound heterozygous recessive mutations. We compared editing efficiency and genotoxicity by biallelic guide RNA versus mutant allele-specific guide RNA in iPSCs derived from a congenital erythropoietic porphyria patient carrying compound heterozygous mutations resulting in UROS gene invalidation. We obtained UROS function rescue and metabolic correction with both guides with the potential of use for porphyria clinical intervention. However, unlike the biallelic one, the mutant allele-specific guide was free of on-target collateral damage. We recommend this design to avoid genotoxicity and to obtain on-target scarless gene correction for recessive disease with frequent cases of compound heterozygous mutations
Données médicales
De nos jours, le paradigme du grand nombre (big data) entraiÌne un renouvellement des repreÌsentations meÌdicales du corps (qui se voit fragmenteÌ en autant de donneÌes comparables et agençables aÌ merci) et de la relation theÌrapeutique. LâaveÌnement de la meÌdecine dite personnaliseÌe, de lâintelligence artificielle et de ses dispositifs algorithmiques sâaccompagne effectivement de nouveaux imaginaires et rheÌtoriques soignantes. Ces dernieÌres sâappliquent aÌ deÌployer un discours propheÌtique sur les deÌcouvertes scientifiques aÌ venir, sur la capaciteÌ des sciences aÌ repousser les maladies et meÌme la mort, aÌ renforcer et normaliser les corps ; lâideÌe dâune meÌdecine toute puissante, en somme. La « veÌriteÌ Â» des corps et la reÌsolution de leurs troubles ne se trouveraient que dans ce qui est penseÌ comme une double objectivation : traitement de donneÌes quantitativement nombreuses et reÌalisation de lâexercice par une machine non doueÌe dâaffects. Ce numĂ©ro revient sur ces rheÌtoriques meÌdicales, de saisir ces promesses intellectuelles et techniques dans le temps long, en les articulant aux relations theÌrapeutiques quâelles induisent. En investissant dâune part les imaginaires soignants, et dâautre part la place des individus souffrants, nous souhaitons enqueÌter sur les ceÌsures que geÌneÌre lâeÌmergence des donneÌes meÌdicales en grand nombre, comme sur les treÌs fortes permanences, jusquâaux discours contemporains, de lâideÌe dâun « progreÌs » neÌcessairement obtenu par une mise aÌ distance, de plus en plus importante, de la meÌdiation humaine, pour saisir les corps et leurs pathologies. Nowadays, the paradigm of large numbers (big data) is leading to a renewal of medical representations of the body (which is fragmented into so many comparable data that can be arranged at will) and of the therapeutic relationship. The advent of so-called personalised medicine, of artificial intelligence and its algorithmic devices is indeed accompanied by new imaginary and rhetorical approaches to care. The latter apply themselves to deploying a prophetic discourse on the scientific discoveries to come, on the capacity of science to repel diseases and even death, to reinforce and normalise bodies; the idea of an all-powerful medicine, in short. The 'truth' of bodies and the resolution of their disorders can only be found in what is thought of as a double objectification: the processing of quantitatively numerous data and the carrying out of the exercise by a machine that is not endowed with affects. This issue revisits these medical rhetoric, to grasp these intellectual and technical promises in the long term, by articulating them to the therapeutic relationships they induce. By looking at the imaginary of care-givers on the one hand, and the place of suffering individuals on the other, we wish to investigate the gaps generated by the emergence of large amounts of medical data, as well as the strong persistence, right up to contemporary discourses, of the idea of a "progress" necessarily obtained by distancing human mediation to an increasing extent, in order to grasp the bodies and their pathologies. En la actualidad, el paradigma de los grandes nĂșmeros (big data) estĂĄ llevando a una renovaciĂłn de las representaciones mĂ©dicas del cuerpo (que se ve fragmentado en datos comparables y organizables a voluntad) y de la relaciĂłn terapĂ©utica. El advenimiento de la llamada medicina personalizada, de la inteligencia artificial y de sus dispositivos algorĂtmicos va, en efecto, acompañado de nuevos imaginarios y retĂłricas de los cuidados. Estas Ășltimas se abocan a desplegar un discurso profĂ©tico sobre los descubrimientos cientĂficos venideros, sobre la capacidad de las ciencias para hacer retroceder a las enfermedades e incluso a la muerte, para reforzar y normalizar los cuerpos; la idea de una medicina todopoderosa, en definitiva. La âverdadâ de los cuerpos y la resoluciĂłn de sus trastornos solo podrĂa encontrarse en lo que se piensa como una doble objetivaciĂłn: el tratamiento de datos cuantitativamente numerosos y la realizaciĂłn del ejercicio por una mĂĄquina desprovista de afectos. Este nĂșmero revisa esta retĂłrica mĂ©dica, para captar estas promesas intelectuales y tĂ©cnicas en una larga duraciĂłn, articulĂĄndolas a las relaciones terapĂ©uticas que inducen. Examinando por un lado los imaginarios del cuidado y, por otro, el lugar de los pacientes, queremos investigar las cesuras generadas por la apariciĂłn de los datos mĂ©dicos en grandes cantidades, asĂ como la fuerte persistencia, hasta los discursos contemporĂĄneos, de la idea de un âprogresoâ que se obtiene necesariamente poniendo cada vez mĂĄs a distancia la mediaciĂłn humana para aprehender los cuerpos y sus patologĂas
CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations
International audienceCRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nucleaseinduced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on-and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies