CRISPR-Cas9 genomic instability risk evaluation and system optimisation for safe and efficient genome editing

L’outil d’édition du génome CRISPR-Cas9 est extrêmement puissant et promet un avenir remarquable en thérapie génique. Toutefois, introduire une cassure double brin dans le génome n’est pas sans risque et peut conduire à des évènements indésirables. En effet, la survenue de modifications non-souhaitées au locus ciblé et l’apparition de sous-produits génère une génotoxicité on-target. Dans une première partie, nous nous sommes intéressés à évaluer le risque d’instabilité génomique induit par CRISPR-Cas9 dans des cellules pertinentes en clinique sur un locus d’intérêt thérapeutique. Nous avons mis en évidence une forme de génotoxicité on-target à large échelle et étudié les conséquences fonctionnelles. Dans une deuxième partie, nous avons cherché à diminuer le risque de survenue de larges réarrangements en modulant la division cellulaire. Nous avons montré que la quiescence des cellules au moment de la cassure double brin les protégeait contre l’apparition de réarrangements génomiques. Dans une dernière partie, nous nous sommes focalisés sur la génotoxicité on-target la plus fréquente, les insertions et délétions induites lors de la réparation par NHEJ quand une correction précise par HDR est souhaitée. Nous avons conçu un système d’import nucléaire de la matrice de correction HDR pour maximiser le ratio modifications précises/imprécises. L’évaluation du risque lié à l’utilisation de CRISPR-Cas9 et l’optimisation de l’outil et de ses alternatives sont cruciaux pour permettre son utilisation clinique la plus adéquate.The CRISPR-Cas9 genome editing tool is extremely powerful and promises a remarkable future in gene therapy. However, introducing a double-strand break into the genome is not without risk and can lead to adverse events. Indeed, the occurrence of undesired modifications at the targeted locus and the appearance of by-products generate the on-target genotoxicity. In a first part, we assessed the risk of genomic instability induced by CRISPR-Cas9 in clinically relevant cells on a locus of therapeutic interest. We have shown a form of large scale on-target genotoxicity and studied the functional consequences. In a second part, we aimed to reduce the risk of occurrence of large rearrangements by modulating cell division. We have demonstrated that cells in a quiescent state at the time of the double-strand break were protected from the appearance of genomic rearrangements. In a last part, we focused on the most frequent on-target genotoxicity, the NHEJ-induced insertions and deletions, when a precise correction by HDR is intended. We have designed a HDR template nuclear import system to maximize the ratio of precise/imprecise modifications. Assessing the risk linked to the use of CRISPR-Cas9 and the optimisation of the technology as well as its alternatives are crucial for safe and efficient genome editing

Visual outcomes of macular melanocytic lesions after early or delayed proton beam therapy

International audiencePurposeDuring their initial management, some macular melanocytic lesions can be closely monitored to wait for a documented growth before advocating a treatment by irradiation. However, the visual outcomes of this strategy have not yet been assessed. This study compares the visual outcomes of macular melanocytic lesions that underwent delayed proton beam therapy (PBT) after an initial observation to those treated early.MethodsA total of 162 patients with suspicious melanocytic lesions whose margins were located within 3 mm of the fovea were recruited from two French ocular oncology centers.ResultsOverall, 82 patients treated with PBT within 4 months after the initial visit (early PBT group) were compared to 24 patients treated with delayed PBT (delayed PBT group) and 56 patients not treated with PBT (observation group). Visual acuity was not significantly different between baseline and last visit in the observation group (p = 0.325). Between baseline and last visit, the median [IQR] loss in visual acuity was significant in both the early (0.7 [0.2; 1.8], p < 0.001) and the delayed (0.5 [0.2; 1.5], p < 0.001) PBT groups. After irradiation, there was no significant difference between the early and delayed PBT groups for visual loss (p = 0.575), diameter reduction (p = 0.190), and thickness lowering (p = 0.892). In multivariate analysis, history of diabetes mellitus and Bruch's membrane rupture remained significantly associated with greater visual loss (p = 0.036 and p = 0.002, respectively).ConclusionFor small lesions in which there is no clear diagnosis of malignant melanoma, an initial close monitoring to document tumor growth does not impact visual prognosis, despite the potential complications associated with the untreated tumor. However, the survival should remain the main outcome of the treatment of these lesions

Non-Cancer Effects following Ionizing Irradiation Involving the Eye and Orbit

International audienceThe eye is an exemplarily challenging organ to treat when considering ocular tumors. It is at the crossroads of several major aims in oncology: tumor control, organ preservation, and functional outcomes including vision and quality of life. The proximity between the tumor and organs that are susceptible to radiation damage explain these challenges. Given a high enough dose of radiation, virtually any cancer will be destroyed with radiotherapy. Yet, the doses inevitably absorbed by normal tissues may lead to complications, the likelihood of which increases with the radiation dose and volume of normal tissues irradiated. Precision radiotherapy allows personalized decision-making algorithms based on patient and tumor characteristics by exploiting the full knowledge of the physics, radiobiology, and the modifications made to the radiotherapy equipment to adapt to the various ocular tumors. Anticipation of the spectrum and severity of radiation-induced complications is crucial to the decision of which technique to use for a given tumor. Radiation can damage the lacrimal gland, eyelashes/eyelids, cornea, lens, macula/retina, optic nerves and chiasma, each having specific dose–response characteristics. The present review is a report of non-cancer effects that may occur following ionizing irradiation involving the eye and orbit and their specific patterns of toxicity for a given radiotherapy modality.</jats:p

Quantitative analysis of choriocapillaris alterations in swept-source optical coherence tomography-angiography during radiation retinopathy

International audiencePurpose: To evaluate choriocapillaris alterations following proton beam therapy irradiation using swept-source optical coherence tomography-angiography, and to assess their correlation with the grade of radiation retinopathy (RR).Methods: Eyes with uveal melanoma evaluated before and after irradiation with proton beam therapy were included, as well as the healthy fellow eye. The gradation of RR was based on a previously published classification. Choriocapillaris flow voids area was analyzed using Phansalkar thresholding. Retinal vascularization was described by foveal avascular zone (FAZ) perimeter, FAZ area, FAZ circularity index, and percentage of nonperfusion area (PAN) in the superficial capillary plexus (SCP) or deep capillary plexus.Results: A total of 157 eyes of 83 patients were analyzed. Overall, there was a significant difference between the control group, the uveal melanoma before proton beam therapy group, and the grades of RR in the uveal melanoma after proton beam therapy group for FAZ perimeter (P < 0.001), FAZ area (P < 0.001), FAZ-circularity index (P < 0.001), PAN-SCP (P < 0.001), PAN-deep capillary plexus (P < 0.001), and choriocapillaris flow voids area (P < 0.001). Moreover, choriocapillaris flow voids area was significantly increased in the early stages of RR (P = 0.003) and was further significantly correlated with FAZ perimeter (P < 0.001), FAZ area (P < 0.001), FAZ-circularity index (P = 0.010), PAN-SCP (P < 0.001), and PAN-deep capillary plexus (P < 0.001).Conclusion: Quantitative optical coherence tomography-angiography alterations in the choriocapillaris microvascularization are an early biomarker of RR and are correlated to the severity of the disease

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells

International audienceCRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers

COMPARATIVE EFFECTIVENESS OF PROTON BEAM VERSUS PHOTODYNAMIC THERAPY TO SPARE THE VISION IN CIRCUMSCRIBED CHOROIDAL HEMANGIOMA

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Low incidence of SARS-CoV-2, risk factors of mortality and the course of illness in the French national cohort of dialysis patients

International audienceThe aim of this study was to estimate the incidence of COVID-19 disease in the French national population of dialysis patients, their course of illness and to identify the risk factors associated with mortality. Our study included all patients on dialysis recorded in the French REIN Registry in April 2020. Clinical characteristics at last follow-up and the evolution of COVID-19 illness severity over time were recorded for diagnosed cases (either suspicious clinical symptoms, characteristic signs on the chest scan or a positive reverse transcription polymerase chain reaction) for SARS-CoV-2. A total of 1,621 infected patients were reported on the REIN registry from March 16th, 2020 to May 4th, 2020. Of these, 344 died. The prevalence of COVID-19 patients varied from less than 1% to 10% between regions. The probability of being a case was higher in males, patients with diabetes, those in need of assistance for transfer or treated at a self-care unit. Dialysis at home was associated with a lower probability of being infected as was being a smoker, a former smoker, having an active malignancy, or peripheral vascular disease. Mortality in diagnosed cases (21%) was associated with the same causes as in the general population. Higher age, hypoalbuminemia and the presence of an ischemic heart disease were statistically independently associated with a higher risk of death. Being treated at a selfcare unit was associated with a lower risk. Thus, our study showed a relatively low frequency of COVID-19 among dialysis patients contrary to what might have been assumed