Bioactive food components for colorectal cancer prevention and treatment: A good match.

Colorectal cancer (CRC) is the third most frequent cancer worldwide, accounts for about 10% of the total cancer cases, and ranks as the second cause of death by cancer. CRC is more prevalent in developed countries in close causal relation with occidental diets. Due to anatomy, the diet has a strong impact on CRC. High contents in meat are acknowledged risk factors whereas a diet rich in fruits and vegetables is an established CRC protective factor. Fruits and vegetables contain numerous Bioactive Food Components (BFCs), physiologically active food compounds, beneficial on health. Preventive and therapeutic benefits of BFCs in cancer have increasingly been reported over the past 20 years. BFCs show both chemopreventive and anti-tumor properties in CRC but more interestingly, abundant research describes BFCs as enhancers of conventional cancer treatments. Despite these promising results, their clinical transferability is slowed down by bioavailability interrogations and their poorly understood hormetic effect. In this review, we would like to reposition BFCs as well-fitted for applications in CRC. We provide a synthetic overview of trustworthy BFC applications in CRC, with a special highlight on combinatory approaches and conventional cancer treatment potentiation strategies.

The Crying Need for a Better Response Assessment in Rectal Cancer.

OPINION STATEMENT: Since total neoadjuvant treatment achieves almost 30% pathologic complete response, organ preservation has been increasingly debated for good responders after neoadjuvant treatment for patients diagnosed with rectal cancer. Two organ preservation strategies are available: a watch and wait strategy and a local excision strategy including patients with a near clinical complete response. A major issue is the selection of patients according to the initial tumor staging or the response assessment. Despite modern imaging improvement, identifying complete response remains challenging. A better selection could be possible by radiomics analyses, exploiting numerous image features to feed data characterization algorithms. The subsequent step is to include baseline and/or pre-therapeutic MRI, PET-CT, and CT radiomics added to the patients' clinicopathological data, inside machine learning (ML) prediction models, with predictive or prognostic purposes. These models could be further improved by the addition of new biomarkers such as circulating tumor biomarkers, molecular profiling, or pathological immune biomarkers.

Impact of Albumin Assays in the Diagnosis of Malnutrition in Hemodialysis Patients: A Cohort Study.

OBJECTIVE: In hemodialysis (HD) patients, malnutrition should be diagnosed by several assessment tools including a plasma albumin concentration of less than 3.8 g/dL or 3.5 g/dL using bromocresol green or immunonephelometry (IN), respectively. However, albumin measurement is not yet standardized and two alternative methods are also commonly used in laboratories: bromocresol purple (BCP) and immunoturbidimetry (IT). This study aimed to revisit the hypoalbuminemia thresholds for BCP and IT, in HD patients. METHODS: Plasma albumin was measured by the four analytical methods during the monthly HD nutritional assessment of 103 prospectively included patients. RESULTS: Significant differences in albumin levels were observed in HD patients depending on the method used. Using BCP or IT with the cut-off at 3.5 g/dL (determined for the general population) we obtained 33% and 9.7% of false hypoalbuminemia in comparison to IN (mean bias of -0.4 g/dL and -0.065 g/dL, respectively). The best hypoalbuminemia threshold for BCP was 3.05 g/dL and 3.4 g/dL for IT. Twenty percent of HD patients were classified as malnourished when albumin was determined by IN. Similar rates were obtained using the new hypoalbuminemia cut-offs for BCP (18.5%) and IT (19.5%). CONCLUSION: To avoid nutritional misclassification of HD patients, we should adjust hypoalbuminemia thresholds when BCP or IT methods are used in laboratories.

Nme1 and Nme2 genes exert metastasis-suppressor activities in a genetically engineered mouse model of UV-induced melanoma.

NME1 is a metastasis-suppressor gene (MSG), capable of suppressing metastatic activity in cell lines of melanoma, breast carcinoma and other cancer origins without affecting their growth in culture or as primary tumours. Herein, we selectively ablated the tandemly arranged Nme1 and Nme2 genes to assess their individual impacts on metastatic activity in a mouse model (HGF:p16-/-) of ultraviolet radiation (UVR)-induced melanoma. Metastatic activity was strongly enhanced in both genders of Nme1- and Nme2-null mice, with stronger activity in females across all genotypes. The study ascribes MSG activity to Nme2 for the first time in an in vivo model of spontaneous cancer, as well as a novel metastasis-suppressor function to Nme1 in the specific context of UVR-induced melanoma.

Circulating Tumor Cell Clusters: United We Stand Divided We Fall.

The presence of circulating tumor cells (CTCs) and CTC clusters, also known as tumor microemboli, in biological fluids has long been described. Intensive research on single CTCs has made a significant contribution in understanding tumor invasion, metastasis tropism, and intra-tumor heterogeneity. Moreover, their being minimally invasive biomarkers has positioned them for diagnosis, prognosis, and recurrence monitoring tools. Initially, CTC clusters were out of focus, but major recent advances in the knowledge of their biogenesis and dissemination reposition them as critical actors in the pathophysiology of cancer, especially metastasis. Increasing evidence suggests that "united" CTCs, organized in clusters, resist better and carry stronger metastatic capacities than "divided" single CTCs. This review gathers recent insight on CTC cluster origin and dissemination. We will focus on their distinct molecular package necessary to resist multiple cell deaths that all circulating cells normally face. We will describe the molecular basis of their increased metastatic potential as compared to single CTCs. We will consider their clinical relevance as prognostic biomarkers. Finally, we will propose future directions for research and clinical applications in this promising topic in cancer.

Intraductal papillary mucinous neoplasms of the pancreas and European guidelines: importance of the surgery type in the decision-making process.

BACKGROUND: The European Consensus 2018 established a new algorithm with absolute and relative criteria for intraductal papillary mucinous neoplasms of the pancreas (IPMN) management. The aim of this study was to validate these criteria and analyse the outcomes in function of the surgical procedure and IPMN subtype. METHODS: Clinical, radiological and surgical data (procedure, morbidity/mortality rates) of patients who underwent surgery for IPMN between 2007 and 2017. The predictive value of the different criteria was analysed. RESULTS: 124 patients (men 67%; mean age 65 years) underwent surgery for IPMN (n = 62 malignant tumours; 50%). Jaundice, cyst ≥4 cm and Wirsung duct size 5-9.9 mm or ≥ 10 mm were significantly associated with malignancy (4.77 < OR < 11.85 p < 0.0001). The positive predictive value of any isolated criterion ranged from 71 to 87%, whereas that of three relative criteria together reached 100%. The mortality and morbidity (grade III-IV complications according to the Dindo-Clavien classification) rates were 3 and 8%, respectively. Morbidity/mortality after duodenopancreatectomy and total pancreatectomy were significantly higher for benign IPMN (p = 0.01). CONCLUSION: Considering the morbidity associated with extended surgery, particularly for benign IPMN, the results of the present study suggest that high-risk surgery should be considered only in the presence of three relative criteria and including the surgery type in the decision-making algorithm.

Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

NME1 (nonmetastatic expressed 1) gene, which encodes nucleoside diphosphate kinase (NDPK) A [also known as nonmetastatic clone 23 (NM23)-H1 in humans and NM23-M1 in mice], is a suppressor of metastasis, but several lines of evidence-mostly from plants-also implicate it in the regulation of the oxidative stress response. Here, our aim was to investigate the physiologic relevance of NDPK A with respect to the oxidative stress response in mammals and to study its molecular basis. NME1-knockout mice died sooner, suffered greater hepatocyte injury, and had lower superoxide dismutase activity than did wild-type (WT) mice in response to paraquat-induced acute oxidative stress. Deletion of NME1 reduced total NDPK activity and exacerbated activation of the stress-related MAPK, JNK, in the liver in response to paraquat. In a mouse transformed hepatocyte cell line and in primary cultures of normal human keratinocytes, MAPK activation in response to H2O2 and UVB, respectively, was dampened by expression of NM23-M1/NM23-H1, dependent on its NDPK catalytic activity. Furthermore, excess or depletion of NM23-M1/NM23-H1 NDPK activity did not affect the intracellular bulk concentration of nucleoside di- and triphosphates. NME1-deficient mouse embryo fibroblasts grew poorly in culture, were more sensitive to stress than WT fibroblasts, and did not immortalize, which suggested that they senesce earlier than do WT fibroblasts. Collectively, these results indicate that the NDPK activity of NM23-M1/NM23-H1 protects cells from acute oxidative stress by inhibiting activation of JNK in mammal models.-Peuchant, E., Bats, M.-L., Moranvillier, I., Lepoivre, M., Guitton, J., Wendum, D., Lacombe, M.-L., Moreau-Gaudry, F., Boissan, M., Dabernat, S. Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

Metabolic correction of congenital erythropoietic porphyria with iPSCs free of reprogramming factors.

Congenital erythropoietic porphyria (CEP) is due to a deficiency in the enzymatic activity of uroporphyrinogen III synthase (UROS); such a deficiency leads to porphyrin accumulation and results in skin lesions and hemolytic anemia. CEP is a candidate for retrolentivirus-mediated gene therapy, but recent reports of insertional leukemogenesis underscore the need for safer methods. The discovery of induced pluripotent stem cells (iPSCs) has opened up new horizons in gene therapy because it might overcome the difficulty of obtaining sufficient amounts of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. In this study, we isolated keratinocytes from a CEP-affected individual and generated iPSCs with two excisable lentiviral vectors. Gene correction of CEP-derived iPSCs was obtained by lentiviral transduction of a therapeutic vector containing UROS cDNA under the control of an erythroid-specific promoter shielded by insulators. One iPSC clone, free of reprogramming genes, was obtained with a single proviral integration of the therapeutic vector in a genomic safe region. Metabolic correction of erythroblasts derived from iPSC clones was demonstrated by the disappearance of fluorocytes. This study reports the feasibility of porphyria gene therapy with the use of iPSCs.

Droplet digital polymerase chain reaction detection of KRAS mutations in pancreatic FNA samples: Technical and practical aspects for routine clinical implementation.

BACKGROUND: Pancreatic adenocarcinoma (PDAC) is associated with a 5-year survival rate of less than 6%, and current treatments have limited efficacy. The diagnosis of PDAC is mainly based on a cytologic analysis of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) samples. However, the collected specimens may prove noncontributory in a significant number of cases, delaying patient management and treatment. The combination of EUS-FNA sample examination and KRAS mutation detection can improve the sensitivity for diagnosis. In this context, the material used for molecular analysis may condition performance. METHODS: The authors prospectively compared the performance of cytologic analysis combined with a KRAS droplet digital polymerase chain reaction (ddPCR) assay for PDAC diagnosis using either conventional formalin-fixed, paraffin-embedded cytologic samples or needle-rinsing fluids. RESULTS: Molecular testing of formalin-fixed, paraffin-embedded cytologic samples was easier to set up, but the authors observed that the treatment of preanalytic samples, in particular the fixation process, drastically reduced ddPCR sensitivity, increasing the risk of false-negative results. Conversely, the analysis of dedicated, fresh needle-rinsing fluid samples appeared to be ideal for ddPCR analysis; it had greater sensitivity and was easily to implement in clinical use. In particular, fluid collection by the endoscopist, transportation to the laboratory, and subsequent freezing did not affect DNA quantity or quality. Moreover, the addition of KRAS mutation detection to cytologic examination improved diagnosis performance, regardless of the source of the sample. CONCLUSIONS: Considering all of these aspects, the authors propose the use of an integrated flowchart for the KRAS molecular testing of EUS-FNA samples in clinical routine.

CRISPR editing to mimic porphyria combined with light: A new preclinical approach for prostate cancer.

Thanks to its very high genome-editing efficiency, CRISPR-Cas9 technology could be a promising anticancer weapon. Clinical trials using CRISPR-Cas9 nuclease to ex vivo edit and alter immune cells are ongoing. However, to date, this strategy still has not been applied in clinical practice to directly target cancer cells. Targeting a canonical metabolic pathway essential to good functioning of cells without potential escape would represent an attractive strategy. We propose to mimic a genetic metabolic disorder in cancer cells to weaken cancer cells, independent of their genomic abnormalities. Mutations affecting the heme biosynthesis pathway are responsible for porphyria, and most of them are characterized by an accumulation of toxic photoreactive porphyrins. This study aimed to mimic porphyria by using CRISPR-Cas9 to inactivate UROS, leading to porphyrin accumulation in a prostate cancer model. Prostate cancer is the leading cancer in men and has a high mortality rate despite therapeutic progress, with a primary tumor accessible to light. By combining light with gene therapy, we obtained high efficiency in vitro and in vivo, with considerable improvement in the survival of mice. Finally, we achieved the preclinical proof-of-principle of performing cancer CRISPR gene therapy.

FGFR3 has tumor suppressor properties in cells with epithelial phenotype.

BACKGROUND: Due to frequent mutations in certain cancers, FGFR3 gene is considered as an oncogene. However, in some normal tissues, FGFR3 can limit cell growth and promote cell differentiation. Thus, FGFR3 action appears paradoxical. RESULTS: FGFR3 expression was forced in pancreatic cell lines. The receptor exerted dual effects: it suppressed tumor growth in pancreatic epithelial-like cells and had oncogenic properties in pancreatic mesenchymal-like cells. Distinct exclusive pathways were activated, STATs in epithelial-like cells and MAP Kinases in mesenchymal-like cells. Both FGFR3 splice variants had similar effects and used the same intracellular signaling. In human pancreatic carcinoma tissues, levels of FGFR3 dropped in tumors. CONCLUSION: In tumors from epithelial origin, FGFR3 signal can limit tumor growth, explaining why the 4p16.3 locus bearing FGFR3 is frequently lost and why activating mutations of FGFR3 in benign or low grade tumors of epithelial origin are associated with good prognosis. The new hypothesis that FGFR3 can harbor both tumor suppressive and oncogenic properties is crucial in the context of targeted therapies involving specific tyrosine kinase inhibitors (TKIs). TKIs against FGFR3 might result in adverse effects if used in the wrong cell context.

Specific High-Sensitivity Enzymatic Reporter UnLOCKing-Mediated Detection of Oncogenic BCR::ABL1 and EGFR Rearrangements.

Advances in molecular medicine have placed nucleic acid detection methods at the center of an increasing number of clinical applications. Polymerase chain reaction (PCR)-based diagnostics have been widely adopted for their versatility, specificity, and sensitivity. However, recently reported clustered regularly interspaced short palindromic repeats-based methods have demonstrated equivalent to superior performance, with increased portability and reduced processing time and cost. In this study, we applied Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology to the detection of oncogenic rearrangements. We implemented SHERLOCK for the detection of BCR::ABL1 mRNA, a hallmark of chronic myeloid leukemia (CML), and EGFR DNA oncogenic alleles, frequently detected in glioblastoma and non-small cell lung cancer (NSCLC). SHERLOCK enabled rapid, sensitive, and variant-specific detection of BCR::ABL1 and EGFR alterations. Compared with the gold-standard PCR-based methods currently used in clinic, SHERLOCK achieved equivalent to greater sensitivity, suggesting it could be a new tool in CML and NSCLC, to detect low level of molecular residual disease.

In vivo gene transfer targeting in pancreatic adenocarcinoma with cell surface antigens.

BACKGROUND: Pancreatic ductal adenocarcinoma is a deadly malignancy resistant to current therapies. It is critical to test new strategies, including tumor-targeted delivery of therapeutic agents. This study tested the possibility to target the transfer of a suicide gene in tumor cells using an oncotropic lentiviral vector. RESULTS: Three cell surface markers were evaluated to target the transduction of cells by lentiviruses pseudotyped with a modified glycoprotein from Sindbis virus. Only Mucin-4 and the Claudin-18 proteins were found efficient for targeted lentivirus transductions in vitro. In subcutaneous xenografts of human pancreatic cancer cells models, Claudin-18 failed to achieve efficient gene transfer but Mucin-4 was found very potent. Human pancreatic tumor cells were modified to express a fluorescent protein detectable in live animals by bioimaging, to perform a direct non invasive and costless follow up of the tumor growth. Targeted gene transfer of a bicistronic transgene bearing a luciferase gene and the herpes simplex virus thymidine kinase gene into orthotopic grafts was carried out with Mucin-4 oncotropic lentiviruses. By contrast to the broad tropism VSV-G carrying lentivirus, this oncotropic lentivirus was found to transduce specifically tumor cells, sparing normal pancreatic cells in vivo. Transduced cells disappeared after ganciclovir treatment while the orthotopic tumor growth was slowed down. CONCLUSION: This work considered for the first time three aspect of pancreatic adenocarcinoma targeted therapy. First, lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. Second, we used a system targeting the tumor cells with cell surface antigens and sparing the normal cells. Finally, the TK/GCV anticancer system showed promising results in vivo. Importantly, the approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors, in comparison with a broad tropism lentivirus. This study will be useful in future designing of targeted therapies for pancreatic cancer.

ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected.

CRISPR-Cas9 is a highly promising technology for clinical development. However, this powerful tool can induce adverse genomic events. The off-target genotoxicity is well described, predictable, detectable, and resolved by the use of new generations of Cas9 nucleases with high fidelity. In contrast, the ON-target genotoxicity due to a DNA double-strand break at the targeted locus is still underestimated. Here, we review several genomic outcomes induced by CRISPR-Cas9 from the insertion/deletion of a few bases to megabase-scale rearrangements. We hope to highlight this barely detectable complex safety concern to promote further studies to understand the mechanisms better, to detect these unwanted events, and to prevent them for the safety management of future CRISPR-Cas9 clinical trials.

Plasma creatinine below limit of quantification in a patient with acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) is an infrequent complication of inflammatory bowel disease and can be exceptionally linked to interstitial nephritis secondary to anti-inflammatory drugs, such as Pentasa® (5-ASA). CASE PRESENTATION: We present an case of an 80-year-old man who presented chronic diarrheas treated by Pentasa®. He developed AKI, evidenced by high plasma creatinine dosed in his local laboratory. At the hospital admission, plasma creatinine was exceptionally undetectable by the enzymatic method while Jaffe's method successfully determined it. Creatinine measurement by the enzymatic method was gradually restored during hospital stay, concomitant with the discontinuation of 5-ASA administration, suggesting that this drug could interfere with creatinine enzymatic assay. Creatinine enzymatic assays combine serial reactions. The last one called Trinder reaction, catalyzed by a peroxidase, uses H2O2 to convert uncolored dye in a colored compound, proportionally to creatinine concentration. We showed that AKI related-plasma accumulation of 5-ASA, could participate in the negative interference observed on creatinine measurement, by scavenging H2O2. Interestingly, all Trinder reaction-based measurements (uric acid, lipase, lactate, triglycerides and cholesterol) were affected. Negative interference of 5-ASA was confirmed by interferogram experiments on all Trinder reaction-dependent assays. CONCLUSION: All Trinder-dependent parameters should be interpreted with the patient's treatment knowledge, in particular salicylate derivatives.

KRAS gene mutation quantification in the resection or venous margins of pancreatic ductal adenocarcinoma is not predictive of disease recurrence.

Pancreatic ductal adenocarcinoma (PDAC) patients eligible for curative surgery undergo unpredictable disease relapse. Even patients with a good pathological response after neoadjuvant treatment (NAT) remain susceptible to recurrent PDAC. Molecular analysis of R0 margins may identify patients with a worse prognosis. The molecular status of mutant KRAS (exon 2, codon 12/13) was analysed retrospectively by digital droplet PCR in tumour areas, venous and resection margins of resected tumours, either undergoing up-front surgery (UFS) or after NAT with a good pathological response. Expectedly, tumour tissues or remnants from patients who underwent NAT presented lower KRAS mutant allele frequencies (MAF) than patients eligible for UFS. Similarly, ypT1 tumour MAFs were greater than the ypT0 tumour remnant MAFs in the NAT group. Mutant KRAS status in margins did not distinguish NAT subgroups. It was not predictive of shorter recurrence-free or overall survival within or between groups. KRAS-double negativity in both venous and resection margins did not identify patients with a better prognosis, regardless of the groups. The cohorts 'sizes were small due to limited numbers of patients meeting the inclusion criteria, but KRAS-positivity or MAFs in resection and venous margins did not carry prognostic value. Comparison of margins from good versus bad responders receiving NAT may provide better clinical value.

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 × 2 Gy three times a week resulted in intermediate survival [48-62 days] compared to 0 Gy (15-24 days), 3 × 2 Gy (41-47 days) and, 5 × 3 Gy (73-83 days). Survival was correlated to tumor growth. Tumor growth and survival were similar after 5 × 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.

Covichem: A biochemical severity risk score of COVID-19 upon hospital admission.

Clinical and laboratory predictors of COVID-19 severity are now well described and combined to propose mortality or severity scores. However, they all necessitate saturable equipment such as scanners, or procedures difficult to implement such as blood gas measures. To provide an easy and fast COVID-19 severity risk score upon hospital admission, and keeping in mind the above limits, we sought for a scoring system needing limited invasive data such as a simple blood test and co-morbidity assessment by anamnesis. A retrospective study of 303 patients (203 from Bordeaux University hospital and an external independent cohort of 100 patients from Paris Pitié-Salpêtrière hospital) collected clinical and biochemical parameters at admission. Using stepwise model selection by Akaike Information Criterion (AIC), we built the severity score Covichem. Among 26 tested variables, 7: obesity, cardiovascular conditions, plasma sodium, albumin, ferritin, LDH and CK were the independent predictors of severity used in Covichem (accuracy 0.87, AUROC 0.91). Accuracy was 0.92 in the external validation cohort (89% sensitivity and 95% specificity). Covichem score could be useful as a rapid, costless and easy to implement severity assessment tool during acute COVID-19 pandemic waves.

Pigments test strips: A rapid companion test to exclude sub-arachnoid haemorrhage.

OBJECTIVES: Subarachnoid haemorrhage (SAH) is characterised by 25% of mortality or induces long-term care. It needs immediate diagnosis with computed tomography (CT) scan. For the inconclusive CT scans, the detection of haem pigments can be performed in the cerebrospinal fluid (CSF). The reference method is spectrophotometry but it requires a large volume of CSF, and specific equipment. Sometimes, urine test strips are used as an alternative method for haem pigments detection. However, this method needs validation in SAH context. The aim of the study was to compare the performance of Multistix® urine test strips for haem pigments detection to the reference spectrophotometry and the final clinical SAH diagnosis. METHODS: We collected 136 CSFs sampled for suspected SAH. We detected haem pigments with urine test strips and spectrophotometry and compared performances for 100 samples. RESULTS: Urine tests strips displayed a high sensitivity (0.97) as compared to the reference spectrophotometry for haem pigments detection. Interestingly, absence of haem pigments fully correlated with absence of SAH. CONCLUSIONS: Negative Multistix® urine test strips could help to exclude SAH diagnosis in combination with clinical data when a spectrophotometer is not available, or as a bedside diagnosis test.

Mutation-Specific Guide RNA for Compound Heterozygous Porphyria On-target Scarless Correction by CRISPR/Cas9 in Stem Cells.

CRISPR/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.