BACKGROUND: Inherited retinal dystrophies (IRD) are one of the main causes of incurable blindness worldwide. IRD are caused by mutations in genes that encode essential proteins for the retina, leading to photoreceptor degeneration and loss of visual function. IRD generates an enormous global financial burden due to the lack of understanding of a significant part of its pathophysiology, molecular diagnosis, and the near absence of non-palliative treatment options. Patient-derived induced pluripotent stem cells (iPSC) for IRD seem to be an excellent option for addressing these questions, serving as exceptional tools for in-depth studies of IRD pathophysiology and testing new therapeutic approaches.
METHODS: From a cohort of 8 patients with PROM1-related IRD, we identified 3 patients carrying the same variant (c.1354dupT) but expressing three different IRD phenotypes: Cone and rod dystrophy (CORD), Retinitis pigmentosa (RP), and Stargardt disease type 4 (STGD4). These three target patients, along with one healthy relative from each, underwent comprehensive ophthalmic examinations and their genetic panel study was expanded through clinical exome sequencing (CES). Subsequently, non-integrative patient-derived iPSC were generated and fully characterized. Correction of the c.1354dupT mutation was performed using CRISPR/Cas9, and the genetic restoration of the PROM1 gene was confirmed through flow cytometry and western blotting in the patient-derived iPSC lines.
RESULTS: CES revealed that 2 target patients with the c.1354dupT mutation presented monoallelic variants in genes associated with the complement system or photoreceptor differentiation and peroxisome biogenesis disorders, respectively. The pluripotency and functionality of the patient-derived iPSC lines were confirmed, and the correction of the target mutation fully restored the capability of encoding Prominin-1 (CD133) in the genetically repaired patient-derived iPSC lines.
CONCLUSIONS: The c.1354dupT mutation in the PROM1 gene is associated to three distinct AR phenotypes of IRD. This pleotropic effect might be related to the influence of monoallelic variants in other genes associated with retinal dystrophies. However, further evidence needs to be provided. Future experiments should include gene-edited patient-derived iPSC due to its potential as disease modelling tools to elucidate this matter in question.

OBJECTIVE: Charcot-Marie-Tooth disease (CMT) severely affects patient activity, and may cause disability. However, no clinical treatment is available to reverse the disease course. The combination of CRISPR/Cas9 and iPSCs may have therapeutic potential against nervous diseases, such as CMT.
METHODS: In the present study, the skin fibroblasts of CMT type 2D (CMT2D) patients with the c.880G>A heterozygous nucleotide mutation in the GARS gene were reprogrammed into iPSCs using three plasmids (pCXLE-hSK, pCXLE-hUL and pCXLE-hOCT3/4-shp5-F). Then, CRISPR/Cas9 technology was used to repair the mutated gene sites at the iPSC level.
RESULTS: An iPSC line derived from the GARS (G294R) family with fibular atrophy was successfully induced, and the mutated gene loci were repaired at the iPSC level using CRISPR/Cas9 technology. These findings lay the foundation for future research on drug screening and cell therapy.
CONCLUSIONS: iPSCs can differentiate into different cell types, and originate from autologous cells. Therefore, they are promising for the development of autologous cell therapies for degenerative diseases. The combination of CRISPR/Cas9 and iPSCs may open a new avenue for the treatment of nervous diseases, such as CMT.

RNA-based therapeutics have entered the mainstream with seemingly limitless possibilities to treat all categories of neurological disease. Here, common RNA-based drug modalities such as antisense oligonucleotides, small interfering RNAs, RNA aptamers, RNA-based vaccines and mRNA drugs are reviewed highlighting their current and potential applications. Rapid progress has been made across rare genetic diseases and neurodegenerative disorders, but safe and effective delivery to the brain remains a significant challenge for many applications. The advent of individualized RNA-based therapies for ultra-rare diseases is discussed against the backdrop of the emergence of this field into more common conditions such as Alzheimer\'s disease and ischaemic stroke. There remains significant untapped potential in the use of RNA-based therapeutics for behavioural disorders and tumours of the central nervous system; coupled with the accelerated development expected over the next decade, the true potential of RNA-based therapeutics to transform the therapeutic landscape in neurology remains to be uncovered.

OBJECTIVE: Current guidelines recommend somatic genomic sequencing for patients with advanced pancreatic cancer to identify targetable alterations amenable to targeted therapy. The benefit of somatic genomic sequencing in pancreatic cancer remains unclear. This study aims to assess the evidence supporting genomic sequencing to inform treatment selection for patients with advanced pancreatic cancer.
METHODS: A systematic review identified prospective studies of exocrine pancreatic cancer patients published before August 2020 which conducted genomic sequencing to inform treatment selection. Outcomes of interest included the proportion of patients with targetable alterations, the proportion that received targeted treatments, and the impact of targeted treatments on overall survival. Meta-analysis for proportions and hazard ratios was performed using Dersimonian and Laird random effect models.
RESULTS: 19 studies (representing 2048 pancreatic cancer patients) were included. Sequencing methodologies, definitions of targetable alterations, and approaches treatment selection varied across studies and were incompletely reported. 590 of 1382 sequenced patients harboured a targetable alteration (random effects meta-analysis estimate of the proportion 0.46, 95% confidence interval 0.32-0.61). The proportion of patients with targetable alterations was highly heterogenous between studies (I2 93%, P < 0.001). 91 of 1390 patients received a matched therapy based on their targetable alterations (random effects meta-analysis estimate of the proportion 0.12, 95% CI 0.06-0.23). One observational study reported an overall survival benefit of matched therapy.
CONCLUSIONS: Genomic sequencing frequently identifies targetable alterations in pancreatic cancers. Further research is required to standardize the definitions of targetable alterations, the approach to treatment matching, and quantify the benefit of targeted therapy.

Achondroplasia (ACH) is the most common genetic form of dwarfism and belongs to dominant monogenic disorder caused by a gain-of-function point mutation in the transmembrane region of FGFR3. There are no effective treatments for ACH. Stem cells and gene-editing technology provide us with effective methods and ideas for ACH research and treatment.
We generated non-integrated iPSCs from an ACH girl\'s skin and an ACH boy\'s urine by Sendai virus. The mutation of ACH iPSCs was precisely corrected by CRISPR-Cas9.
Chondrogenic differentiation ability of ACH iPSCs was confined compared with that of healthy iPSCs. Chondrogenic differentiation ability of corrected ACH iPSCs could be restored. These corrected iPSCs displayed pluripotency, maintained normal karyotype, and demonstrated none of off-target indels.
This study may provide an important theoretical and experimental basis for the ACH research and treatment.

OBJECTIVE: K-MASTER project is a Korean national precision medicine platform that screened actionable mutations by analyzing next-generation sequencing (NGS) of solid tumor patients. We compared gene analyses between NGS panel from the K-MASTER project and orthogonal methods.
METHODS: Colorectal, breast, non-small cell lung, and gastric cancer patients were included. We compared NGS results from K-MASTER projects with those of non-NGS orthogonal methods (KRAS, NRAS, and BRAF mutations in colorectal cancer [CRC]; epidermal growth factor receptor [EGFR], anaplastic lymphoma kinase [ALK] fusion, and reactive oxygen species 1 [ROS1] fusion in non-small cell lung cancer [NSCLC], and Erb-B2 receptor tyrosine kinase 2 (ERBB2) positivity in breast and gastric cancers).
RESULTS: In the CRC cohort (n=225), the sensitivity and specificity of NGS were 87.4% and 79.3% (KRAS); 88.9% and 98.9% (NRAS); and 77.8% and 100.0% (BRAF), respectively. In the NSCLC cohort (n=109), the sensitivity and specificity of NGS for EGFR were 86.2% and 97.5%, respectively. The concordance rate for ALK fusion was 100%, but ROS1 fusion was positive in only one of three cases that were positive in orthogonal tests. In the breast cancer cohort (n=260), ERBB2 amplification was detected in 45 by NGS. Compared with orthogonal methods that integrated immunohistochemistry and in situ hybridization, sensitivity and specificity were 53.7% and 99.4%, respectively. In the gastric cancer cohort (n=64), ERBB2 amplification was detected in six by NGS. Compared with orthogonal methods, sensitivity and specificity were 62.5% and 98.2%, respectively.
CONCLUSIONS: The results of the K-MASTER NGS panel and orthogonal methods showed a different degree of agreement for each genetic alteration, but generally showed a high agreement rate.

Hutchinson-Gilford Progeria (acute premature aging) is caused by a de novo point mutation in the lamin A gene. Recently, this mutation has been accurately corrected by base editing in patient cell lines and in a mouse model, resulting in nearly complete reversal to a normal phenotype. This success opens the perspective for clinical applications in Progeria and other diseases.

Clustered Regularly Interspersed Short Palindromic Repeat-CRISPR-Associated (CRISPR-Cas) system has improved the ability to edit and control gene expression as desired. Genome editing approaches are currently leading the biomedical research with improved focus on direct nuclease dependent editing. So far, the research was predominantly intended on genome editing over the DNA level, recent adapted techniques are initiating to secure momentum through their proficiency to provoke modifications in RNA sequence. Integration of this system besides to lateral flow method allows reliable, quick, sensitive, precise and inexpensive diagnostic. These interesting methods illustrate only a small proportion of what is technically possible for this novel technology, but several technological obstacles need to be overcome prior to the CRISPR-Cas genome editing system can meet its full ability. This chapter covers the particulars on recent advances in CRISPR-Cas9 genome editing technology including diagnosis and technical advancements, followed by molecular mechanism of CRISPR-based RNA editing and diagnostic tools and types, and CRISPR-Cas-based biosensors.

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

Calpainopathies are inherited limb-girdle muscular dystrophies, most often following an autosomal recessive (AR) transmission. Autosomal dominant (AD) forms with less severe presentation are increasingly reported. Calpainopathies with autosomal recessive (AR) mutations of the calpain3 gene (CAPN3) are associated with limb girdle muscular dystrophy type R1 (LGMD-R1, OMIM 253600) also referred to as LGMD-2A according to the old nomenclature. LGMD-R1 is the commonest form of all LGMDs, with an estimated prevalence of 10 to 70 cases per million inhabitants, that is a cohort of between 670 and 4,200 patients in France theoritically. Patients present a symmetrical proximal axial myopathy manifesting itself between the first and second decade. The clinical course is variable. The level of Creatine- Kinase (CK) is usually high and there is no cardiac involvement. From a therapeutic perspective, the autosomal recessive form of calpainopathy is quite suitable to gene replacement strategies; the viability of recombinant AAV-mediated calpain 3 transfer has been demonstrated in animal models and clinical trials are expected in the coming years. Meanwhile, natural history studies are needed to prepare for future clinical trials.
UNASSIGNED: Calpaïnopathies - État des lieux et perspectives thérapeutiques.
UNASSIGNED: Les calpaïnopathies sont des dystrophies musculaires des ceintures héréditaires, le plus souvent avec une transmission autosomique récessive (AR). Des formes autosomiques dominantes (AD) de présentation moins sévère sont de plus en plus rapportées. Les calpaïnopathies avec mutations autosomiques récessives du gène de la calpaïne 3 (CAPN3) sont associées à la dystrophie musculaire des ceintures de type R1 (OMIM 253600) ou LGMD-2A, selon l’ancienne nomenclature. La LGMD-R1 est la plus fréquente de toutes les formes de LGMD, sa prévalence étant estimée entre 10 et 70 cas par million d’habitants. Il existerait ainsi entre 670 et 4 200 patients atteints de LGMD-R1 en France. Les patients présentent une myopathie proximale symétrique et axiale se manifestant entre la première et la deuxième décennie. L’évolution est variable. Le taux de Créatine-Phospho-Kinase sérique est élevé et il n’y a pas d’atteinte cardiaque. Au niveau thérapeutique, la forme autosomique récessive de calpaïnopathie se prête à des stratégies de remplacement de gène. La viabilité d’un transfert de calpaïne 3 médié par un AAV recombinant a été démontrée dans des modèles animaux et un passage en clinique est attendu dans les prochaines années. En attendant, des études d’histoire naturelle sont nécessaires afin de préparer les futurs essais cliniques.