Feasibility of Self-Performed Lung Ultrasound with Remote Teleguidance for Monitoring at Home COVID-19 Patients

During the COVID-19 pandemic, use of telemedicine with the aim of reducing the rate of viral transmission increased. This proof-of-concept observational study was planned to test the feasibility of a home-based lung ultrasound (LUS) follow-up performed by patients with mild COVID-19 infection on themselves. We enrolled patients presenting to the emergency department with SARS-CoV-2 infection without signs of pneumonia and indication to discharge. Each patient received a brief training on how to perform LUS and a handheld ultrasound probe. Then, patients were contacted on a daily basis, and LUS images were acquired by the patients themselves under “teleguidance” by the investigator. Twenty-one patients were enrolled with a median age of 44 years. All evaluations were of sufficient quality for a follow up. Probability of a better LUS quality was related to higher degree (odds ratio, OR, 1.42, 95% CI 0.5–3.99) and a lower quality to evaluation time (from 0.71, 95% CI 0.55–0.92 for less than 7 min, to 0.52, 95% CI 0.38–0.7, between 7 and 10 min, and to 0.29, 95% CI 0.2–0.43, for evaluations longer than 10 min). No effect related to gender or age was detected. LUS performed by patients and remotely overseen by expert providers seems to be a feasible and reliable telemedicine tool

VSV-G-Enveloped Vesicles for Traceless Delivery of CRISPR-Cas9

The method of delivery of CRISPR-Cas9 into target cells is a strong determinant of efficacy and specificity in genome editing. Even though high efficiency of Cas9 delivery is necessary for optimal editing, its long-term and high levels of expression correlate with increased off-target activity. We developed vesicles (VEsiCas) carrying CRISPR-SpCas9 ribonucleoprotein complexes (RNPs) that are efficiently delivered into target cells through the fusogenic glycoprotein of the vesicular stomatitis virus (VSV-G). A crucial step for VEsiCas production is the synthesis of the single guide RNA (sgRNA) mediated by the T7 RNA polymerase in the cytoplasm of producing cells as opposed to canonical U6-driven Pol III nuclear transcription. In VEsiCas, the absence of DNA encoding SpCas9 and sgRNA allows rapid clearance of the nuclease components in target cells, which correlates with reduced genome-wide off-target cleavages. Compared with SpCas9 RNPs electroporation, which is currently the method of choice to obtain transient SpCas9 activity, VEsiCas deliver the nuclease with higher efficiency and lower toxicity. We show that a wide variety of cells can be edited through VEsiCas, including a variety of transformed cells, induced pluripotent stem cells (iPSCs), and cardiomyocytes, in vivo. VEsiCas is a traceless CRISPR-Cas9 delivery tool for efficient and safe genome editing that represents a further advancement toward the therapeutic use of the CRISPR-Cas9 technology

Author Correction: Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Genome editing strategies to restore altered splicing events

Cystic fibrosis is an autosomal recessive disease caused by mutations in the CFTR gene. A significant number of mutations (~13%) alter the correct splicing of the CFTR gene, causing the transcription of aberrant transcripts resulting in the production of a non-functional CFTR channel. We focus our research on two intronic CF causing mutations, 3272-26A>G and 3849+10kbC>T that create a new acceptor and donor splice site, respectively, generating in the inclusion of intronic portions into the mRNA. We developed a new genome editing approach to permanently correct the abovementioned mutations by means of CRISPR nucleases. We exploited the use of either Streptococcus pyogenes Cas9, SpCas9, or Acidaminococcus sp. BV3L6, AsCas12a, to edit the aberrant splicing sites and restore the production of the correct transcript, avoiding modifications of the CFTR coding sequence. A comparative analysis between SpCas9 and AsCas12a revealed that the use of AsCas12a with a single crRNA efficiently edits the target loci, producing correctly spliced mRNAs in both 3272-26A>G and 3849+10kbC>T mutations. Furthermore, this genetic repair strategy proved to be highly specific, exhibiting a strong discrimination between the mutated and the wild-type allele and no detectable off-target activity with genome-wide analysis. The selected crRNAs were tested in patients derived primary airway cells and intestinal organoids compound heterozygous for the 3272-26A>G or 3849+10kbC>T mutations, that are considered relevant CF models for translational research. The efficient splicing repair and the complete recovery of CFTR channel activity observed confirmed the goodness of the proposed gene editing strategy. These results demonstrated that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.status: publishe

Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing

Since the early days of its conceptualization and application, human gene transfer held the promise of a permanent solution to genetic diseases including cystic fibrosis (CF). This field went through alternated periods of enthusiasm and distrust. The development of refined technologies allowing site specific modification with programmable nucleases highly revived the gene therapy field. CRISPR nucleases and derived technologies tremendously facilitate genome manipulation offering diversified strategies to reverse mutations. Here we discuss the advancement of gene therapy, from therapeutic nucleic acids to genome editing techniques, designed to reverse genetic defects in CF. We provide a roadmap through technologies and strategies tailored to correct different types of mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, and their applications for the development of experimental models valuable for the advancement of CF therapies

Correction of β-thalassemia by CRISPR/Cas9 editing of the α-globin locus in human hematopoietic stem cells

International audienceAbstract β-thalassemias (β-thal) are a group of blood disorders caused by mutations in the β-globin gene (HBB) cluster. β-globin associates with α-globin to form adult hemoglobin (HbA, α2β2), the main oxygen-carrier in erythrocytes. When β-globin chains are absent or limiting, free α-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis. Clinical data show that severity of β-thal correlates with the number of inherited α-globin genes (HBA1 and HBA2), with α-globin gene deletions having a beneficial effect for patients. Here, we describe a novel strategy to treat β-thal based on genome editing of the α-globin locus in human hematopoietic stem/progenitor cells (HSPCs). Using CRISPR/Cas9, we combined 2 therapeutic approaches: (1) α-globin downregulation, by deleting the HBA2 gene to recreate an α-thalassemia trait, and (2) β-globin expression, by targeted integration of a β-globin transgene downstream the HBA2 promoter. First, we optimized the CRISPR/Cas9 strategy and corrected the pathological phenotype in a cellular model of β-thalassemia (human erythroid progenitor cell [HUDEP-2] β0). Then, we edited healthy donor HSPCs and demonstrated that they maintained long-term repopulation capacity and multipotency in xenotransplanted mice. To assess the clinical potential of this approach, we next edited β-thal HSPCs and achieved correction of α/β globin imbalance in HSPC-derived erythroblasts. As a safer option for clinical translation, we performed editing in HSPCs using Cas9 nickase showing precise editing with no InDels. Overall, we described an innovative CRISPR/Cas9 approach to improve α/β globin imbalance in thalassemic HSPCs, paving the way for novel therapeutic strategies for β-thal

Author Correction: Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.status: Published onlin

Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The 3272-26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR gene, generating new acceptor and donor splice sites respectively. Here we develop a genome editing approach to permanently correct these genetic defects, using a single crRNA and the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets. The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272-26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel. These results demonstrate that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.status: publishe

Functional restoration of a CFTR splicing mutation through RNA delivery of CRISPR adenine base editor

: Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The 2789+5G>A CFTR mutation is a quite frequent defect causing an aberrant splicing and a non-functional CFTR protein. Here we used a CRISPR adenine base editing (ABE) approach to correct the mutation in the absence of DNA double-strand breaks (DSB). To select the strategy, we developed a minigene cellular model reproducing the 2789+5G>A splicing defect. We obtained up to 70% editing in the minigene model by adapting the ABE to the PAM sequence optimal for targeting 2789+5G>A with a SpCas9-NG (NG-ABE). Nonetheless, the on-target base correction was accompanied by secondary (bystander) A-to-G conversions in nearby nucleotides, which affected the wild-type CFTR splicing. To decrease the bystander edits, we used a specific ABE (NG-ABEmax), which was delivered as mRNA. The NG-ABEmax RNA approach was validated in patient-derived rectal organoids and bronchial epithelial cells showing sufficient gene correction to recover the CFTR function. Finally, in-depth sequencing revealed high editing precision genome-wide and allele-specific correction. Here we report the development of a base editing strategy to precisely repair the 2789+5G>A mutation resulting in restoration of the CFTR function, while reducing bystander and off-target activities