Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

Intra-subject variability in oscillometry correlates with acute rejection and CLAD post-lung transplant.

Background: Chronic lung allograft dysfunction (CLAD) is the major cause of death post-lung transplantation, with acute cellular rejection (ACR) being the biggest contributing risk factor. Although patients are routinely monitored with spirometry, FEV1 is stable or improving in most ACR episodes. In contrast, oscillometry is highly sensitive to respiratory mechanics and shown to track graft injury associated with ACR and its improvement following treatment. We hypothesize that intra-subject variability in oscillometry measurements correlates with ACR and risk of CLAD. Methods: Of 289 bilateral lung recipients enrolled for oscillometry prior to laboratory-based spirometry between December 2017 and March 2020, 230 had ≥ 3 months and 175 had ≥ 6 months of follow-up. While 37 patients developed CLAD, only 29 had oscillometry at time of CLAD onset and were included for analysis. These 29 CLAD patients were time-matched with 129 CLAD-free recipients. We performed multivariable regression to investigate the associations between variance in spirometry/oscillometry and the A-score, a cumulative index of ACR, as our predictor of primary interest. Conditional logistic regression models were built to investigate associations with CLAD. Results: Multivariable regression showed that the A-score was positively associated with the variance in oscillometry measurements. Conditional logistic regression models revealed that higher variance in the oscillometry metrics of ventilatory inhomogeneity, X5, AX, and R5-19, was independently associated with increased risk of CLAD (p < 0.05); no association was found for variance in %predicted FEV1. Conclusion: Oscillometry tracks graft injury and recovery post-transplant. Monitoring with oscillometry could facilitate earlier identification of graft injury, prompting investigation to identify treatable causes and decrease the risk of CLAD.

Characterization of chronic lung allograft dysfunction phenotypes using spectral and intrabreath oscillometry.

Background: Chronic lung allograft dysfunction (CLAD) is the major cause of death beyond 2 years after lung transplantation and develops in 50% of all patients by 5 years post-transplant. CLAD is diagnosed on the basis of a sustained drop of 20% for at least 3 months in the forced expiratory volume (FEV1), compared to the best baseline value achieved post-transplant. CLAD presents as two main phenotypes: bronchiolitis obliterans syndrome (BOS) is more common and has better prognosis than restrictive allograft syndrome (RAS). Respiratory oscillometry is a different modality of lung function testing that is highly sensitive to lung mechanics. The current study investigated whether spectral and intrabreath oscillometry can differentiate between CLAD-free, BOS- and RAS-CLAD at CLAD onset, i.e., at the time of the initial 20% drop in the FEV1. Methods: A retrospective, cross-sectional analysis of 263 double lung transplant recipients who underwent paired testing with oscillometry and spirometry at the Toronto General Pulmonary Function Laboratory from 2017 to 2022 was conducted. All pulmonary function testing and CLAD diagnostics were performed following international guidelines. Statistical analysis was conducted using multiple comparisons. Findings: The RAS (n = 6) spectral oscillometry pattern differs from CLAD-free (n = 225) by right-ward shift of reactance curve similar to idiopathic pulmonary fibrosis whereas BOS (n = 32) has a pattern similar to obstructive lung disease. Significant differences were found in most spectral and intrabreath parameters between BOS, RAS, and time-matched CLAD-free patients. Post-hoc analysis revealed these differences were primarily driven by BOS instead of RAS. While no differences were found between CLAD-free and RAS patients with regards to spectral oscillometry, the intrabreath metric of reactance at end-inspiration (XeI) was significantly different (p < 0.05). BOS and RAS were differentiated by spectral oscillometry measure R5, and intrabreath resistance at end expiration, ReE (p < 0.05 for both). Conclusion: Both spectral and intrabreath oscillometry can differentiate BOS-CLAD from CLAD-free states while intrabreath oscillometry, specifically XeI, can uniquely distinguish RAS-CLAD from CLAD-free. Spectral and intrabreath oscillometry offer complementary information regarding lung mechanics in CLAD patients to help distinguish the two phenotypes and could prove useful in prognostication.

Hepatitis B splice-generated protein antibodies in Syrian chronic hepatitis B patients: incidence and significance.

BACKGROUND: Previous studies have suggested hepatitis B splice-generated protein (HBSP), when expressed, is involved in the pathogenesis of HBV infection. OBJECTIVES: We aimed to evaluate anti-HBSP incidence and association with several HBV infection parameters in a group of Syrian chronic hepatitis B patients. PATIENTS AND METHODS: Eighty treatment-naïve HBsAg-positive adult chronic hepatitis B patients' sera were included in our prospective targeted study. Liver function, virological and histological tests results were obtained from patients' medical files. Three variants of a 20-mer HBSP-derived peptide were designed based on HBV genome sequences obtained from Syrian patients' sera (GenBank Accession No. JN257148-JN257217). Microtiter plate wells were coated with the synthetic peptides and used to detect anti-HBSP antibodies by an optimized indirect enzyme-linked immunosorbent assay (ELISA). Samples were considered positive when showed optical density (OD) values higher than the cut-off value for at least one peptide variant. RESULTS: Seven out of eighty (9%) CHB patients were positive for anti-HBSP antibodies. Mean OD values were not significantly different between HBeAg-positive and -negative patients (P > 0.05). OD values showed weak positive correlation with ALT and AST values (P < 0.05), and weak to moderate positive correlation with liver biopsy staging ranks (P < 0.05). No significant correlation was revealed with viral load values or liver biopsy grading ranks (P > 0.05). CONCLUSIONS: We introduced an anti-HBSP antibodies ELISA, designed for locally circulating HBV strains. Correlation observed of Anti-HBSP with liver fibrosis staging regardless of viral replication and liver inflammation suggests anti-HBSP antibodies as possible indicator for HBV-associated liver fibrosis.