Taylor Liu

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

X-linked Severe Combined Immunodeficiency (SCID-X1) due to IL2RG mutations is potentially fatal in infancy where 'emergency' life-saving stem cell transplant may only achieve incomplete immune reconstitution following transplant. Salvage therapy SCID-X1 patients over 2 years old (NCT01306019) is a non-randomized, open-label, phase I/II clinical trial for administration of lentiviral-transduced autologous hematopoietic stem cells following busulfan (6 mg/kg total) conditioning. The primary and secondary objectives assess efficacy in restoring immunity and safety by vector insertion site analysis (VISA). In this ongoing study (19 patients treated), we report VISA in blood lineages from first eight treated patients with longer follow up found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of the High Mobility Group AT-hook 2 gene. All eight patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but without disturbance of hematopoiesis. Our molecular analysis demonstrated a cryptic splice site within the chicken β-globin hypersensitivity 4 insulator element in the vector generating truncated mRNA transcripts from many transcriptionally active gene containing forward-oriented intronic vector insert. A two base-pair change at the splice site within the lentiviral vector eliminated splicing activity while retaining vector functional capability. This highlights the importance of functional analysis of lentivectors for cryptic splicing for preclinical safety assessment and a redesign of clinical vectors to improve safety.

BACKGROUND: Active esophageal cooling reduces the incidence of endoscopically identified severe esophageal lesions during radiofrequency (RF) catheter ablation of the left atrium for the treatment of atrial fibrillation. A formal analysis of the atrioesophageal fistula (AEF) rate with active esophageal cooling has not previously been performed. OBJECTIVES: The authors aimed to compare AEF rates before and after the adoption of active esophageal cooling. METHODS: This institutional review board (IRB)-approved study was a prospective analysis of retrospective data, designed before collecting and analyzing the real-world data. The number of AEFs occurring in equivalent time frames before and after adoption of cooling using a dedicated esophageal cooling device (ensoETM, Attune Medical) were quantified across 25 prespecified hospital systems. AEF rates were then compared using generalized estimating equations robust to cluster correlation. RESULTS: A total of 14,224 patients received active esophageal cooling during RF ablation across the 25 hospital systems, which included a total of 30 separate hospitals. In the time frames before adoption of active cooling, a total of 10,962 patients received primarily luminal esophageal temperature (LET) monitoring during their RF ablations. In the preadoption cohort, a total of 16 AEFs occurred, for an AEF rate of 0.146%, in line with other published estimates for procedures using LET monitoring. In the postadoption cohort, no AEFs were found in the prespecified sites, yielding an AEF rate of 0% (P < 0.0001). CONCLUSIONS: Adoption of active esophageal cooling during RF ablation of the left atrium for the treatment of atrial fibrillation was associated with a significant reduction in AEF rate.

BACKGROUND: T-wave alternans (TWA) is a promising electrocardiogram (ECG) predictor of sudden cardiac arrest, yet needs specialized recordings for conventional spectral analysis. Modified moving average (MMA) analysis is a new approach that can measure TWA from routine ECGs, thus widening its applicability. However, MMA-TWA has not been calibrated against spectral TWA nor outcome in high risk patients. We hypothesized that spectral and MMA-TWA would both predict arrhythmia-free survival on long-term prospective follow-up. METHODS AND RESULTS: In 41 patients with left ventricular systolic dysfunction (ejection fraction 31 +/- 13%), we studied TWA simultaneously using spectral and MMA during pacing (< 110 beats/min). MMA amplified TWA over spectral analyses (13.0 +/- 8.28 microV vs 1.96 +/- 5.15 microV, P < 0.001). On 542 +/- 311 days' follow-up, from clinic visits, telephonic interviews, and device interrogations, there were 11 deaths or sustained ventricular arrhythmias ('events'). Positive spectral TWA (>or=1.9 microV) identified patients with from those without events (P = 0.02). Receiver-operating characteristics for MMA-TWA showed that the cutpoint >or= 10.75 microV was optimal for the combined endpoint. Kaplan-Meier analysis using this MMA-TWA cutpoint trended to predict events (P = 0.06), while MMA combined with spectral TWA identified events (P = 0.01). CONCLUSIONS: MMA amplifies TWA compared to traditional spectral analyses, but both likely reflect similar pathophysiology. Validation in larger populations will enable MMA-TWA to be widely applied to stratify risk for sudden cardiac arrest.